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1. Choose database Nucleotide O Protein All Fields human E All Fields v hemoglobin E All Fields v complete E Add search parameters 8 Start search C Append wildcard to search words Rows 50 Search results Filter Accession Definition Modification Date AM270166 Aspergillus niger contig An08c0110 complete genome 2007 03 24 AM711867 Clavibacter michiganensis subsp michiganensis NCPPB 2007 05 18 AP008209 Oryza sativa japonica cultivar group genomic DNA c 2007 05 19 BA000016 Clostridium perfringens str 13 DNA complete genome 2007 05 19 BC029387 Homo sapiens hemoglobin gamma G mRNA cDNA clon 2007 02 08 BC130457 Homo sapiens hemoglobin gamma G mRNA cDNA clon 2007 01 04 BC130459 Homo sapiens hemoglobin gamma G mRNA cDNA clon 2007 01 04 BC139602 Danio rerio hemoglobin beta embryonic 2 mRNA cDNA 2007 04 18 q BC142787 Danio rerio hemoglobin beta embryonic 1 mRNA cDNA 2007 06 11 IBX842577 Mycobacterium tuberculosis H37Rw complete genome 2006 11 14 v Download and Open Download and Save Total number of hits 245 Open at NCBI Ch Figure 2 11 NCBI search view Click Start search 8 to commence the search in NCBI 2 4 1 Searching for matching objects When the search is complete the list of hits is shown If the desired complete human hemoglobin DNA sequence is found the sequence can be viewed by double clicking it in the list of hits f
2. 30 Er FPI ose hee Ea DES ES SDRAM A 30 1 7 1 Installing plug iNS 2 c005 da 0 ap ek a koe E oe ee ee dE SA E 30 1 7 2 Uninstalling plug ins 2 2 ee es 31 1 7 3 Updating PUESTOS cn cee eae a weed ss 32 1 4 Pe s ara tae Fo oe eh a ee oe oe ee oe ee 32 1 8 Network configuration 1 2 eee 4 33 1 9 The format of the user manual lt lt lt lt 34 Lo ECRM odos ds ES 30 10 CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 11 Welcome to CLC Protein Workbench a software package supporting your daily bioinformatics work We strongly encourage you to read this user manual in order to get the best possible basis for working with the software package This software is for research purposes only CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 12 1 1 Contact information The CLC Protein Workbench is developed by CLC bio A S Science Park Aarhus Finlandsgade 10 12 8200 Aarhus N Denmark herp www Clen O tom VAT no DK 28 30 50 87 Telephone 45 70 22 55 09 Fax 45 70 22 55 19 E mail info clcbio com If you have questions or comments regarding the program you are welcome to contact our Support function E mail support clcbio com 1 2 Download and installation The CLC Protein Workbench is developed for Windows Mac OS X and Linux The software for either platform can be downloaded from http ww
3. at the top 3 3 Zoom and selection in View Area The mode toolbar items in the right side of the Toolbar apply to the function of the mouse pointer When e g Zoom Out is selected you zoom out each time you click in a view where zooming is relevant texts tables and lists cannot be zoomed The chosen mode is active until another mode toolbar item is selected Fit Width and Zoom to 100 do not apply to the mouse pointer FE amp mM ze 7 le CA A Fit Width 100 Pan SOCATA Zoom In Zoom Cut Figure 3 16 The mode toolbar items 3 3 1 Zoom In There are four ways of Zooming In Click Zoom In 550 in the toolbar click the location in the view that you want to zoom in on or Click Zoom In 50 in the toolbar click and drag a box around a part of the view the view now zooms in on the part you selected or Press on your keyboard The last option for zooming in is only available if you have a mouse with a scroll wheel or Press and hold Ctrl 38 on Mac Move the scroll wheel on your mouse forward When you choose the Zoom In mode the mouse pointer changes to a magnifying glass to reflect the mouse mode Note You might have to click in the view before you can use the keyboard or the scroll wheel to ZOOM lf you press the Shift button on your keyboard while clicking in a View the zoom function is reversed Hence clicking on a sequence in this way while the Zoom In mode toolbar item is selected zooms out instead
4. END USER LICENSE AGREEMENT FOR CLC BIO SOFTWARE CLC Genomics Workbench 1 0 1 Recitals 1 1 This End User License Agreement EULA is a legal agreement between you either an individual person or a single legal entity who will be referred to in this EULA as You and CLC bio A S CVR no 28 30 50 87 for the software products that accompanies this EULA including any associated media printed materials and electronic documentation the Software Product I accept these terms If you experience any problems please contact The CLC Support Team Proxy Settings Previous Finish Quit Workbench Figure 1 6 Read the license agreement carefully Please read the License agreement carefully before clicking I accept these terms and Finish CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 19 1 4 2 Download a license When you purchase a license you will get a license ID from CLC bio Using this option you will get a license based on this ID When you have clicked Next you will see the dialog shown in 1 7 At the top enter the ID paste using Ctrl V or 38 V on Mac License Wizard ball a CLC Protein Workbench Download a license Please copy pas bio r Lice PUREA ID i Ta nd choo ne U WO pira ad you license pda n pro des request the license sei will che de the License Order I vailable For ani ad on this computer License Order ID CLC LICENSE SRENMNSTED 0D43CA9EDF90000D844A4C0C480
5. Example data Nucleotide a f F Extra README R Recycle bin 1 x Alignments and Trees A General Sequence Analyses KA Nucleotide Analyses tag Restriction Sites a Protein Analyses GLAST Search A Database Search i Processes Toolbox Idle 1 element s are selected Figure 2 1 The user interface as it looks when you start the program for the first time Windows version of CLC Protein Workbench The interface is similar for Mac and Linux At this stage the important issues are the Navigation Area and the View Area The Navigation Area to the left is where you keep all your data for use in the program Most analyses of CLC Protein Workbench require that the data is saved in the Navigation Area There are several ways to get data into the Navigation Area and this tutorial describes how to import existing data The View Area is the main area to the right This is where the data can be viewed In general CHAPTER 2 TUTORIALS 38 a View is a display of a piece of data and the View Area can include several Views The Views are represented by tabs and can be organized e g by using drag and drop 2 1 1 Creating a a folder When CLC Protein Workbench is started there is one element in the Navigation Area called CLC Data This element is a Location A location points to a folder on your computer where your data for use with CLC Protei
6. Primers Protein analyses Protein orthologs RNA secondary structure Sequencing data al 2 Previot gt Next Finis X Cancel Figure 17 9 Choosing sequence ATP8a1 MRNA for restriction map analysis lf a sequence was selected before choosing the Toolbox action this sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements 17 2 1 Selecting sorting and filtering enzymes Clicking Next lets you define which enzymes to use as basis for finding restriction sites on the sequence At the top you can choose to Use existing enzyme list Clicking this option lets you select an enzyme list which is stored in the Navigation Area See section 17 4 for more about creating and modifying enzyme lists Below there are two panels e To the left you see all the enzymes that are in the list select above If you have not chosen to use an existing enzyme list this panel shows all the enzymes available e To the right there is a list of the enzymes that will be used Select enzymes in the left side panel and add them to the right panel by double clicking or clicking the Add button E If you e g wish to use EcoRV and BamHI select these two enzymes and add them to the right side panel The CLC Protein Workbench comes with a standard set of enzymes based on http www rebase neb com You can customize
7. Chal gatc FokI lt NA gt 3 N6 met cg 5 S meth toca ge 5 5 meth Hhal NsiI Sacll 03 a 02 O 0 0 0 0 A ow Figure 17 10 Selecting enzymes If you need more detailed information and filtering of the enzymes either place your mouse cursor on an enzyme for one second to display additional information see figure 17 21 or use the view of enzyme lists see 17 4 All enzymes Filter 3 Name Overh Methyl Pop PstI 3 N6 meth Poe ES Kpnl 3 N6 meth ee Sacl 3 S methyl Pete Sphi 3 HEEK Apal 3 S methyl pee Sacll S methyl Nsil Enzyme Sacll Chal Recognition site pattern CCGCGG Suppliers GE Healthcare Qbiogene American Allied Biochemical Inc Nippon Gene Co Ltd Takara Bio Inc New England Biolabs Toyobo Biochemicals Molecular Biology Resources Banll Promega Corporation EURx Ltd Figure 17 11 Showing additional information about an enzyme like recognition sequence or a list of commercial vendors 17 2 2 Number of cut sites Clicking Next confirms the list of enzymes which will be included in the analysis and takes you to the dialog shown in figure 17 12 CHAPTER 17 RESTRICTION SITE ANALYSES 2 2 E g Restriction Site Analysis x 1 Select DNA RNA Number of cut sites sequence s 2 Enzymes to be considered in calculation 3 Number of cut sites Display enzyme
8. If a sequence was selected before choosing the Toolbox action the sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements If you want to adjust the parameters for finding open reading frames click Next 15 6 1 Open reading frame parameters This opens the dialog displayed in figure 15 8 The adjustable parameters for the search are CHAPTER 15 NUCLEOTIDE ANALYSES 231 e Start codon a MN Find Open Reading Frames LES 1 Select nucleotide Projects Selected Elements 1 sequences CLC Data xx ATP8al genomic sequence HE Example Data xx XxX ATP8al mRNA E3 Cloning EE Primers Protein analyses Ej Protein orthologs ES RNA secondary structure FS Sequencing data E 4 HE Q zenter search term gt A s Previous Finish x Cancel Figure 15 7 Create Reading Frame dialog r q Find Open Reading Frames amp 3 1 Select nucleotide les aramet sequences 2 Set parameters Start Codon AUG Any All start codons in genetic code 5 Other AUG CUG UUG 4 Both strands Open ended sequence Genetic code 1 Standard v Minimum length codons 100 3 V Include stop codon in result WE Gema a ea Figure 15 8 Create Reading Frame dialog AUG Most commonly used start codon Any Find all open reading frames
9. Liso Gel electrophoresis cr e e dee a dr we eS RR ee ew we DS 2 215 1 4 Restriction enzyme lists ee ee 2 8 18 Sequence alignment 282 18 1 Create an alignment lt 0 283 18 2 View alignments lt lt bean eh a ean aa A RAR 288 18 3 Edit alignmentS lt lt ee 4 0 292 Loe JOWMSNENMICIIS idas rm aa A A RARA A 294 18 5 Pairwise comparison 4464642402484 aaa aa 296 18 6 Bioinformatics explained Multiple alignments 299 19 Phylogenetic trees 301 19 1 Inferring phylogenetic trees 0 301 19 2 Bioinformatics explained phylogenetics 306 IV Appendix 310 A Comparison of workbenches 311 B Graph preferences 316 C Working with tables 318 Gal PU ODES 2 ee kee eee ee TERRAS TEA E 319 D BLAST databases 321 D 1 Peptide sequence databases oaoa ee a 321 D 2 Nucleotide sequence databases 321 D 3 Adding more databases 0 ee 322 E Proteolytic cleavage enzymes 324 F Formats for import and export 326 Pal List of bioinformatic data formats 326 F 2 List of graphics data formats ai 32 e aa a a aaa AR 8 329 G IUPAC codes for amino acids 330 CONTENTS H IUPAC codes for nucleotides Custom codon frequency tables Bibliography
10. Note Annotations are included if you export the sequence in GenBank Swiss Prot EMBL or CLC format When exporting in other formats annotations are not preserved in the exported file 10 3 1 Viewing annotations Annotations can be viewed in a number of different ways e As arrows or boxes in the sequence views Linear and circular view of sequences ret 0 Alignments i Graphical view of sequence lists i BLAST views only the query sequence at the top can have annotations 8 e In the table of annotations E e In the text view of sequences In the following sections these view options will be described in more detail In all the views except the text view annotations can be added modified and deleted This is described in the following sections View Annotations in sequence views Figure 10 6 shows an annotation displayed on a sequence The various sequence views listed in section 10 3 1 have different default settings for showing annotations However they all have two groups in the Side Panel in common e Annotation Layout e Annotation Types CHAPTER 10 VIEWING AND EDITING SEQUENCES 134 CDS 20 l HUMHBB GGCCCTGTTCTGATCATGGGCCCTTCCTAACACTGCATGACTACCTTA CDS HUMHBB TTCTTGTTAGGATCCAAGCAACGGATTCTGCTGGAGCTGTCGTTTTTT CDS 140 HUMHBB CTGGGTGTGTCTCCAACAAGTCCTGAGCACACATAACTGGAAACAATG Figure 10 6 An annotation showing a coding region on a genomic dna sequence k Sequence
11. select the two sequences by lt Ctrl gt click 38 click on Mac or lt Shift gt click Export E choose where to export to choose GenBank gbk format enter name the new file Save Export of dependent elements When exporting e g an alignment CLC Protein Workbench can export the alignment including all the sequences that were used to create it This way when sending your alignment with the dependent sequences your colleagues can reproduce your findings with adjusted parameters if desired To export with dependent files select the element in Navigation Area File in Menu Bar Export with Dependent Elements enter name of of the new file choose where to export to Save The result is a folder containing the exported file with dependent elements stored automatically in a folder on the desired location of your desk Export history To export an element s history select the element in Navigation Area Export E select History PDF pdf choose where to export to Save The entire history of the element is then exported in pdf format The CLC format CLC Protein Workbench keeps all bioinformatic data in the CLC format Compared to other formats the CLC format contains more information about the object like its history and comments The CLC format is also able to hold several elements of different types e g an alignment a graph and a phylogenetic tree This means that if you are exporting your data t
12. 186 CHAPTER 13 3D MOLECULE VIEWING 18 ES CAA26204 BLAST amp Rows 103 Summary oF hits From query C4426204 Filter Description E value Score Bit score 3 366 66 3 36E 66 7 48E 66 SEO Figure 13 1 Itis possible to open a structure file directly from the output of a conducted BLAST search by clicking the Open Structure button 13 2 Viewing structure files An example of a 3D structure is shown in figure 13 2 Figure 13 2 3D view Structure files can be opened viewed and edited in several ways Structures can be rotated and moved using the mouse and keyboard Pan mode must be enabled in order to rotate and move the sequence Note It is only possible to view one structure file at a time in order to limit the amount of memory used 13 2 1 Moving and rotating Structure files are simply rotated by holding down the left mouse button while moving the mouse This will rotate the structure in the direction the mouse is moved The structures can be freely rotated in all directions Holding down the Ctrl on Windows or 38 on Mac key on the keyboard while dragging the mouse CHAPTER 13 3D MOLECULE VIEWING 188 moves the structure in the direction the mouse is moved This is particularly useful if the view is zoomed to cover only a small region of the protein structure Zoom in 545 and zoom out 42 on the structure is do
13. ColE1 6646 Circular Basic NCBI Entrez NCBI uc CREB1 2964 Linear Basic NCBI Entrez NCBI ax JEPAC 3261 Linear Basic NCBI Entrez NCBI ue FYN 2647 Linear Basic NCBI Entrez NCBI ae GNAT1 3367 Linear Basic NCBI Entrez NCBI ram mm 343 DNA RNA molecules Figure 7 2 Data stored in the Vector NTI Local Database accessed through Vector NTI Explorer File Import Vector NTI Database Edit Search View Toolbox Workspace Help g Show Ctrl 0 Extract Sequences New Show PA Close Ctrl W 2 Close Tab Area Close All Views Ctrl Shift W Close Other Tabs Save ctrl S E Save As Ctrl Shift S ES Import Ctrl ES Import VectorNTI Data ES Export Ctrl E Export with Dependent Elements Export Graphics Ctrl G Location b P Page Setup amp Print Ctrl P S Exit Alt F4 Figure 7 3 Import the whole Vector NTI Database This will bring up a dialog letting you choose to import from the default location of the database or you can specify another location If the database is installed in the default folder like e g C VNTI Database press Yes If not click No and specify the database folder manually When the import has finished the data will be listed in the Navigation Area of the Workbench as shown in figure 7 4 If something goes wrong during the import process please report the problem to sup port clcbio com To circumvent the problem see the following section on how to import parts of the
14. Elf CLC Data Ke 09429 gt Example Data e P39524 Xx ATP8a1 genomit Su P57792 XX ATPSal mRNA ys Q29449 fht ATPSal sue QONTI2 Cloning ye Q95X33 Primers Protein analyse Protein ortholog SEE ATPBal orth As EM ns mm Ss su e RNA secondary Sequencing dat Figure 2 25 The alignment dialog displaying the six protein sequences CHAPTER 2 TUTORIALS 95 It is possible to add and remove sequences from Selected Elements list Since we had already selected the eight proteins just click Next to adjust parameters for the alignment Clicking Next opens the dialog shown in figure 2 26 a G Create Alignment 1 Select sequences of same MES amete type 2 Set parameters Gap settings Gap open cost 10 Gap extension cost 1 End gap cost As any other w Alignment Fast less accurate Slow very accurate Redo alignments Use Fixpoints PA aaas Previous Next XX Cancel Figure 2 26 The alignment dialog displaying the available parameters which can be adjusted Leave the parameters at their default settings An explanation of the parameters can be found by clicking the help button Alternatively a tooltip is displayed by holding the mouse cursor on the parameters Click Finish to start the alignment process which is shown in the Toolbox under the Processes tab When the program is finished calculating it displays the alignment see fig 2 27 FEE ATPas
15. end 5 Enzymes producing an overhang at the 5 end There is a checkbox for each group which can be used to hide show all the enzymes in a group 17 1 2 Manage enzymes The list of restriction enzymes contains per default 20 of the most popular enzymes but you can easily modify this list and add more enzymes by clicking the Manage enzymes button This will display the dialog shown in figure 17 6 f g Manage enzymes eS pe 1 Please choose enzymes AA us Enzyme list v Use existing enzyme list Popular enzymes X o Enzymes in Popular en Enzymes shown in Side Panel Filter Filter Name Overhang Methylation Popula Name Overhang Methylation Popula BamHI 5 gatc N4 methy a EcoRI 5 aatt N6 methy BglII 5 gate N4 methy E Smal Blunt N4 methy ee EcoRI 5 aatt N6 methy E _ Sall 5 tega N6 methy EcoRV Blunt N6 methy PstI 3 taca N6 methy HindIII 5 agct N6 methy ZI Xhol 5 tcga N6 methy eee PstI 3 tgca N6 methy T JIEcorRV Blunt N6 methy Sall 5 tcga N6 methy BglII 5 gate N4 methy Smal Blunt N4 methy Xbal 5 ctag N6 methy XbaI 5 ctag N6 methy Peer HindIII 5 agct N6 methy ek XhoI 5 tcga N6 methy BamHI 5 gatc N4 methy Lalar S ra Save Save as new enzyme list E Figur
16. Apal 3 S methyl pee Sacll S methyl pee Nsil Enzyme Sacll Chal Recognition site pattern CCGCGG Suppliers GE Healthcare Qbiogene American Allied Biochemical Inc Nippon Gene Co Ltd Takara Bio Inc New England Biolabs Toyobo Biochemicals Molecular Biology Resources Promega Corporation EURx Ltd Figure 17 8 Showing additional information about an enzyme like recognition sequence or a list of commercial vendors At the bottom of the dialog you can select to save this list of enzymes as a new file In this way you can save the selection of enzymes for later use When you click Finish the enzymes are added to the Side Panel and the cut sites are shown on the sequence CHAPTER 17 RESTRICTION SITE ANALYSES 210 If you have specified a set of enzymes which you always use it will probably be a good idea to save the settings in the Side Panel see section 3 2 7 for future use 17 2 Restriction site analysis from the Toolbox Besides the dynamic restriction sites you can do a more elaborate restriction map analysis with more output format using the Toolbox Toolbox Restriction Sites 3 Restriction Site Analysis of This will display the dialog shown in figure 17 9 g Restriction Site Analysis eS 1 Select DNA RNA M i o ull isa sequence s Projects Selected Elements 1 CLC_Data xx ATP8a1 mRNA Example Data Xc ATP8al genomic sequence xx 55 Cloning
17. CHAPTER 17 RESTRICTION SITE ANALYSES 213 q Restriction Site Analysis e 1 Select DNA RNA Result handling sequence s 2 Enzymes to be considered in calculation 3 Number of cut sites 4 Result handling ies a a a Y Add restriction sites as annotations to sequence s y Create restriction map Create list of cutting enzymes Result handling Open Save Log handling Make log A Previous Ne wf Einish XX Cancel Figure 17 13 Choosing to add restriction sites as annotations or creating a restriction map As a table of restriction sites as shown in figure 17 15 If more than one sequence were selected the table will include the restriction sites of all the sequences This makes it easy to compare the result of the restriction map analysis for two sequences As a table of fragments which shows the sequence fragments that would be the result of cutting the sequence with the selected enzymes see figure1 7 16 As a virtual gel simulation which shows the fragments as bands on a gel see figure 17 18 For more information about gel electrophoresis see section 17 3 The following sections will describe these output formats in more detail In order to complete the analysis click Finish see section 9 1 for information about the Save and Open options 17 2 4 Restriction sites as annotation on the sequence If you chose to add the restriction sites as annotation t
18. CLC Protein Workbench User manual Manual for CLC Protein Workbench 5 8 Windows Mac OS X and Linux February 23 2012 This software is for research purposes only CLC bio Finlandsgade 10 12 DK 8200 Aarhus N gt Denmark o il bio Contents 1 2 Introduction Introduction to CLC Protein Workbench EE ACU s a nasia dkk tawes ee eee eee Se eee 1 2 D wnload and installation i s s s 2 s a wea we we eo oe we EDS we a 1 3 System requirements noanoa 45 a a RES a a a ER O MOOM OS sou aaa AA A 1 5 About CLC Workbenches 0603 sa sal A sas 1 6 When the program is installed Getting started ft PUEDE lt lt cia ee oa ae aa e aaa e e 1 8 Network configuration lt lt lt be bebe sas e ss a E 139 Theformat ofthe user manual lt i e sa es eae eae ek ewa oe de Tutorials 2 1 Tutorial Getting started sui aa aa RR A 2 2 Tutorial View sequence aoao a ee asa 2 3 Tutorial Side Panel Settings aono noanoa a a 2 4 Tutorial GenBank search and download a osoo a 0 0 eee eee 29 MOREL NC s s s wasa ga ee Aw Rw Eee REA SA E 2 6 Tutorial Tips for specialized BLAST searches 2 1 Tutorial Proteolytic cleavage detection 2 8 Tutorial Align protein Sequences a 2 9 Tutorial Create and modify a phylogenetic tree 2 10 TUNA TING resStiction SIES ticas rara aa 10 12
19. Description positive Greatest hit length Accession hit length W Figure 12 9 An overview BLAST table summarizing the results for a number of query sequences list with the selected query sequences This can be useful in work flows where BLAST is used as a filtering mechanism where you can filter the table to include e g sequences that have a certain top hit and then extract those In the overview table the following information is shown e Query Since this table displays information about several query sequences the first column is the name of the query sequence e Number of hits The number of hits for this query sequence e For the following list the value of the best hit is displayed together with accession number CHAPTER 12 BLAST SEARCH 169 and description of this hit Lowest E value Greatest identity Greatest positive Greatest hit length Greatest bit score If you wish to save some of the BLAST results as individual elements in the Navigation Area open them and click Save As in the File menu 12 2 3 BLAST graphics The BLAST editor shows the sequences hits which were found in the BLAST search The hit sequences are represented by colored horizontal lines and when hovering the mouse pointer over a BLAST hit sequence a tooltip appears listing the characteristics of the sequence As default the query sequence is fitted to the window width but it is possible to zoom in the window
20. Figure 16 13 The different available scales in Protein info in CLC Protein Workbench The level of hydrophobicity is calculated on the basis of the different scales The different scales add different values to each type of amino acid The hydrophobicity score is then calculated as the sum of the values in a window which is a particular range of the sequence The window length can be set from 5 to 25 residues The wider the window the less fluctuations in the hydrophobicity scores For more about the theory behind hydrophobicity see 16 5 3 In the following we will focus on the different ways that CLC Protein Workbench offers to display the hydrophobicity scores We use Kyte Doolittle to explain the display of the scores but the different options are the same for all the scales Initially there are three options for displaying the hydrophobicity scores You can choose one two or all three options by selecting the boxes See figure 16 14 Atp8at ATP8al MPTMRRTVSEIRSRAEGYEKTDDVSEKTSLADOEEVRTIFINOPOLTKFCNNHVS 40 jA tp8a1 ATPBa1 TAKYNVITFLPRFLYSOFRRAANSFFLFIALLOQIPDVSPTGRYTTLVPLEFILA DT TT a Atp8a1 ATPSai WAAITKE 1 EDIKRHKADNAVNKKOTOVLRNGAWE VHWEKVNVGDIVI IKGKEYI v Hopp Weod x JR y Figure 16 14 The different ways of displaying the hydrophobicity scores using the Kyte Doolittle scale Coloring the letters and their background When choosing coloring of letters or coloring of their background t
21. Sec Selenocysteine Ser Serine Thr Threonine Trp Tryptophan Tyr Tyrosine Val Valine ASX Aspartic acid or Asparagine Asparagine Glx Glutamic acid or Glutamine Glutamine Xaa Any amino acid 331 Appendix H IUPAC codes for nucleotides Single letter codes based on International Union of Pure and Applied Chemistry The information is gathered from http www iupac org and http www ebi ac uk zen Eucorials da Wee Code Description Adenine Cytosine Guanine Thymine Uracil Purine A or G Pyrimidine C T or U CorA T U or G T U or A CorG C T U or G not A A T U or G not C A T U or C not G A C or G not T not U Any base A C G T or U Zz lt IODOWO0S lt XZS lt VIE SO O gt 332 Appendix Custom codon frequency tables You can edit the list of codon frequency tables used by CLC Protein Workbench Note Please be aware that this process needs to be handled carefully otherwise you may have to re install the Workbench to get it to work In the Workbench installation folder under res there is a folder named codonfreg This folder contains all the codon frequency tables organized into subfolders in a hierarchy In order to change the tables you simply add delete or rename folders and the files in the folders If you wish to add new tables please use the existing ones as template Restart the Workbench to have the changes take effect Please note that when upd
22. dependent objects 108 folder 107 graph in csv format 113 graphics 109 history 108 list of formats 326 INDEX multiple files 107 preferences 94 Side Panel Settings 92 tables 329 Export visible area 110 Export whole view 110 Expression analysis 312 Extensions 30 External files import and export 109 Extinction coefficient 212 Extract sequences 145 FASTA file format 327 Feature request 28 Feature table 214 Features see Annotations File system local BLAST database 1 4 Filtering restriction enzymes 269 2 1 2 9 Find in GenBank file 142 in sequence 127 results from a finished process 8 Find open reading frames 230 Fit to pages print 100 Fit Width 77 Fixpoints for alignments 286 Floating license 24 Floating license use offline 25 Floating Side Panel 97 Folder create new tutorial 38 Follow selection 123 Footer 101 Format of the manual 35 FormatDB 1 4 Fragment table 2 4 Fragment select 129 Fragments separate on gel 2 6 Free end gaps 284 fsa file format 329 G C content 126 313 Gap compare number of 298 delete 292 extension cost 284 fraction 289 313 insert 292 open cost 284 Gb Division 141 343 gbk file format 329 GCG Alignment file format 328 GCG Sequence file format 32 gck file format 329 GCK Gene Construction Kit file format 327 Gel separate sequences without restriction en zyme digestion 2 6 tabular view of fragments
23. e BLAST Database Select a database already available in one of your designated BLAST database folders Read more in section 12 4 When a database or a set of sequences has been selected click Next This opens the dialog seen in figure 12 7 See section 12 1 1 for information about these limitations There is one setting available for local BLAST jobs that is not relevant for remote searches at the NCBI e Number of processors You can specify the number of processors which should be used if your Workbench is installed on a multi processor system CHAPTER 12 BLAST SEARCH 167 1 Select sequences of same PSetpara pedi type 2 Choose program and target 3 Set BLAST parame ters Choose parameters Number ofthreads 1 E v Filter low Complexity Choose filter C Mask lower case Expect 10 Word size 3H Matrix BLOSUM62 v ap cos xistenc xtension 1 Max number of hit sequences 250H JS Previous Next X cancel Figure 12 7 Examples of parameters that can be set before submitting a BLAST search 12 1 4 BLAST a partial sequence against a local database You can search a database using only a part of a sequence directly from the sequence view select the region that you wish to BLAST right click the selection BLAST Selection Against Local Database This will go directly to the dialog shown in figure 12 6 and the rest of the options are the same as when performing a BLAST search
24. e Minimum pattern length Here the minimum length of patterns to search for can be specified CHAPTER 14 GENERAL SEQUENCE ANALYSES 217 e Maximum pattern length Here the maximum length of patterns to search for can be specified e Noise Specify noise level of the model This parameter has influence on the level of degeneracy of patterns in the sequence s The noise parameter can be 1 2 5 or 10 percent e Number of different kinds of patterns to predict Number of iterations the algorithm goes through After the first iteration we force predicted pattern positions in the first run to be member of the background In that way the algorithm finds new patterns in the second iteration Patterns marked Pattern1 have the highest confidence The maximal iterations to go through is 3 e Include background distribution For protein sequences it is possible to include information on the background distribution of amino acids from a range of organisms Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish This will open a view showing the patterns found as annotations on the original sequence see figure 14 21 If you have selected several Sequences a corresponding number of views will be opened Pattern1 Pattern1 3VCNKNGOTA EDLAWSYGFP ECARFLTMIK CMQTARSSGE Figure 14 21 Sequence view displaying two discovered patterns 14 6 2 Pattern search output If the analysis is perform
25. f EB Export Graphics Es 1 Output options IS 2 Save in file 3 Export size Choose resolution Screen resolution 530x3072 pixels 9 MB memory usage Low resolution 286x1660 pixels 2 MB memory usage Medium resolution 1145x6640 pixels 43 MB memory usage High resolution 4582x26561 pixels 696 MB memory usage Camas ore ET Figure 7 12 Parameters for bitmap formats size of the graphics file You can adjust the size the resolution of the file to four standard sizes Screen resolution e Low resolution Medium resolution e High resolution CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 113 The actual size in pixels is displayed in parentheses An estimate of the memory usage for exporting the file is also shown If the image is to be used on computer screens only a low resolution is sufficient If the image is going to be used on printed material a higher resolution iS necessary to produce a good result Parameters for vector formats For pdf format clicking Next will display the dialog shown in figure 7 13 this is only the case if the graphics is using more than one page q Export Graphics ES 1 Output options E 2 Save in file 3 Page setup Page setup parameters Orientation Portrait Paper Size A4 Horizontal Pagecount Not Applicable Vertical Pagecount Not Applicable Header Text Footer Text Show Pagenumber Yes E Page Setup ener Si
26. links 159 overview of 136 show hide 133 table of 136 types of 133 view on sequence 133 viewing 133 Annotations add links to 139 Antigenicity 242 313 Append wildcard search 149 152 155 Arrange layout of sequence 38 views in View Area 3 Assembly 311 Atomic composition 213 Audit 90 Backup 108 Batch edit element properties 69 Batch processing 340 INDEX log of 120 Bibliography 338 Bioinformatic data export 10 formats 102 326 bl2seq see Local BLAST BLAST 312 against a local Database 165 against NCBI 162 create database from file system 1 4 create database from Navigation Area 1 4 create local database 1 4 database file format 329 database management 1 5 graphics output 169 list of databases 321 parameters 163 search 161 162 specify server URL 94 table output 1 0 tips for specialized searches 4 tutorial 44 4 7 URL 94 BLAST database index 1 4 BLAST DNA sequence BLASTn 162 BLASTx 162 tBLASTx 162 BLAST Protein sequence BLASTp 163 tBLASTn 163 BLAST result search in 1 2 BLAST search Bioinformatics explained 1 6 BLOSUM scoring matrices 204 Bootstrap values 309 Borrow floating license 25 Browser import sequence from 104 Bug reporting 28 C G content 126 CDS translate to protein 129 Chain flexibility 126 Cheap end gaps 284 ChIP Seq analysis 311 cif file format 186 329 Circular view of sequence 130 312 Clc file format 10
27. 12 15 15 21 29 30 33 34 CONTENTS Core Functionalities User interface opal 3 2 3 3 3 4 3 5 3 6 Navigation Area a a a VEW NEG pareada A Zoom and selection in View Area coimas ee a a RAR feio plo and StAtUs BDO O RD DD rra Workspace 4 sara aaa LISCO SHONQUES a a ioa nanos edad a eee KE AR aa Searching your data 4 1 4 2 4 3 4 4 What kind of information can be searched 08 28 ee eae a eR ce adhe edhe tee Ce Ree eee eee et we we eG Advanced search 2 0 0 ee A Search index res did teehee ew het ew E ee oe oes bee User preferences and settings 5 1 General preferences ee 2 4 4 5 2 Default view preferences 4 4 5 3 Advanced preferences ee ee aaa 5 4 Export import Of preferences 4 5 5 View settings for the Side Panel lt lt 0 Printing 6 1 Selecting which part of the view to print 0 0 lt lt 0 0 Due Paos eeke krean EnEn benka AAA 6 3 Print preview oaaao a a a aaa Import export of data and graphics f 1 Bioinformatic data formats 2 0 4 e fo CMN a sos ss ss Tes Export geraphics TO HIS a lt sac oe as A 7 4 Export graph data points to a file fer Copy paste view output osos ew
28. 2 a a a 1 1 243 16 4 2 Antigenicity graphs along sequence a 5050208 244 16 5 MVYCIOONGUIGNY rra aa ARA 244 16 5 1 Hydrophobicity plot im Ga we casara e 245 16 5 2 Hydrophobicity graphs along sequence 246 16 5 3 Bioinformatics explained Protein hydrophobicity 247 16 6 Pfam domain search lt 2 4 249 16 6 1 Pfam search parameters a 250 16 6 2 Download and installation of additional Pfam databases 251 16 7 Secondary structure prediction lt 2 251 16 8 Protein report 2 2 eee lt lt 4 4 4 4 253 16 8 1 Protein report output 2c cca cada dbo iras 254 16 9 Reverse translation from protein into DNA 0 00082 2 wena 255 16 9 1 Reverse translation parameters 256 16 9 2 Bioinformatics explained Reverse translation 257 16 10 Proteolytic cleavage detection lt lt eee eee ee ee es 259 16 10 1 Proteolytic cleavage parameters 2 00 2 eee een nee 259 16 10 2 Bioinformatics explained Proteolytic cleavage 262 CLC Protein Workbench offers a number of analyses of proteins as described in this chapter 233 CHAPTER 16 PROTEIN ANALYSES 234 16 1 Signal peptide prediction Signal peptides target proteins to the extracellular environment either through d
29. 29 Statistics about sequence 312 protein 211 sequence 208 Status Bar 8 79 illustration 62 Str file format 329 Structure scanning 314 Structure prediction 251 Style sheet preferences 95 Support mail 12 Surface probability 126 svg format export 111 Swiss Prot 152 search see UniProt Swiss Prot file format 32 Swiss Prot TrEMBL 312 Swp file format 329 System requirements 15 Tab delimited file format 329 Tab file format 327 Table of fragments 2 4 Tabs use of 69 Tag based expression profiling 311 TaqMan primers 314 tar file format 329 Tar file format 329 Taxonomy batch edit 69 tBLASTn 163 tBLASTx 162 Terminated processes 8 Text format 128 user manual 35 view sequence 142 Text file format 329 tif format export 111 Tips for BLAST searches 47 TMHMM 241 Toolbar illustration 62 preferences 91 Toolbox 8 9 illustration 62 show hide 9 INDEX Topology layout trees 305 Trace colors 125 Trace data 311 Translate a selection 126 along DNA sequence 125 annotation to protein 129 CDS 230 coding regions 230 DNA to RNA 225 nucleotide sequence 228 ORF 230 protein 255 RNA to DNA 226 to DNA 313 to protein 228 313 Translation of a selection 126 show together with DNA sequence 125 Transmembrane helix prediction 241 313 TrEMBL search 152 Trim 311 TSV file format 327 Tutorial Getting started 37 txt file format 329 UIPAC codes amino a
30. ATP8al QLTKFCNNHVSTAKYNVITFLPRFLYSQFRRAANSFFLFIALLQ Strand Helix ATP8al QIPDVSPTGRYTTLVPLLFILAVAAIKE IEDIKRHKADNAVNK Figure 16 19 Alpha helices and beta strands shown as annotations on the sequence Undesired alpha helices or beta sheets can be removed through the Delete Annotation gr right click mouse menu See section 10 3 4 16 8 Protein report CLC Protein Workbench is able to produce protein reports that allow you to easily generate different kinds of information regarding a protein Actually a protein report is a collection of some of the protein analyses which are described elsewhere in this manual To create a protein report do the following Right click protein in Navigation Area Toolbox Protein Analyses ia Create Protein Report This opens dialog Step 1 where you can choose which proteins to create a report for When the correct one is chosen click Next In dialog Step 2 you can choose which analyses you want to include in the report The following list shows which analyses are available and explains where to find more details e Sequence statistics See section 14 4 for more about this topic e Plot of charge as function of pH See section 16 2 for more about this topic e Plot of hydrophobicity See section 16 5 for more about this topic e Plot of local complexity See section 14 3 for more about this topic e Dot plot against self See section 14 2 for more about this topic e Secondary structure
31. Certain amino acid substitutions change of one amino acid to another happen often whereas other substitutions are very rare For instance tryptophan W which is a relatively rare amino acid will only on very rare occasions mutate into a leucine L Based on evolution of proteins it became apparent that these changes or substitutions of amino acids can be modeled by a scoring matrix also refereed to as a substitution matrix See an example of a scoring matrix in table 14 1 This matrix lists the substitution scores of every single amino acid A score for an aligned amino acid pair is found at the intersection of the corresponding column and row For example the substitution score from an arginine R to a lysine K is 2 The diagonal show scores for amino acids which have not changed Most substitutions changes have a negative score Only rounded numbers are found in this matrix The two most used matrices are the BLOSUM Henikoff and Henikoff 1992 and PAM Dayhoff and Schwartz 1978 CHAPTER 14 GENERAL SEQUENCE ANALYSES 205 oa ESEPEPESELO PILA 4 a v PPPPPRPEIPEE EP LE OL p Y Y PELE RN Fa i Pa Pi a a le tae te Li PELERAES m4 Y A j cw Yi N s Aia MD ARA oi dade T MM A hy ARANA ma e ADD N Se e Ma E Mey k ia PERESELESEPLIPE Er re ER Figure 14 12 The dot plot A a low complexity region in the sequence The seque
32. Examples 0 01 1 238 5 1 23E 4 1 23E 5 Figure 5 5 Number formatting of tables The examples below the text field are updated when you change the value so that you can see the effect After you have changed the preference you have to re open your tables to see the effect 5 2 2 Import and export Side Panel settings If you have created a special set of settings in the Side Panel that you wish to share with other CLC users you can export the settings in a file The other user can then import the settings To export the Side Panel settings first select the views that you wish to export settings for Use Ctri click click on Mac or Shift click to select multiple views Next click the Export button Note that there is also another export button at the very bottom of the dialog but this will export the other settings of the Preferences dialog see section 5 4 A dialog will be shown see figure 5 6 that allows you to select which of the settings you wish to export When multiple views are selected for export all the view settings for the views will be shown in the dialog Click Export and you will now be able to define a save folder and name for the exported file The settings are saved in a file with a vsf extension View Settings File To import a Side Panel settings file make sure you are at the bottom of the View panel of the Preferences dialog and click the Import button Note that there is also another import button
33. FERHABT 100 134 Tsol 100 151 133 151 146 104 179 196 Figure 17 16 The result of the restriction analysis shown as annotations e Overhangs If there is an overhang this is displayed with an abbreviated version of the fragment and its overhangs The two rows of dots represent the two strands of the fragment and the overhang is visualized on each side of the dots with the residue s that make up the overhang If there are only the two rows of dots it means that there is no overhang e Left end The enzyme that cuts the fragment to the left 5 end e Right end The enzyme that cuts the fragment to the right 3 end e Conflicting enzymes If more than one enzyme cuts at the same position or if an enzyme s recognition site is cut by another enzyme a fragment is displayed for each possible combination of cuts At the same time this column will display the enzymes that are in conflict If there are conflicting enzymes they will be colored red to alert the user If the same experiment were performed in the lab conflicting enzymes could lead to wrong results For this reason this functionality is useful to simulate digestions with complex combinations of restriction enzymes If views of both the fragment table and the sequence are open clicking in the fragment table will select the corresponding region on the sequence 17 2 7 Gel The restriction map can also be shown as a gel This is de
34. Most recent common ancestor Operational Taxonomical Units Orangutan Human Pygmy chimpanzee Chimpanzee Gorilla Internal Node vertice Hypothetical Taxonomical Unit Figure 19 5 A proposed phylogeny of the great apes Hominidae Different components of the tree are marked see text for description The ordering of the nodes determine the tree topology and describes how lineages have diverged over the course of evolution The branches of the tree represent the amount of evolutionary divergence between two nodes in the tree and can be based on different measurements A tree is completely specified by its topology and the set of all edge lengths The phylogenetic tree in figure 19 5 is rooted at the most recent common ancestor of all Hominidae species and therefore represents a hypothesis of the direction of evolution e g that CHAPTER 19 PHYLOGENETIC TREES 307 the common ancestor of gorilla chimpanzee and man existed before the common ancestor of chimpanzee and man In contrast an unrooted tree would represent relationships without assumptions about ancestry 19 2 2 Modern usage of phylogenies Besides evolutionary biology and systematics the inference of phylogenies is central to other areas of research As more and more genetic diversity is being revealed through the completion of multiple genomes an active area of research within bioinformatics is the development of comparative machine learning algorithms that can simul
35. Select sequences of same Meios type 2 Set parameters Set order of concatenation top first ss 094296 a ss P39524 7a OCS Ce Coe a ea Figure 14 18 Setting the order in which sequences are joined Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The result is shown in figure 14 19 Gene Joined Sequence Figure 14 19 The result of joining sequences is a new sequence containing the annotations of the joined sequences they each had a HBB annotation 14 6 Pattern Discovery With CLC Protein Workbench you can perform pattern discovery on both DNA and protein sequences Advanced hidden Markov models can help to identify unknown sequence patterns across single or even multiple sequences In order to search for unknown patterns CHAPTER 14 GENERAL SEQUENCE ANALYSES 216 Select DNA or protein sequence s Toolbox in the Menu Bar General Sequence Analyses 4 Pattern Discovery 2 or right click DNA or protein sequence s Toolbox General Sequence Analyses A Pattern Discovery 2 If a sequence was selected before choosing the Toolbox action the sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements You can perform the analysis on several DNA or several protein sequences at a time lf the analysis is performed on
36. Step two in the Hydrophobicity Plot allows you to choose hydrophobicity scale and the window size The Window size is the width of the window where the hydrophobicity is calculated The wider the window the less volatile the graph You can chose from a number of hydrophobicity scales which are further explained in section 16 5 3 Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The result can be seen in figure 16 12 Hydrophobicity plot of ATP8al il i iti dl i WW MNT AY PRN T 0 100 700 300 400 500 600 700 800 5900 1 Position Figure 16 12 The result of the hydrophobicity plot calculation and the associated Side Panel CHAPTER 16 PROTEIN ANALYSES 246 See section B in the appendix for information about the graph view 16 5 2 Hydrophobicity graphs along sequence Hydrophobicity graphs along sequence can be displayed easily by activating the calculations from the Side Panel for a sequence right click protein sequence in Navigation Area Show Sequence open Protein info in Side Panel or double click protein sequence in Navigation Area Show Sequence open Protein info in Side Panel These actions result in the view displayed in figure 16 13 Protein info k Evte Doolikkle Cornette k Engelman Eisenberg k Rose k Janin k Hopp Woads t Welling k Kolaskar Tongaonkar k Surface Probability Chain Flexibility Find
37. Takara Bio Inc Banll New England Biolabs Toyobo Biochemicals Molecular Biology Resources Promega Corporation EURx Ltd Figure 1 21 Showing additional information about an enzyme like recognition sequence or a list of commercial vendors 17 4 2 View and modify enzyme list An enzyme list is shown in figure 17 22 The list can be sorted by clicking the columns ES All enzymes O Rows 1362 Table of restriction enzymes Filter r s S 1 E Name Recognition sequence Overhang Suppliers Methylation sensitivity Star activity Column width i EcoRV gatatc Blunt GE Healthc N methyladenosine Yes Automatic Y o E x BglII agatct 5 gate GE Healthc N4 methylcytosine No eae Sall ategac 5 toga GE Healthc N6 methyladenosine Yes Name Xhol ctcgag 5 tega GE Healthc N methyladenosine No Recognition sequence HindIII aagctt 5 agct GE Healthc N methyladenosine Yes Xbal tctaga 5 ctag GE Healthc N6 methyladenosine Yes Overhang EcoRI gaattc 5 aatt GE Healthc N 6 methyladenosine Yes Suppliers PstI ctgcag 3 tgca GE Healthc N methyladenosine Yes i Ea v M BamHI ggatcc 5 gate GE Healthc N4 methylcytosine Yes e es Clal atcgat 5 cg GE Healthc N methyladenosine No C Recognizes palindrome NotI gcggecge 5 ggcc GE Healthc N4 methylcytosine No Star activity NdeI catatg 5 ta GE Healthc N methyladenosine Yes SacI gagcte 3 agct GE Healthc 5 methyl
38. This means that a good E value which gives a confident prediction is much less than 1 E values around 1 is what is expected by chance Thus the lower the E value the more specific the search for domains will be Only positive numbers are allowed Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish This will open a view showing the found domains as annotations on the original sequence see figure 16 17 If you have selected several Sequences a corresponding number of views will be opened E 22 7 ATPBal DIKDIDSLMRISGRIECESPNRHLYDFVGNIRLDGHGTVPLGADQILLRGA Glycos transf 2 Atp8a1 _ ATPBal DTKLMQNSTSPPLKLSNVERITNVQILILFCILIAMSLVCSVGSA IWNRRH Figure 16 17 Domains annotations based on Pfam Each found domain will be represented as an annotation of the type Region More information on each found domain is available through the tooltip including detailed information on the identity score which is the basis for the prediction For a more detailed description of the provided scores through the tool tip look at http pfam sanger ac uk help tabview tab5 16 6 2 Download and installation of additional Pfam databases Additional databases can be downloaded as a resource using the Plug in manager E see section 1 7 4 If you are not able to download directly from the Plug in manager 16 7 Secondary structure prediction An important issue when trying to understand protei
39. This opens the dialog shown in figure 17 19 a Create new enzyme list es 1 Please choose enzymes RISE ASAS Name Overhang Methylation Popularity Name Overhang Methylation Popularity HindIII 5 agct N6 methyl tee a EcoRV Blunt N6 methyl 40k Smal Blunt N4 methyl EcoRI 5 aatt N6 methyl eeer Xbal 5 ctag N6 methyl SmaI Blunt N4 methy l Sall 5 tcga N6 methyl Sall 5 tcga N6 methyl aer EcoRV Blunt N6 methyl PstI 3 tgca N6 methyl EcoRI 5 aatt N6 methyl ter XhoI 5 tega N6 methy l tt BglII 5 gatc N4 methyl BglII 5 gate N4 methyl eee Xhol 5 tega N6 methyl xb N6 methyl eee PstI 3 tgca N6 methyl HindIII 5 agct N6 methyl BamHI 5 gatc N4 methyl BamHI 5 gatc N4 methyl rt KpnI 3 gtac N6 methyl Ncol 5 catg N4 methyl eee NotI 5 gacc N4 methyl ee SacI 3 aget S methylc Ncol 5 catg N4 methyl er KpnI 3 gtac N6 methyl Sacl 3 aget S methylc NotI 5 ggcc N4 methyl Ndel 5 ta N6 methyl eee Fa ES a E ca A E _ a L ES n j Y ok XX Cancel Pl Figure 17 19 Choosing enzymes for the new enzyme list At the top you can choose to Use existing enzyme list Clicking this option lets you select an enzyme list which is stored in the Navigation Area See section 17 4 for more
40. Yang and Rannala 1997 Yang Z and Rannala B 1997 Bayesian phylogenetic inference using DNA sequences a Markov Chain Monte Carlo Method Mol Biol Evol 14 7 717 724 Part V Index 339 Index 3D molecule view 180 export graphics 193 navigate 187 output 193 rotate 187 zoom 187 454 sequencing data 311 AB1 file format 327 Abbreviations amino acids 330 ABI file format 327 About CLC Workbenches 27 Accession number display 6 ace file format 329 ACE file format 328 Add annotations 137 312 sequences to alignment 294 Adjust selection 129 Advanced preferences 93 Advanced search 86 Algorithm alignment 282 neighbor joining 308 UPGMA 307 Align alignments 285 protein sequences tutorial 54 sequences 313 Alignment see Alignments Alignments 282 313 add sequences to 294 compare 296 create 283 edit 292 fast algorithm 284 join 294 multiple Bioinformatics explained 299 remove sequences from 293 view 288 view annotations on 133 Aliphatic index 211 aln file format 329 Alphabetical sorting of folders 65 Ambiguities reverse translation 258 Amino acid composition 213 Amino acids abbreviations 330 UIPAC codes 330 Annotation select 129 Annotation Layout in Side Panel 133 Annotation types define your own 137 Annotation Types in Side Panel 133 Annotations add 137 copy to other sequences 293 edit 137 139 in alignments 293 introduction to 133
41. can be very time consuming and computationally demanding Increasing the window size will make the dot plot more smooth Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish CHAPTER 14 GENERAL SEQUENCE ANALYSES 199 a EB Create Dot Plot Es p 1 Select one or two set parameters sequences of same type 2 Set parameters Distance correction and window size Score model BLOSUM62 w Window size 9 2 A Previous gt Next Y Erin XX Cancel Figure 14 4 Setting the dot plot parameters 14 2 2 View dot plots A view of a dot plot can be seen in figure 14 5 You can select Zoom in 55 in the Toolbar and click the dot plot to zoom in to see the details of particular areas ATP8a1 vs 094296 1100 E E 1000 900 800 700 600 ATP8al 500 400 300 200 5574 100 200 400 600 800 1000 1200 094296 Figure 14 5 A view is opened showing the dot plot The Side Panel to the right let you specify the dot plot preferences The gradient color box can be adjusted to get the appropriate result by dragging the small pointers at the top of the box Moving the slider from the right to the left lowers the thresholds which can be directly seen in the dot plot where more diagonal lines will emerge You can also choose another color gradient by clicking on the gradient box and choose from the list Adjusting the sliders above the
42. e For more refined and systematic search for motifs can be performed through the Toolbox This will generate a table and optionally add annotations to the sequences The two approaches are described below 14 7 1 Dynamic motifs In the Side Panel of sequence views there is a group called Motifs see figure 14 22 Motifs Show Found 1 motif Labels Nolabels Include reverse motif F Exclude unknown regions attE1 0 attE2 0 SP6 0 TF tO CM 1 T3 0 pGEX 5 00 T7 terminator 0 His tag 0 Select All Deselect All Manage Motifs Figure 14 22 Dynamic motifs in the Side Panel The Workbench will look for the listed motifs in the sequence that is open and by clicking the check box next to the motif it will be shown in the view as illustrated in figure 14 23 A A TCE 440 460 AAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC 480 500 520 GGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGG Figure 14 23 Showing dynamic motifs on the sequence This case shows the CMV promoter primer Sequence which is one of the pre defined motifs in CLC Protein Workbench The motif is per default shown as a faded arrow with no text The direction of the arrow indicates the strand of the motif CHAPTER 14 GENERAL SEQUENCE ANALYSES 219 Placing the mouse cursor on the arrow will display additional information about the motif as illustrated in figure 14 24 sCCCCATTGACGCA
43. in a image editor e g GIMP http www gimp org e Crop edit the screen shot e Save in your preferred file format and or print CHAPTER 13 3D MOLECULE VIEWING 194 Linux e Set up your 3D view e e g use GIMP to take the screen shot http www gimp org e Crop edit the screen shot e Save in your preferred file format and or print Chapter 14 General sequence analyses Contents 14 1 Shuffle sequence 0 00 eee eee a 195 14 2 Dot plotS sa AAA AAA 197 doca BIOS dot OS 4242 4 gale oe tee ES ewe ee SEE RE 197 14 2 2 View dotpiots cesos ea eee PEGA EEE TAM ee E 199 14 2 3 Bioinformatics explained Dot plots 2 0 08 0s 200 14 2 4 Bioinformatics explained Scoring matrices 204 14 3 Local complexity plot 1 2 ee ee a 207 14 4 Sequence statistics 8 eee a 208 14 4 1 Bioinformatics explained Protein statistics 4 211 14 5 Join sequences 0 088 ee eee ee ee ee aaa 214 14 6 Pattern Discovery 2 nononono 215 14 6 1 Pattern discovery search parameters 2 05028 ee 216 14 6 2 Pattern search output wo amp ee ee EG we e ee de 217 14 7 Motif Search lt 2 2 ma 217 TAT Dynamic MOS e w we ee ee ds a ee we a 218 14 7 2 Motif search fromthe Toolbox 2 ee ee ee ee ee 220 14 7 3 Java regular expressions aoao oaoa aoao 221 14 7 4 Create motif list
44. structure in a different way First of all you can have more than one criterion in the filter Criteria can be added or removed by clicking the Add S or Remove E buttons At the top you can choose whether all the criteria should be fulfilled Match all or if just one of the needs to be fulfilled Match any For each filter criterion you first have to select which column it should apply to Next you choose an operator For numbers you can choose between e equal to lt smaller than e gt greater than e lt gt not equal to e abs value lt absolute value smaller than This is useful if it doesn t matter whether the number is negative or positive e abs value gt absolute value greater than This is useful if it doesn t matter whether the number is negative or positive For text based columns you can choose between e contains the text does not have to be in the beginning e doesn t contain APPENDIX C WORKING WITH TABLES 320 e the whole text in the table cell has to match also lower upper case Once you have chosen an operator you can enter the text or numerical value to use If you wish to reset the filter simply remove E all the search criteria Note that the last one will not disappear it will be reset and allow you to start over Figure C 3 shows an example of an advanced filter which displays the open reading frames larger than 400 that are placed on the negative strand Fin
45. the Esc key For renaming annotations instead of folders or elements see section 10 3 3 3 1 7 Delete elements Deleting a folder or an element can be done in two ways right click the element Delete 4 or select the element press Delete key This will cause the element to be moved to the Recycle Bin ff where it is kept until the recycle bin is emptied This means that you can recover deleted elements later on For deleting annotations instead of folders or elements see section 10 3 4 Restore Deleted Elements The elements in the Recycle Bin lj can be restored by dragging the elements with the mouse into the folder where they used to be If you have deleted large amounts of data taking up very much disk space you can free this disk space by emptying the Recycle Bin ff Edit in the Menu Bar Empty Recycle Bin ie Note This cannot be undone and you will therefore not be able to recover the data present in the recycle bin when it was emptied 3 1 8 Show folder elements in a table A location or a folder might contain large amounts of elements It is possible to view their elements in the View Area select a folder or location Show 2 in the Toolbar Contents H An example is shown in figure 3 6 When the elements are shown in the view they can be sorted by clicking the heading of each of the columns You can further refine the sorting by pressing Ctrl on Mac while clicking the heading of another co
46. 1157 1084 1113 1136 1161 413 396 Figure 18 15 A pairwise comparison table The following settings are present in the side panel e Contents Upper comparison Selects the comparison to show in the upper triangle of the table Upper comparison gradient Selects the color gradient to use for the upper triangle Lower comparison Selects the comparison to show in the lower triangle Choose the same comparison as in the upper triangle to show all the results of an asymmetric comparison Lower comparison gradient Selects the color gradient to use for the lower triangle Diagonal from upper Use this setting to show the diagonal results from the upper comparison Diagonal from lower Use this setting to show the diagonal results from the lower comparison No Diagonal Leaves the diagonal table entries blank e Layout Lock headers Locks the sequence labels and table headers when scrolling the table Sequence label Changes the sequence labels CHAPTER 18 SEQUENCE ALIGNMENT 299 e Text format Text size Changes the size of the table and the text within it Font Changes the font in the table Bold Toggles the use of boldface in the table 18 6 Bioinformatics explained Multiple alignments Multiple alignments are at the core of bioinformatical analysis Often the first step in a chain of bioinformatical analyses is to construct a multiple alignment of a number of
47. 129 Selection mode in the toolbar 77 Selection adjust 129 Selection expand 129 Selection location on sequence Separate sequences on gel 2 6 using restriction enzymes 2 6 Sequence alignment 282 analysis 195 display different information 6 extract from sequence list 145 find 127 information 141 join 214 layout 123 lists 143 logo 313 logo Bioinformatics explained 290 INDEX new 142 region types 130 search 127 select 129 shuffle 195 statistics 208 view 122 view as text 142 view circular 130 view format 6 web info 158 Sequence logo 289 Sequencing data 311 Sequencing primers 314 Share data 63 311 Share Side Panel Settings 92 Shared BLAST database 1 3 Shortcuts 80 Show results from a finished process 8 Show dialogs 90 Show hide Toolbox 9 Shuffle sequence 195 312 Side Panel tutorial 40 Side Panel Settings export 92 import 92 Share with others 92 Side Panel location of 91 Signal peptide 234 235 313 SignalP 234 Bioinformatics explained 235 Single base editing in sequences 130 Single cutters 267 SNP detection 311 Solexa see Illumina Genome Analyzer SOLID data 311 Sort sequences alphabetically 293 sequences by similarity 293 Sort folders 65 Source element 117 Species display name 67 Staden file format 327 Standard layout trees 305 Standard Settings CLC 96 348 Star activity 2 8 Start Codon 230 Startup problems
48. 2 Set program parameters 3 Set input parameters Choose parameters Limit by entrez query Made Y Low complexity Human repeats Choose filter Mask For lookup Mask lower case Expect 10 Word size 3 y Matrix BLOSUM62 v Gap cost Existence 11 Extension 1 w JS epee See Semen Xo Figure 2 13 The BLAST search is limited to homo sapiens ORGN The remaining parameters are left as default 2 5 2 Inspecting the results The output is shown in figure 2 14 and consists of a list of potential homologs that are sorted by their BLAST match score and shown in descending order below the query sequence ATPBal 2QU AT8BA1 HUMAN NTI2 ATBA2_H 8198 AT8B2_H score 1567 8 bits 4050 Expect DEDO TFOZATOBA M identities 779 144 68 Positives 933 1144 82 Gaps 22 1144 2 3920 478B1_ HUMAN e M i 2GIAT11B_ HUMAN B196 AT114 HUMAN IB4S AT11C HUMAN CA23JATOBS HUMAN 0423 478B3_ HUMAN J11O ATPSA HUMAN 202114 THA ULIAAA KI 4 lis IT Eigg NE Figure 2 14 Output of a BLAST search By holding the mouse pointer over the lines you can get information about the sequence sp Q9NTIZIATEA HUMAN Probable phospholipicttransporting ATPase IB ATPase class 2 ML 1 Try placing your mouse cursor over a potential homologous sequence You will see that a context box appears containing information about the sequence and the match scores obtained from the BLAST a
49. 2 4 Gel electrophoresis 2 5 315 marker 2 8 view 2 6 view preferences 2 6 when finding restriction sites 2 3 GenBank view sequence in 142 file format 327 search 148 312 search sequence in 158 tutorial 43 Gene Construction Kit file format 327 Gene expression analysis 312 Gene finding 230 General preferences 89 General Sequence Analyses 195 Genetic code reverse translation 25 Getting started tutorial 37 gff file format 329 Google sequence 158 Graph export data points in csv format 113 Graph Side Panel 316 Graphics data formats 329 export 109 gzip file format 329 Gzip file format 329 Half life 212 Handling of results 118 Header 101 Heat map 312 Help 29 Hide show Toolbox 9 High throughput sequencing 311 History 116 export 108 preserve when exporting 11 7 source elements 11 7 INDEX Homology pairwise comparison of sequences in alignments 298 Hydrophobicity 244 313 Bioinformatics explained 247 Chain Flexibility 248 Cornette 126 248 Eisenberg 126 247 Emini 126 Engelman GES 126 247 Hopp Woods 126 248 Janin 126 248 Karplus and Schulz 126 Kolaskar Tongaonkar 126 248 Kyte Doolittle 126 247 Rose 248 Surface Probability 248 Welling 126 248 ID license 19 Ilumina Genome Analyzer 311 Import bioinformatic data 103 104 existing data 38 FASTA data 38 from a web page 104 list of formats 326 preferences 94 raw sequence 104 Side
50. 280 Modules 30 Molecular weight 211 Motif list 223 Motif search 217 223 314 Mouse modes 6 Move content of a view elements in Navigation Area 65 sequences in alignment 293 msf file format 329 Multiple alignments 299 313 Multiselecting 65 Name 141 Navigate 3D structure 187 Navigation Area 62 create local BLAST database 1 4 illustration 62 NCBI 148 search for structures 154 search sequence in 158 search tutorial 43 NCBI BLAST add more databases 322 Negatively charged residues 213 Neighbor Joining algorithm 308 Neighbor joining 314 Nested PCR primers 314 Network configuration 33 Network drive shared BLAST database 1 3 Never show this dialog again 90 New feature request 28 folder 65 folder tutorial 38 sequence 142 New sequence create from a selection 129 Newick file format 328 Next Generation Sequencing 311 nexus file format 329 Nexus file format 327 328 NGS 311 nhr file format 329 NHR file format 329 Non standard residues 125 Nucleotide info 125 345 sequence databases 321 Nucleotides UIPAC codes 332 Numbers on sequence 123 nwk file format 329 nxs file format 329 094 file format 329 Open consensus sequence 288 from clipboard 104 Open reading frame determination 230 Open ended sequence 230 Order primers 314 ORF 230 Organism 141 Origins from 117 Overhang of fragments from restriction digest 2 4 Overhang find rest
51. 289 Foreground color Colors the letters using a gradient where the right side color is used for highly conserved positions and the left side color is used for positions that are less conserved Background color Sets a background color of the residues using a gradient in the same way as described above Graph Displays the conservation level as a graph at the bottom of the alignment The bar default view show the conservation of all sequence positions The height of the graph reflects how conserved that particular position is in the alignment If one position is 100 conserved the graph will be shown in full height Learn how to export the data behind the graph in section 7 4 x Height Specifies the height of the graph x Type The type of the graph Line plot Displays the graph as a line plot Bar plot Displays the graph as a bar plot Colors Displays the graph as a color bar using a gradient like the foreground and background colors x Color box Specifies the color of the graph for line and bar plots and specifies a gradient for colors e Gap fraction Which fraction of the sequences in the alignment that have gaps The gap fraction is only relevant if there are gaps in the alignment Foreground color Colors the letter using a gradient where the left side color is used if there are relatively few gaps and the right side color is used if there are relatively many gaps Background color Sets a backgrou
52. 5 5 hours Extinction coefficient This measure indicates how much light is absorbed by a protein at a particular wavelength The extinction coefficient is measured by UV spectrophotometry but can also be calculated The amino acid composition is important when calculating the extinction coefficient The extinction coefficient is calculated from the absorbance of cysteine tyrosine and tryptophan using the following equation Ext Protein count Cystine Ext Cystine count Tyr xExt T yr count Trp Ext Trp CHAPTER 14 GENERAL SEQUENCE ANALYSES 213 where Ext is the extinction coefficient of amino acid in question At 280nm the extinction coefficients are Cys 120 Tyr 1280 and Trp 5690 This equation is only valid under the following conditions e pH 6 5 e 6 0 M guanidium hydrochloride e 0 02 M phosphate buffer The extinction coefficient values of the three important amino acids at different wavelengths are found in Gill and von Hippel 1989 Knowing the extinction coefficient the absorbance optical density can be calculated using the following formula Ext Protei Absorbancel Protein A olecular weig Two values are reported The first value is computed assuming that all cysteine residues appear as half cystines meaning they form di sulfide bridges to other cysteines The second number assumes that no di sulfide bonds are formed Atomic composition Amino acids are indeed very simple compounds All 20 amino acid
53. A soe Delete ae A paTH10 Tue Jun Edit b A pATHI I Tue JUN Sioe Sorin ae p THE Tue Jun smoensted Cloninm ae p THS Tue Jun smoensted Clonirn HC pBLCATE Tue Jun smoensted Plasmi ae pELCATS Tue Jun smoensted Plasmii HE pBLCATS Tue Jun smoensted Clonin Pest46 DEVGGEALGF Pest46 LLVVYPWT OF P68046 FFDSFGDLS 4 Move to Recycle Bin Figure 3 7 Changing the common name of five sequences Length feed 7599 Mame Description Latin Mame Taxonomy Common Mame Linear act PEBDES E ast pasosa O wer Peso46 E IEEE mM act Pas225 Ea PF68225 VDEVGGEALI P68225 RLLVWYPWT PF68225 RFFESFGDL E Jam EJES Il Common Mame gt Lis Oe Him Ei Ela Li Mme Li El 10 Figure 3 8 A View Area can enclose several views each view is indicated with a tab see right view which shows protein P68225 Furthermore several views can be shown at the same time in this example four views are displayed This chapter deals with the handling of views inside a View Area Furthermore it deals with rearranging the views Section 3 3 deals with the zooming and selecting functions 3 2 1 Open view Opening a view can be done in a number of ways double click an element in the Navigation Area CHAPTER 3 USER INTERFACE 1 or select an element in the Navigation Area File Show Select the desired way to view the element or select an element in the
54. AY738615 Homo sapiens hemoglobin delta beta Fusion protein HBD HBB gene 180 HUMDINUC Human dinucleotide repeat polymorphism at the D115439 and HBB loci 190 HUMHBB Human beta globin region on chromosome NH 000044 Homo sapiens androgen receptor dihydrotestosterone receptor testi 4314 IPERH2BD P maniculatus deer mouse beta 2 globin Hbb b2 DNA 3 region 194 PERH3BC P maniculatus deer mouse beta 3 globin Hbb b3 DNA 3 region 196 sequence list 0 Ty EIEEE EE Figure 7 16 Selected elements in a Folder Content view When the elements are selected do the following to copy the selected elements right click one of the selected elements Edit Copy 55 Then right click in the cell AZ Paste 7 The outcome might appear unorganized but with a few operations the structure of the view in CLC Protein Workbench can be produced Except the icons which are replaced by file references in Excel Note that all tables can also be Exported ES directly in Excel format Chapter 8 History log Contents 8 1 Element history 2 4665 c a8 ee ee ss dade ds E Owe A AA 116 8 1 1 Sharing data with history 2 117 CLC Protein Workbench keeps a log of all operations you make in the program If e g you rename a sequence align sequences create a phylogenetic tree or translate a sequence you can always go back and check what you have done In this way you ar
55. An explanation of how a particular function is activated is illustrated by and bold E g select the element Edit Rename Chapter 2 Tutorials Contents 2 1 Tutorial Getting started lt 2 37 2 1 1 Creating a a folder en sicario A dom E 38 2 1 2 Import dat esmero A 38 2 2 Tutorial View sequence 0 ee eee eee ee aa 38 2 3 Tutorial Side Panel Settings 0 0 0 2 ee eee ee es 40 2 3 1 Saving the settings inthe Side Panel 42 2 3 2 Applying saved settings aoao aooaa oa oa a a a a ae 43 2 4 Tutorial GenBank search and download lt lt lt lt 43 2 4 1 Searching for matching objects e a cc 44 24 2 Saving the sequence ee ee a 2 44 2 5 Tutorial BLAST search 288i 88 eee ee eH eee eee eRe ada 44 2 5 1 Performing the BLAST search 2 6 eee ee ee eee a ee ew ES 45 2 5 2 Inspecting the results vs 46 2 5 3 Using the BLAST table view 0 0000 a eee 47 2 6 Tutorial Tips for specialized BLAST searches 0 80808 47 2 6 1 Locate a protein sequence on the chromosome 48 2 6 2 BLAST for primer binding sites 0 02058 eee eee 50 2 0 3 Finding remote protein homologues 50 505806 51 2 6 4 Further reading 2 8 gc ee a Ge a ee ae ede eee a 51 2 7 Tutorial Proteolytic cleavage detection lt 2 52 2 8 Tu
56. Area The Side panel in the right side of the view allows you to adjust the way the tree is displayed 2 9 1 Tree layout Using the Side Panel in the right side of the view you can change the way the tree is displayed Click Tree Layout and open the Layout drop down menu Here you can choose between standard and topology layout The topology layout can help to give an overview of the tree if some of the branches are very short When the sequences include the appropriate annotation it is possible to choose between the accession number and the species names at the leaves of the tree Sequences downloaded from GenBank for example have this information The Labels preferences allows these different node annotations as well as different annotation on the branches The branch annotation includes the bootstrap value if this was selected when the tree was calculated It is also possible to annotate the branches with their lengths CHAPTER 2 TUTORIALS of 2 10 Tutorial Find restriction sites This tutorial will show you how to find restriction sites and annotate them on a sequence There are two ways of finding and showing restriction sites In many cases the dynamic restriction sites found in the Side Panel of sequence views will be useful since it is a quick and easy way of showing restriction sites In the Toolbox you will find the other way of doing restriction site analyses This way provides more control of the analysis and gives you mor
57. Found 7 reading frames Fri Nov 17 PERH2BA Found 4 reading Frames Fri Nov 17 PERH2BB Found 7 reading frames Fri Now 17 PERH2BD Found 8 reading frames Fri Now 17 PERH3BA Found 3 reading frames Fri Now 17 PERH3BC Found 7 reading frames Fri Nov 17 Figure 9 4 An example of a batch log when finding open reading frames The log will either be saved with the results of the analysis or opened in a view with the results depending on how you chose to handle the results Part II Bioinformatics Chapter 10 Viewing and editing sequences Contents 10 1 View Sequence 0 88 ee eee ee ee ee a 122 10 1 1 Sequence settings in Side Panel 0 eee 123 10 1 2 Restriction sites in the Side Panel lt 129 10 1 3 Selecting parts of the sequence onoono 129 10 1 4 Editing the sequence oo 130 10 1 5 Sequence region TypeS 130 10 2 CMN sus os 130 10 2 1 Using split views to see details of the circular molecule 132 10 2 2 Mark molecule as circular and specify starting point 152 10 3 Working with annotations lt 133 10 3 1 Viewing annotations eee ee 133 10 3 2 Adding annotations lt ia ue a sa kw a aa a 137 10 3 3 Edit annotations 24 w aw a AAA 139 10 3 4 Removing annotations 140 10
58. G R W P D ER A Sbjct 290 TLASVLDCTVTPMGSRMLKRWLHMPVRDTRVLLERQOTIGA 330 Figure 12 17 Blast aligning in both directions The initial word match is marked green N ab G T 12 m og O N Sequence 1 N D O D 5 T 16 O 0 0 Sequence 1 Figure 12 18 Each dot represents a word match Increasing the threshold of T limits the search space significantly 12 5 4 Which BLAST program should use Depending on the nature of the sequence it is possible to use different BLAST programs for the database search There are five versions of the BLAST program blastn blastp blastx tblastn tblastx Option Query Type DB Type Comparison Note Nucleotide Nucleotide Nucleotide Nucleotide blastp Protein Protein tblastn Protein Nucleotide Protein Protein EN database is translated into protein blastx Nucleotide Protein Protein Protein The queries are translated Bee Mesos A A TS O tolastx Nucleotide Nucleotide Protein Protein The queries and database are A A A rensleted into proton The most commonly used method is to BLAST a nucleotide sequence against a nucleotide database blastn or a protein sequence against a protein database blastp But often another BLAST program will produce more interesting hits E g if a nucleotide sequence is translated CHAPTER 12 BLAST SEARCH 180 before the search it is more likely to find better and more accurate hits than just a blastn search One of the reasons for this is
59. J D Kiemer L Fausb ll A and Brunak S 2005 Non classical protein secretion in bacteria BMC Microbiol 5 58 Bendtsen et al 2004b Bendtsen J D Nielsen H von Heijne G and Brunak S 2004b Improved prediction of signal peptides SignalP 3 0 J Mol Biol 340 4 7183 795 Blobel 2000 Blobel G 2000 Protein targeting Nobel lecture Chembiochem 1 86 102 Clote et al 2005 Clote P Ferr F Kranakis E and Krizanc D 2005 Structural RNA has lower folding energy than random RNA of the same dinucleotide frequency RNA 11 5 578 591 Cornette et al 1987 Cornette J L Cease K B Margalit H Spouge J L Berzofsky J A and DeLisi C 1987 Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins J Mol Biol 195 3 659 685 Crooks et al 2004 Crooks G E Hon G Chandonia J M and Brenner S E 2004 WebLogo a sequence logo generator Genome Res 14 6 1188 1190 Dayhoff and Schwartz 1978 Dayhoff M O and Schwartz R M 1978 Atlas of Protein Sequence and Structure volume 3 of 5 suppl pages 353 358 Nat Biomed Res Found Washington D C 334 BIBLIOGRAPHY 335 Dempster et al 1977 Dempster A Laird N Rubin D et al 1977 Maximum likelihood from incomplete data via the EM algorithm Journal of the Royal Statistical Society 39 1 1 38 Eddy 2004 Eddy S R 2004 Where did the BLOSUM62
60. Panel Settings 92 using copy paste 104 Index for searching 88 Infer Phylogenetic Tree 301 Insert gaps 292 Installation 12 Invert sequence 228 Isoelectric point 211 IUPAC codes nucleotides 332 Join alignments 294 sequences 214 Jpg format export 111 Keywords 141 Label of sequence 123 Landscape Print orientation 100 344 Lasergene sequence file format 327 Latin name batch edit 69 Length 141 License 15 ID 19 starting without a license 27 License server 24 License server access offline 25 Limited mode 27 Links from annotations 139 Linux installation 14 installation with RPM package 15 List of restriction enzymes 2 8 List of sequences 143 Load enzyme list 268 Local BLAST 165 Local BLAST Database 174 Local BLAST database management 1 5 Local BLAST Databases 172 Local complexity plot 207 312 Local Database BLAST 165 Locale setting 90 Location search in 86 of selection on sequence 7 7 path to 63 Side Panel 91 Locations multiple 311 Log of batch processing 120 Logo sequence 289 313 ma4 file format 329 Mac OS X installation 13 Manage BLAST databases 175 Manipulate sequences 312 315 Manual editing auditing 90 Manual format 34 Marker in gel view 2 8 Maximize size of view 4 Maximum likelihood 314 Menu Bar illustration 62 MFold 314 mmCGCIF file format 329 Mode toolbar 6 Modification date 141 INDEX Modify enzyme list
61. Processes 8 Properties batch edit 69 Protease cleavage 259 Protein charge 240 313 cleavage 259 hydrophobicity 24 7 Isoelectric point 211 report 253 312 346 report output 254 signal peptide 234 statistics 211 structure prediction 251 translation 255 Proteolytic cleavage 259 313 Bioinformatics explained 262 tutorial 52 Proteolytic enzymes cleavage patterns 324 Proxy server 33 ps format export 111 psi file format 329 PubMed references search 159 PubMed references search 312 Quick start 29 Rasmol colors 125 Reading frame 230 Realign alignment 313 Rebase restriction enzyme database 2 8 Rebuild index 88 Recycle Bin 68 Redo alignment 285 Redo Undo 2 Reference sequence 311 References 338 Region types 130 Remove annotations 140 sequences from alignment 293 terminated processes 8 Rename element 68 Report program errors 28 Report protein 312 Request new feature 28 Residue coloring 125 Restore deleted elements 68 size of view 4 Restriction enzmyes filter 269 271 279 from certain suppliers 269 2 1 279 Restriction enzyme list 278 Restriction enzyme star activity 2 8 Restriction enzymes methylation 269 2 1 279 number of cut sites 267 INDEX overhang 269 2 1 279 separate on gel 2 6 sorting 267 Restriction sites 313 enzyme database Rebase 2 8 select fragment 129 number of 2 1 on sequence 124 266 parameters 2 0 tutorial 5
62. Protein Workbench should be used to open CLC files and click Next e Choose whether you would like to create desktop icon for launching CLC Protein Workbench and click Next e Choose if you would like to associate clc files to CLC Protein Workbench If you check this option double clicking a file with a clc extension will open the CLC Protein Workbench e Wait for the installation process to complete choose whether you would like to launch CLC Protein Workbench right away and click Finish When the installation is complete the program can be launched from your Applications folder or from the desktop shortcut you chose to create If you like you can drag the application icon to the dock for easy access 1 2 4 Installation on Linux with an installer Navigate to the directory containing the installer and execute it This can be done by running a command similar to sh CLCProteinWorkbench 5 JRE sh If you are installing from a CD the installers are located in the linux directory Installing the program is done in the following steps e On the welcome screen click Next e Read and accept the License agreement and click Next e Choose where you would like to install the application and click Next For a system wide installation you can choose for example opt or usr local If you do not have root privileges you can choose to install in your home directory e Choose where you would like to create symbolic links to the progra
63. Results handling 118 Reverse complement 227 313 Reverse sequence 228 Reverse translation 255 313 Bioinformatics explained 257 Right click on Mac 34 RNA secondary structure 314 RNA translation 228 RNA Seq analysis 311 rnami file format 329 Rotate 3D structure 187 Safe mode 29 Save changes in a view 2 sequence 44 style sheet 95 view preferences 95 workspace 9 Save enzyme list 268 SCF2 file format 327 SCF3 file format 327 Score BLAST search 170 Scoring matrices Bioinformatics explained 204 BLOSUM 204 PAM 204 Scroll wheel to zoom in 76 to zoom out 6 Search 86 in one location 86 BLAST 161 162 for structures at NCBI 154 GenBank 148 GenBank file 142 handle results from GenBank 150 handle results from NCBI structure DB 156 handle results from UniProt 153 347 hits number of 90 in a sequence 127 in annotations 127 in Navigation Area 84 Local BLAST 165 local data 311 options GenBank 149 options GenBank structure search 155 options UniProt 152 own motifs 223 parameters 149 152 155 patterns 215 217 Pfam domains 249 PubMed references 159 sequence in UniProt 159 sequence on Google 158 sequence on NCBI 158 sequence on web 158 TrEMBL 152 troubleshooting 88 UniProt 152 Secondary structure predict RNA 314 Secondary structure prediction 251 313 Select exact positions 127 in sequence 129 paris of a sequence 129 workspace 9 Select annotation
64. Suffix Import Export Description ACE ace X X No chromatogram or quality score CLC cle X X Rich format including all information Zip export Zip X Selected files in CLC format Zip import zip gzip tar X Contained files folder structure F 1 3 Alignment formats File type Suffix Import Export Description CLC cle X X Rich format including all information Clustal Alignment aln X X GCG Alignment msf X X Nexus NXs nexus X X Phylip Alignment phy X X Zip export zip X Selected files in CLC format Zip import zip gzip tar X Contained files folder structure F 1 4 Tree formats File type Suffix Import Export Description CLC cle X X Rich format including all information Newick wk X X Nexus nXS nexus X X Zip export Zip X Selected files in CLC format Zip import Zip gzip tar X Contained files folder structure APPENDIX F FORMATS FOR IMPORT AND EXPORT F 1 5 Miscellaneous formats File type Suffix BLAST Database phr nhr CLC clc CSV CSV Excel xIs xIsx GFF gff mmCIF cif PDB pdb Tab delimited txt Text txt Zip export Zip Zip import 329 Import Export Description X X zip gzip tar X Link to database imported Rich format including all information All tables All tables and reports See http www clcbio com annotate with gff 3D structure 3D structure All tables All data in a textual format Selected files in CLC format Contained files folder structure Note The Workbench can
65. The algorithm assumes that the distance data has the so called molecular clock property i e the divergence of sequences occur at the same constant rate at all parts of the tree This means that the leaves of UPGMA trees all line up at the extant sequences and that a root is estimated as part of the procedure Arabidopsis thaliana Arabidopsis thaliana Saccharomyces cerevisiae Schizosaccharomyces pombe 100 Mus musculus Bos taurus Homo sapiens 201 Mus musculus Bos taurus Homo sapiens Saccharomyces cerevisiae Schizosaccharomyces pombe Arabidopsis thaliana Arabidopsis thaliana Figure 19 6 Algorithm choices for phylogenetic inference The bottom shows a tree found by the neighbor joining algorithm while the top shows a tree found by the UPGMA algorithm The latter algorithm assumes that the evolution occurs at a constant rate in different lineages Neighbor Joining The neighbor joining algorithm Saitou and Nei 1987 on the other hand builds a tree where the evolutionary rates are free to differ in different lineages i e the tree does not have a particular root Some programs always draw trees with roots for practical reasons but for neighbor joining trees no particular biological hypothesis is postulated by the placement of the root The method works very much like UPGMA The main difference is that instead of using pairwise distance this method subtracts the distance to all other nodes from the pairwise distance Th
66. Translate part of a nucleotide sequence If you want to make separate translations of all the coding regions of a nucleotide sequence you can check the option Translate CDS and ORF in the translation dialog see figure 15 6 If you want to translate a specific coding region which is annotated on the sequence use the following procedure Open the nucleotide sequence right click the ORF or CDS annotation Translate CDS ORF 1 choose a translation table OK If the annotation contains information about the translation this information will be used and you do not have to specify a translation table The CDS and ORF annotations are colored yellow as default 15 6 Find open reading frames The CLC Protein Workbench Find Open Reading Frames function can be used to find all open reading frames ORF in a sequence or by choosing particular start codons to use it can be used as a rudimentary gene finder ORFs identified will be shown as annotations on the sequence You have the option of choosing a translation table the start codons to use minimum ORF length as well as a few other parameters These choices are explained in this section To find open reading frames select a nucleotide sequence Toolbox in the Menu Bar Nucleotide Analyses Ga Find Open Reading Frames xx or right click a nucleotide sequence Toolbox Nucleotide Analyses 4 Find Open Reading Frames x This opens the dialog displayed in figure 15 7
67. V Index 332 333 334 339 Part Introduction Chapter 1 Introduction to CLC Protein Workbench Contents 1 1 Contact information 2 eee 2 12 1 2 Download and installation lt 12 1 2 1 Program download o s o ss essere da 12 1 2 2 Installation on Microsoft Windows n nonoo e 28528 eee 12 1 2 3 Installation on Mac OSX aoaaa E EE E TO 13 1 2 4 Installation on Linux with an installer noa oaoa o 582506 14 1 2 5 Installation on Linux with an RPM package 15 1 3 System requirements 2 15 LA CUICENSES e224 cu eae wee eee eee CESS AA 15 1 4 1 Request an evaluation license a ee ee ee ee es 16 1 4 2 Download a license lt lt lt 19 1 43 Importa license from a file lt lt 21 LAA MC MC usaras a AAA 21 1 4 5 Configure license server connection lt 24 1 4 6 Limited mode isa se cde owe a a SO A amp 21 1 5 About CLC Workbenches 0 lt lt lt 27 1 5 1 New program feature request 28 Lose Report program errorS css E TES DS 28 1 5 3 CLC Sequence Viewer vs Workbenches 29 1 6 When the program is installed Getting started 29 LO TIRO assinar owe ea hee Ea da 29 1 6 2 Import of example data
68. View Double click the tab of the View Restore View Double click the View title EL Reverse zoom function Shift Shift Click in view Select multiple elements Ctrl ab Click elements Select multiple elements Shift Shift Click elements ements in this context refers to elements and folders in the Navigation Area selections on sequences and rows in tables Chapter 4 Searching your data Contents 4 1 What kind of information can be searched 080 088 ee eae 83 42 Quick search ice ee eee bee eee eee ee E ER eH 84 fie Quick search TESUNS moe baw be Aes eo ed bw ewe a 84 4 2 2 Special search expressions 85 4 2 3 Quick search history 0 86 4 3 Advanced search 2 0 02 eee ee ee 2 86 44 Seatech index aos ma AAA 88 There are two ways of doing text based searches of your data as described in this chapter e Quick search directly from the search field in the Navigation Area e Advanced search which makes it easy to make more specific searches In most cases quick search will find what you need but if you need to be more specific in your search criteria the advanced search is preferable 4 1 What kind of information can be searched Below is a list of the different kinds of information that you can search for applies to both quick search and the advanced search e Name The name of a sequence an alignment or any other kind
69. Windows Server 2008 e Mac OS X 10 5 or newer PowerPC G4 G5 or Intel CPU required e Linux RedHat 5 or later SuSE 10 or later e 32 or 64 bit e 256 MB RAM required e 512 MB RAM recommended e 1024 x 768 display recommended 1 4 Licenses When you have installed CLC Protein Workbench and start for the first time you will meet the license assistant shown in figure 1 1 The following options are available They will be described in detail in the following sections e Request an evaluation license The license is a fully functional time limited license see below e Download a license When you purchase a license you will get a license ID from CLC bio Using this option you will get a license based on this ID e Import a license from a file If CLC bio has provided a license file or if you have downloaded a license from our web based licensing system you can import it using this option e Upgrade license lf you already have used a previous version of CLC Protein Workbench and you are entitled to upgrading to the new CLC Protein Workbench 5 8 select this option to get a license upgrade CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 16 License Wizard fez p CLC Protein Workbench You need a license In order to use this application you need a valid license Please choose how you would like to obtain a license For your workbench Request an Evaluation License Choose this option if you would like
70. a aa ee eS 60 61 62 69 16 18 19 80 83 83 84 86 88 89 89 90 93 94 95 98 99 100 101 CONTENTS 8 History log 8 1 Element history sicwscnsaascidsciadiams sda ee Oe a OS 9 Batching and result handling 9 1 Howto handle results of analyses a III Bioinformatics 10 Viewing and editing sequences 10 1 View sequence e sra AA 20 ROS aw atta eek eRe he oe eee oe Se ee eS ERA GX 10 3 Working with annotations 2 26 548682 eee CARERS eR EH TE A E 2 MC MM A s Gee oe ee AAA US VIEW eee wk ae eee Hee ee eee ee ee ee ee ee et 10 6 Creating a new sequence lt he 0 10 7 Seguence LS esoo AAA 11 Online database search 11 1 GenBank search 0 0 ce ee ee ee ek ee ek a 11 2 UniProt Swiss Prot TrEMBL search a 11 3 Search for structures at NCBI 0 0 2 0 0 eee ee ee es 11 4 Sequence web info 24 4 5 486 0848 a ew KEEL ARERR SO 12 BLAST Search 12 1 Running BLAST searches 2 16888 cms ew ee NE ew E A 12 2 Output from BLAST searches o a ee 42 0 Local BLASI databases oa ce duh ee be ee Bw eee eae we Rw Se Ee E 12 4 Manage BLAST databases 0 0 0 2 eee ee ee ee 12 5 Bioinformatics explained BLAST 0 00 00 wee ee ee ee 13 3D molecule viewing 193 1 IMPONE SUUCUUING THES lt lt s sos acra Re oe we de eck Gwe we ew Ge ek ES 13 2 Viewing structure files 13 3 Sele
71. a list of available resources see figure 1 27 Currently the only resources available are PFAM databases for use with CLC Protein Workbench and CLC Main Workbench Because procedures for downloading installation uninstallation and updating are the same as for plug ins see section 1 7 1 and section 1 7 2 for more information CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 33 Updates available nip CLC Plugins Updates are available for your plug ins and or resources Use the list below to select which updates you would like to install IF you prefer you can install the updates manually through the plugin and resource manager Additional Alignments Version 1 03 Size 12 5 MB Updated bo At new versions of the CLC Workbenches Figure 1 26 Plug in updates Manage Plug ins and Resources Q 5 gt Manage Plug ins Download Plug ins Manage Resources Download Resources PFAM 100 A Version 1 01 Top 100 occuring protein domains G PF AM 100 Size 5 MB Download and Install E Version 1 0 PFAM 500 D inti Version 1 0 sop Top 500 occuring protein domains PFAM Full Version 1 0 Complete PFAM database Mi Figure 1 27 Resources available for download 1 8 Network configuration If you use a proxy server to access the Internet you must configure CLC Protein Workbench to use this Otherwise you will not be able to perform any online activities e g searching GenBank CLC Protein
72. a sequence was selected before choosing the Toolbox action this sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove elements from the selected elements Click Next to adjust dot plot parameters Clicking Next opens the dialog Shown in figure 14 4 Notice Calculating dot plots take up a considerable amount of memory in the computer Therefore you see a warning if the sum of the number of nucleotides amino acids in the sequences is higher than 8000 If you insist on calculating a dot plot with more residues the Workbench may shut down allowing you to save your work first However this depends on your computer s memory configuration Adjust dot plot parameters There are two parameters for calculating the dot plot e Distance correction only valid for protein sequences In order to treat evolutionary transitions of amino acids a distance correction measure can be used when calculating the dot plot These distance correction matrices substitution matrices take into account the likeliness of one amino acid changing to another e Window size A residue by residue comparison window size 1 would undoubtedly result in a very noisy background due to a lot of similarities between the two sequences of interest For DNA sequences the background noise will be even more dominant as a match between only four nucleotide is very likely to happen Moreover a residue by residue comparison window size 1
73. action they are now listed in the Selected Elements window of the dialog Use the arrows to add or remove alignments from the selected elements Click Next opens the dialog shown in figure 18 11 o q Join Alignments 1 Select alignments of Me ameters type same y 2 Set parameters Set order of concatenation top first IEE alignment 2 aj W IEE alignment 1 vi A Previous gt Next Y Emish XX cone o Figure 18 11 Selecting order of concatenation To adjust the order of concatenation click the name of one of the alignments and move it up or down using the arrow buttons Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The result is seen in figure 18 12 CHAPTER 18 SEQUENCE ALIGNMENT 296 4 100 200 sequence A from alignment 1 _ _ _ _ _ _ ________________ 110 sequence B from alignment _ _ _ _ __ 110 sequence A from alignment 2 _ _ il sequence B from alignment 2 e 111 v Es a Figure 18 12 The joining of the alignments result in one alignment containing rows of sequences corresponding to the number of uniquely named sequences in the joined alignments 18 4 1 How alignments are joined Alignments are joined by considering the sequence names in the individual alignments If two sequences from different alignments have identical names they are considere
74. among sites The number of categories used in the dicretization of the gamma distribution as well as the gamma distribution parameter may be adjusted by the user as the gamma distribution is restricted to have mean 1 there is only one parameter in the distribution e Estimation estimation is done according to the maximum likelihood principle that is a search is performed for the values of the free parameters in the model assumed that results in the highest likelihood of the observed alignment Felsenstein 1981 By ticking the estimate substitution rate parameters box maximum likelinood values of the free parameters in the rate matrix describing the assumed substitution model are found If the Estimate topology box is selected a search in the space of tree topologies for that which best explains the alignment is performed If left un ticked the starting topology is kept fixed at that of the starting tree The Estimate Gamma distribution parameter is active if rate variation has been included in the model and in this case allows estimation of the Gamma distribution parameter to be switched on or off If the box is left un ticked the value is fixed at that given in the Rate variation part In the absence of rate variation estimation of substitution parameters and branch lengths are carried out according to the expectation maximization algorithm Dempster et al 1977 With rate variation the maximization algorithm is performed The topology space i
75. and the file is imported as the type specified Force import as external file This option should be used if a file is imported as a bioinformatics file when it should just have been external file It could be an ordinary text file which is imported as a sequence Import using drag and drop It is also possible to drag a file from e g the desktop into the Navigation Area of CLC Protein Workbench This is equivalent to importing the file using the Automatic import option described above If the file type is not recognized it will be imported as an external file Import using copy paste of text If you have e g a text file or a browser displaying a sequence in one of the formats that can be imported by CLC Protein Workbench there is a very easy way to get this sequence into the Navigation Area Copy the text from the text file or browser Select a folder in the Navigation Area Paste 71 This will create a new sequence based on the text copied This operation is equivalent to saving the text in a text file and importing it into the CLC Protein Workbench If the sequence is not formatted i e if you just have a text like this ATGACGAATAGGAGTTC TAGCTA you can also paste this into the Navigation Area Note Make sure you copy all the relevant text otherwise CLC Protein Workbench might not be able to interpret the text 1 1 2 Import Vector NTI data There are several ways of importing your Vector NTI data into the CLC Workbench
76. and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents 16 2 Protein charge In CLC Protein Workbench you can create a graph in the electric charge of a protein as a function of pH This is particularly useful for finding the net charge of the protein at a given pH This knowledge can be used e g in relation to isoelectric focusing on the first dimension of 2D gel electrophoresis The isoelectric point pl is found where the net charge of the protein is zero The calculation of the protein charge does not include knowledge about any potential post translational modifications the protein may have The pKa values reported in the literature may differ slightly thus resulting in different looking graphs of the protein charge plot compared to other programs In order to calculate the protein charge Select a protein sequence Toolbox in the Menu Bar Protein Analyses la Create Protein Charge Plot or right click a protein sequence Toolbox Protein Analyses la Create Protein Charge Plot L This opens the dialog displayed in figure 16 6 If a sequence
77. are displayed 189 134 JD OUUU isso ona eed cased bee wwe RG Ee 193 In order to understand protein function it is often valuable to see the actual three dimensional structure of the protein This is of course only possible if the structure of the protein has been resolved and published CLC Protein Workbench has an integrated viewer of structure files Structure files are usually deposited at the Protein DataBank PDB www rcsb org where protein structure files can be searched and downloaded 13 1 Importing structure files In order to view the three dimensional structure files there are different ways to import these The supported file formats are PDB and mmCIF which both can be downloaded from the Protein DataBank http www rcsb org and imported through the import menu see section 7 1 1 Another way to import structure files is if a structure file is found through a direct search at the GenBank structure database http www ncbi nlm nih gov entrez query fcgi db Structure Read more about search for structures in section 11 3 It is also possible to make a BLAST search against the PDB database In the latter case structure files can be directly downloaded to the navigation area by clicking the Open structure button below all the BLAST hits Downloading structure files from a conducted BLAST search is only possible if the results are shown in a BLAST table See figure 13 1 How to conduct a BLAST search can be seen in section 12 1 1
78. at the very bottom of the dialog but this will import the other settings of the Preferences dialog see section 5 4 The dialog asks if you wish to overwrite existing Side Panel settings or if you wish to merge the CHAPTER 5 USER PREFERENCES AND SETTINGS 93 x q Select Settings To Export x v Non compact 4 No annotations Y No restriction sites A Export XX Cancel A Cancel Figure 5 6 Exporting all settings for circular views imported settings into the existing ones see figure 5 7 CO How do you want to import o Merge into existing styles Overwrite existing styles X Cancel Figure 5 7 When you import settings you are asked if you wish to overwrite existing settings or if you wish to merge the new settings into the old ones Note If you choose to overwrite the existing settings you will loose all the Side Panel settings that you have previously saved To avoid confusion of the different import and export options here is an overview e Import and export of bioinformatics data such as sequences alignments etc described in section 1 1 e Graphics export of the views which creates image files in various formats described in section 7 3 e Import and export of Side Panel Settings as described above e Import and export of all the Preferences except the Side Panel settings This is described in the previous section 5 3 Advanced preferences
79. bin 0 Y ay738615 CCTTTAGTGATGGCCTGGCTCACCTGG F 10000 puma C Double stranded Tool box H t Alignments and Trees Kd General Sequence Analyses Numbers on sequences ELEA Nucleotide Analyses Relative to 1 57 Rmelgiction Sites da A Protein AMalyses Numbers on plus strand e Follow selection a Database Search a Processes Toolbox Idle 1 elementis ape selected Status Bar Figure 3 1 The user interface consists of the Menu Bar Toolbar Status Bar Navigation Area Toolbox and View Area 3 1 Navigation Area The Navigation Area is located in the left side of the screen under the Toolbar see figure 3 2 It is used for organizing and navigating data Its behavior is similar to the way files and folders are usually displayed on your computer ta HS CLC_Data E gt Example Data i a Cloning vectors FE Extra aa Nucleotide GF Protein ES RNA E im e Qs centr searchter gt JA Figure 3 2 The Navigation Area CHAPTER 3 USER INTERFACE 63 3 1 1 Data structure The data in the Navigation Area is organized into a number of Locations When the CLC Protein Workbench is started for the first time there is one location called CLC_Data unless your computer administrator has configured the installation otherwise A location represents a folder on the computer The data shown under a location in the Navigation Area is stored on the computer in the folder which the location points to This is explai
80. bioinformatics methods 19 1 Inferring phylogenetic trees For a given set of aligned sequences see chapter 18 it is possible to infer their evolutionary relationships In CLC Protein Workbench this may be done either by using a distance based method see Bioinformatics explained in section 19 2 or by using the statistically founded maximum likelinood ML approach Felsenstein 1981 Both approaches generate a phylogenetic tree The tools are found in Toolbox Alignments and trees To generate a distance based phylogenetic tree choose Create Tree HE and to generate a maximum likelihood based phylogenetic tree choose Maximum Likelihood Phylogeny In both cases the dialog displayed in figure 19 1 will be opened 301 CHAPTER 19 PHYLOGENETIC TREES 302 E q Create Tree 1 Select alignments of Select alia ments Of sa E ee Projects Selected Elements 1 a omni IEE alignment 1 23 Example Data E3 Cloning Primers Protein analyses 5 Protein orthologs RNA secondary str Sequencing data b Q lt enter search term gt A E Previous gt Next Finish X Cancel Figure 19 1 Creating a Tree If an alignment was selected before choosing the Toolbox action this alignment is now listed in the Selected Elements window of the dialog Use the arrows to add or remove elements from the Navigation Area Click Next to adjust parameters 19 1 1 Phyloge
81. can add or change the locations in this list using the Manage BLAST Databases tool see section 12 4 CHAPTER 12 BLAST SEARCH 1 5 g El Create BLAST Database S xs J 1 Choose where to run Set database abe obs 2 Select sequences of same 3 Set database properties Database Name Orthologs Description set of ortholog proteins Location C Users smoensted CLCdatabases w DA Cem EE Figure 12 13 Providing a name and description for the database and the location to save the files to Click Finish to create the BLAST database Once the process is complete the new database will be available in the Manage BLAST Databases dialog see section 12 4 and when running local BLAST see section 12 1 3 12 4 Manage BLAST databases The BLAST database available as targets for running local BLAST searches see section 12 1 3 can be managed through the Manage BLAST Databases dialog see figure 12 14 Toolbox BLAST Manage BLAST Databases e BLAST Database Manager BLAST database locations home joeuser CLCdatabases a home joeuser blastdbs Add Location Remove Location Refresh Locations BLAST databases overview Name Description Date Sequences Type Total size Location 1000 residues fungupdate fungnew 08 02 2011 17634 DNA 51683 home joeuser pataa Protein sequences d 04 05 2011 974785 Protei
82. can be either numerical or graphical Why is that In clear cut examples there are no doubt yes this is a signal peptide But in borderline cases it is often convenient to have more information than just a yes no answer Here a graphical output can aid to interpret the correct answer An example is shown in figure 16 5 The graphical output from SignalP neural network comprises three different scores C S and Y Two additional scores are reported in the SignalP3 NN output namely the S mean and the D score but these are only reported as numerical values For each organism class in SignalP Eukaryote Gram negative and Gram positive two different neural networks are used one for predicting the actual signal peptide and one for predicting the position of the signal peptidase SPase cleavage site The S score for the signal peptide prediction is reported for every single amino acid position in the submitted sequence with high scores indicating that the corresponding amino acid is part of a signal peptide and low scores indicating that the amino acid is part of a mature protein CHAPTER 16 PROTEIN ANALYSES 239 SignalP NN prediction gram networks SFMA ECOLI C score S score Y score Score MES INE I EG I YMKLRF SSALAAAL FAATGSYAAVVDGGT HFEGELVNAACSVNTDSADQVVT LGQYR 0 10 20 30 40 50 60 70 Position Figure 16 5 Graphical output from the SignalP method of Swiss Prot entry SFMA_ECOLT Initially this seemed like a borde
83. corresponding selection providing an easy way for you to focus on the same region in both views 10 2 2 Mark molecule as circular and specify starting point You can mark a DNA molecule as circular by right clicking its name in either the sequence view or the circular view In the right click menu you can also make a circular molecule linear A circular molecule displayed in the normal sequence view will have the sequence ends marked with a The starting point of a circular sequence can be changed by make a selection starting at the position that you want to be the new starting point right click the selection Move Starting Point to Selection Start Note This can only be done for sequence that have been marked as circular CHAPTER 10 VIEWING AND EDITING SEQUENCES 133 10 3 Working with annotations Annotations provide information about specific regions of a sequence A typical example is the annotation of a gene on a genomic DNA sequence Annotations derive from different Sources Sequences downloaded from databases like GenBank are annotated e In some of the data formats that can be imported into CLC Protein Workbench sequences can have annotations GenBank EMBL and Swiss Prot format The result of a number of analyses in CLC Protein Workbench are annotations on the sequence e g finding open reading frames and restriction map analysis You can manually add annotations to a sequence described in the section 10 3 2
84. daa ADA 07 33 0 Meto aaa a E EA AAA TT 3 3 7 Changing compactness a 17 3 4 Toolbox and Status Bar lt 78 3 4 1 Processes o ee rara 18 S o TOON ce twee eee aa eh AAA 19 C435 SOUS EG iat tte dass de inad taa eaa ed se haw ee as 19 3 5 MoMA Essas ET ES RAE E 19 CHAPTER 3 USER INTERFACE 62 3 5 1 Create Workspace 6c ec ee eh GBH ERS HE KR A 19 3 5 2 Select Workspace ee a 19 3 5 3 Delete Workspace ee 80 3 6 List of shortcuts 6 ec aaa aaa a A 80 This chapter provides an overview of the different areas in the user interface of CLC Protein Workbench As can be seen from figure 3 1 this includes a Navigation Area View Area Menu Bar Toolbar Status Bar and Toolbox CLC Protein Workbench 3 0 Current workspace Default SEE File Edit Search view Toolbox Workspace Help s E t A Casa AE e vir ANA Es TK E IN i 7 Show New Import Export Graphics Print Copy Workspace Search Fit Width 10096 Selection Zoom In Zoom Menu Bar Wavigation Ares acre AY738615 Toolbar ta EIS S E Sequences A de SE DOG PERH2BD Navigation Are A 208 HUMHBB v Sequence layout ge 733615 Spacing 206 NM 000044 7 Ras A No spacing 3 sequence list E X PERH3BC O No wrap View Area f Protein Hj Extra Auto wrap 3 E README O Fixed yap s Recycle
85. database It will take a few more steps but you will most likely be able to import this way Importing parts of the database Instead of importing the whole database automatically you can export parts of the database from Vector NTI Explorer and subsequently import into the Workbench First export a selection CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 106 EE vector NTI Data aa Proteins EE Nucleotide OE ADCY 2 Adenoz 50 ADRALA j 256 BaculoDirect Linear DMA i cem 206 BaculoDirect Linear DNA Clonir an e BPw1 sn E ER OF j an e CDE E CAF Figure 4 The Vector NTI Data folder containing all imported sequences of the Vector NTI Database of files as an archive as shown in figure 7 5 Exploring Local Vector NTI Database DNA RNA Edit View Analyses Align Database Assemble Tools Help Ge O Ea ta Order Open ase DNA RNA Molecules Edit Linear Basic NCBI Entrez NCBI E New 2 35937 Linear Basic NCBI Entrez NCBIE Import A 2306 Linear Basic NCBI Entrez NCBI E Molecule into Text file Linear Basic Invitrogen Invitro Gateway cloning Sequence into Tert file E Ce AE cs Launch TOPO wizard 5 Circular Basic NCBI Entrez NCBI E Selection into Archive Linear Basic NCBI Entrez NCBIE Delete with Descendants from DB 2226 Linear Basic NCBI Entrez NCBIE 6 Circular Basic NCBI Entrez MCBI E Exclude from Subset Linear Basic NCBI Entrez NCBI E lt Delete from Database Linear Basic NCBI Entre
86. e g which pages to print The Print preview window is for preview only the layout of the pages must be adjusted in the Page setup Chapter 7 Import export of data and graphics Contents 7 1 Bioinformatic data formats ananass osoasa 102 GLL Import of bioinformatic dala a os do a a da we aw dE dd 103 e er import Vect r NTI data lt lt lt sisi 104 7 1 3 Export of bioinformatics data a o aoao oaoa a a a a a a a a 107 7 2 External files lt 2 2 4 109 7 3 Export graphics to files lt lt lt lt tt 1 109 7 3 1 Which part of the view to export 110 1 3 2 Save location and file formats 110 7 3 3 Graphics export parameters le 112 2 Exporting protein repots cassar ME Bow woe 113 7 4 Export graph data points to a file 2 ee ee lt lt lt 4 113 7 5 Copy paste view output 0 2 ee et 4 115 CLC Protein Workbench handles a large number of different data formats All data stored in the Workbench are available in the Navigation Area The data of the Navigation Area can be divided into two groups The data is either one of the different bioinformatic data formats or it can be an external file Bioinformatic data formats are those formats which the program can work with e g sequences alignments and phylogenet
87. env nr Environmental samples month New or revised GenBank sequences Simply add another database as a new line with the first item being the database name taken from http www ncbi nlm nih gov staff tao URLAPI remote blastdblist html and the second part is the name to display in the Workbench Restart the Workbench and the new database will be visible in the BLAST dialog Appendix E Proteolytic cleavage enzymes Most proteolytic enzymes cleave at distinct patterns Below is a compiled list of proteolytic enzymes used in CLC Protein Workbench 324 APPENDIX E PROTEOLYTIC CLEAVAGE ENZYMES 325 Name PA PPP AA Cyanogen bromide CNBr M h e AspNendopeptidase o DO E RR Bse o eo e quo E BE 2 E ARE op e Trypsin KR Trypsin MRR Trypsin BK Toypsint KY Tyesi RK Toyesint RRR Chymotrypsin high spec ff motP o gt Chymotrypsin high spec J je W _ not MP anda Speo A E a ii A tae ENANA low Chymotrypsin low spec _ M not ner Pe Y Chymotrypsin low spec tro CA M IN W O o lodosobenzoate a W E pee F L M or V Foto o HRR P oP o CEE E O ha Factor Xa G CC ee Troman Prev Tobacco Fin Wis LY T E ET Appendix F Formats for import and export F 1 List of bioinformatic data formats Below is a list of bioinformatic data formats i e formats for importing and exporting sequences alignments and trees 326 APPENDIX F FORMATS FOR IMPORT A
88. existing alignment At the top you can see a fixpoint that has already been added on g 100 HBA ANAPE HBA_ANSSE ED Se HBB_ANAPP HBB_AQUCH HBB_CALJA 100 200 HBA ANAPE E HBA ANSSE HBA_ACCGE HBB_ANAPP HBB AQUCH im HBB_CALJA Figure 18 7 Realigning using fixpoints In the top view fixpoints have been added to two of the sequences In the view below the alignment has been realigned using the fixpoints The three top sequences are very similar and therefore they follow the one sequence number two from the top that has a fixpoint aligned to each other Advanced use of fixpoints Fixpoints with the same names will be aligned to each other which gives the opportunity for great control over the alignment process It is only necessary to change any fixpoint names in very Special cases One example would be three sequences A B and C where sequences A and B has one copy of a domain while sequence C has two copies of the domain You can now force sequence A to align to the first copy and sequence B to align to the second copy of the domains in sequence C This is done by inserting fixpoints in sequence C for each domain and naming them fp and fp2 CHAPTER 18 SEQUENCE ALIGNMENT 288 for example Now you can insert a fixpoint in each of sequences A and B naming them fp1 and fp2 respectively Now when aligning the three sequences us
89. files into the Navigation Area select one or more of the search results Ctrl C 38 C on Mac select location or folder in the Navigation Area Ctrl V Note Search results are downloaded before they are saved Downloading and saving several files may take some time However since the process runs in the background displayed in the Toolbox under the Processes tab it is possible to continue other tasks in the program Like the search process the download process can be stopped paused and resumed 11 2 3 Save UniProt search parameters The search view can be saved either using dragging the search tab and and dropping it in the Navigation Area or by clicking Save E When saving the search only the parameters are saved not the results of the search This is useful if you have a special search that you perform from time to time Even if you don t save the search the next time you open the search view it will remember the parameters from the last time you did a search 11 3 Search for structures at NCBI This section describes searches for three dimensional structures from the NCBI structure database http www ncbi nlm nih gov Structure MMDB mmdb shtml For manipu lating and visualization of the downloaded structures see section 13 The NCBI search view is opened in this way Search Search for structures at NCBI He CHAPTER 11 ONLINE DATABASE SEARCH or Ctrl B 3 Bon Mac This opens the view shown in
90. fixpoint on each of the two residues select the region and realign it using the fixpoints Now the two residues are aligned with each other and everything in the selected region around them is adjusted to accommodate this change 18 4 Join alignments CLC Protein Workbench can join several alignments into one This feature can for example be used to construct supergenes for phylogenetic inference by joining alignments of several disjoint genes into one spliced alignment Note that when alignments are joined all their annotations are carried over to the new spliced alignment Alignments can be joined by CHAPTER 18 SEQUENCE ALIGNMENT 295 select alignments to join Toolbox in the Menu Bar Alignments and Trees 2 Join Alignments or select alignments to join right click either selected alignment Toolbox Align ments and Trees Join Alignments Ez This opens the dialog shown in figure 18 10 E q Join Alignments 1 Select alignments of Select align ents or sa pe type same y Projects Selected Elements 2 CLC Data PE alignment 2 Example Data Cloning 55 Primers 4 7 Protein analyses Protein orthologs iai i alignment 1 RNA secondary str 3 Sequencing data e ES EJ Q lt enter search term gt A Figure 18 10 Selecting two alignments to be joined If you have selected some alignments before choosing the Toolbox
91. fragment mass The molecular weight is not necessarily directly correlated to the fragment length as amino acids have different molecular masses For that reason it is also possible to limit the search for proteolytic cleavage sites to mass range Example If you have one protein sequence but you only want to show which enzymes cut between two and four times Then you should select The enzymes has more cleavage sites than 2 and select The enzyme has less cleavage sites than 4 In the next step you should simply select all enzymes This will result in a view where only enzymes which cut 2 3 or 4 times are presented Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The result of the detection is displayed in figure 16 26 ac ATPBal le a i Trypsin Trypsin ryP 1 dis E Sequence layout Atp8al Trypsin Trypsin Trypsin Trypsin E Spacing E No spacing v 3 No wrap ATP8a1 ETNLKIRQGLPATSDIKDIDSLMRISGRIECESPNRHL TO Auto wrap EE ATP8a1 proteo Rows 26 Table of remaining fragments based on parameter settings Filter All v ES 2 Automatic w a Start End posi Length Mass C end Name Fragment N end Name Show column 28 37 10 1 147 19 4 27 K START TSLADQEEVR T Trypsin 7 Start position 38 48 11 1 302 52 9 75 R Trypsin TIFINQPQLTK F Trypsin ioe i i V End position 49 58 10 1 120 24 9 22 K Trypsin FCNNHVSTA
92. generating a sequence of the same expected single amino acid frequency e Dipeptide shuffling Shuffle method generating a sequence of the exact same dipeptide frequency e Dipeptide sampling from first order Markov chain Resampling method generating a sequence of the same expected dipeptide frequency For further details of these algorithms see Clote et al 2005 In addition to the shuffle method you can specify the number of randomized sequences to output Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish This will open a new view in the View Area displaying the shuffled sequence The new sequence is not saved automatically To save the sequence drag it into the Navigation Area or press ctrl S S on Mac to activate a save dialog 14 2 Dot plots Dot plots provide a powerful visual comparison of two sequences Dot plots can also be used to compare regions of similarity within a sequence This chapter first describes how to create and second how to adjust the view of the plot 14 2 1 Create dot plots A dot plot is a simple yet intuitive way of comparing two sequences either DNA or protein and is probably the oldest way of comparing two sequences Maizel and Lenk 1981 A dot plot is a 2 dimensional matrix where each axis of the plot represents one sequence By sliding a fixed size window over the sequences and making a sequence match by a dot in the matrix a diagonal lin
93. gradient box is also practical when producing an output for printing Too much background color might not be desirable By crossing one slider over the other the two sliders change side the colors are inverted allowing for a white background If you choose a color gradient which includes white Se figure 14 5 CHAPTER 14 GENERAL SEQUENCE ANALYSES 200 PRECES ya PEGS 140 q 130 120 1104 Sequence 2 10 a 30 40 50 ED TD Em Bo 100 110 im 130 140 Sequence Figure 14 6 Dot plot with inverted colors practical for printing 14 2 3 Bioinformatics explained Dot plots Realization of dot plots Dot plots are two dimensional plots where the x axis and y axis each represents a sequence and the plot itself shows a comparison of these two sequences by a calculated score for each position of the sequence If a window of fixed size on one sequence one axis match to the other sequence a dot is drawn at the plot Dot plots are one of the oldest methods for comparing two sequences Maizel and Lenk 1981 The scores that are drawn on the plot are affected by several issues e Scoring matrix for distance correction Scoring matrices BLOSUM and PAM contain substitution scores for every combination of two amino acids Thus these matrices can only be used for dot plots of protein sequences e Window size The single residue comparison bit by bit comparison window size 1 in dot plots
94. import external files too This means that all kinds of files can be imported and displayed in the Navigation Area but the above mentioned formats are the only ones whose contents can be shown in the Workbench F 2 List of graphics data formats Below is a list of formats for exporting graphics All data displayed in a graphical format can be exported using these formats Data represented in lists and tables can only be exported in pdf format see section 7 3 for further details Format Suffix Portable Network Graphics png JPEG jpg Tagged Image File tif PostScript ps Encapsulated PostScript eps Portable Document Format pdf Scalable Vector Graphics SVE Type bitmap bitmap bitmap vector graphics vector graphics vector graphics vector graphics Appendix G IUPAC codes for amino acids Single letter codes based on International Union of Pure and Applied Chemistry The information is gathered from http www ebi ac uk 2can tutorials aa html 330 APPENDIX G One letter abbreviation Mm TOmOooOo VU Z D gt x NU lt lt SA WCCO VU TZ Ss IUPAC CODES FOR AMINO ACIDS Three letter Description abbreviation Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid Cys Cysteine Gin Glutamine Glu Glutamic acid Gly Glycine HIS Histidine Xle Leucine or Isoleucineucine Leu Leucine ILe Isoleucine Lys Lysine Met Methionine Phe Phenylalanine Pro Proline Pyl Pyrrolysine
95. import of preferences lt lt lt lt lt 94 5 4 1 The different options for export and importing 94 5 5 View settings for the Side Panel lt ee eee 95 5 5 1 Fidating Side Panel o caciozornesiao aras 97 The first three sections in this chapter deal with the general preferences that can be set for CLC Protein Workbench using the Preferences dialog The next section explains how the settings in the Side Panel can be saved and applied to other views Finally you can learn how to import and export the preferences The Preferences dialog offers opportunities for changing the default settings for different features of the program The Preferences dialog is opened in one of the following ways and can be seen in figure 5 1 Edit Preferences 175 or Ctrl K 36 on Mac 5 1 General preferences The General preferences include e Undo Limit As default the undo limit is set to 500 By writing a higher number in this field more actions can be undone Undo applies to all changes made on sequences alignments or trees See section 3 2 5 for more on this topic 89 CHAPTER 5 USER PREFERENCES AND SETTINGS 90 EB Preferences Lea inde Suppo Undo limit 500 Rude Su ppor Enable audit of manual sequence modifications EN Search Number of hits ca Number of hits normal search 50 Number of hits NCBI Uniprot 50 a View c
96. intervals with unknown endpoints some cover more than one interval etc In the following all of these will be referred to as regions Regions are generally illustrated by markings often arrows on the sequences An arrow pointing to the right indicates that the corresponding region is located on the positive strand of the sequence Figure 10 2 is an example of three regions with separate colors HBE1 Figure 10 2 Three regions on a human beta globin DNA sequence HUMHBB Figure 10 3 shows an artificial sequence with all the different kinds of regions 10 2 Circular DNA A sequence can be shown as a circular molecule select a sequence in the Navigation Area Show in the Toolbar As Circular Q or If the sequence is already open Click Show As Circular at the lower left part of the view This will open a view of the molecule similar to the one in figure 10 4 CHAPTER 10 VIEWING AND EDITING SEQUENCES 131 20 40 Gene Gene 1 Gene Gene CLCCECCLCE LCCLCCLCOL CCLCCLCCLO GLEGCLGCLCEG LCCLCCLCCL CC ED Bl 100 Gene Gene Gene LCELCCLCCL CCLCCL COLCOCLCCLUCELCO LCCLCCLOCL CCLCCLCCOLCOCL 120 140 Gene I Gene Gene COPLCOELCELO EOSCGLECUECAEECUECGLCECL CCLCCLCCLC CLCCLCCLCC LC 160 180 200 Gene CLCCLCCLCC LCCLECCLCCL CCOLCCLCCLC CLCELCELEC LECCLCCLCCL ce 220 M0 260 Gene Genel LCCELCOELCEL GELCCLCCLC CLECCLCELCO LECLCCLCCL CELCELCELC EL 280 300 CCLCCLCCLC CCLCCLCCLC CCLCCLCCLC CCLECLCCLC CCLCCLCCLC C
97. like the green promoter region annotation in figure 2 3 and zoomed to see the residues In this tutorial we want to have an overview of the whole sequence Hence click Zoom Out 5 in the Toolbar click the sequence until you can see the whole sequence lf you have downloaded the example data this will be placed as a folder in CLC Data CHAPTER 2 TUTORIALS 39 E CLC Protein Workbench 3 0 Current workspace Default SEs File Edit Search View Toolbox Workspace Help da SL e a o Dad ES ER DER LPP Show New Import Export Graphics Print Copy Workspace Search Fit Width 10096 Selection Zoom In Zoom Out ac HUMDINUC Es ta y a CLC Data My folder HUMDINUC ACAAATTGATTAATGATAGTGCTATC lt x HUMDINUC Sequence layout 43 Recycle bin 1 T Spacing HUMDINUC CTCTTGCATTTAGAGTTTAACTGGTA No spacing 60 No wrap HUMDINUC CCTACTTCCAAAAGGGAAACAGAATT Atowrap 80 100 Fixed wrap HUMDINUC AGAAAAGAAAATGTGGTTCCAGAAAG 10000 120 Double stranded Alignments and Trees General Sequence Analyses HUMDINUC GAAGAAAAAGAACACACACACACACA 4 Numbers on sequences A Nucleotide Analyses 140 PE EO 1 ag Restriction Sites F Protein Analyses HUMDINUC CACACACACACACACACACACACTGC A SILL a Follow selection Database Search 160 180 Processes Toolbox Idle 1 element s are selected Figure 2 2 The HUMDINUC file is imported and opened Ge Ee E Er od
98. maT NM 177965 2 Homo sapiens chromosome 8 open reading frame 37 C8orf37 38 2 38 2 4 7 2 100 UEGM Genomic sequences show first NT 010859 14 Homo sapiens chromosome 18 genomic contig reference assembly 339 602 85 1e 90 100 NW 926940 1 Homo sapiens chromosome 18 genomic contig alternate assembly 339 602 85 1e 90 100 NT 011109 15 Homo sapiens chromosome 19 genomic contig reference assembly 262 375 73 3e 67 94 NW 927217 1 Homo sa piens chromosome 19 genomic contig alternate assembly 62 375 73 3e 67 94 Figure 12 20 BLAST table view A table view with one row per hit showing the accession number and description field from the sequence file together with BLAST output scores and the start and stop positions for the query and hit sequence are listed The strand and orientation for query sequence and hits are also found here In most cases the table view of the results will be easier to interpret than tens of sequence alignments CHAPTER 12 BLAST SEARCH 183 gt ref NM 173209 1 UE GM Homo sapiens TGFB induced factor TALE family homeobox TGIF transcript variant 5 mRNA Length 1382 Sort alignments for this subject sequence by Evalue Score Percent identity LEFY start position Subject start position Score 339 bits 171 Expect le 50 Identities 171 171 100 Gaps 0171 0 Stcrand Plus Plus Query Sbjct Query 1113 ATTIGCACATGGGATIGCTAAAACAGCTICCIGITACIGAGATGICITCAATGGAATACA AAA ATITGCACA
99. molecular phylogeny The data is most commonly represented in the form of DNA or protein sequences but can also be in the form of e g restriction fragment length polymorphism RFLP Methods for constructing molecular phylogenies can be distance based or character based Distance based methods Two common algorithms both based on pairwise distances are the UPGMA and the Neighbor Joining algorithms Thus the first step in these analyses is to compute a matrix of pairwise distances between OTUs from their sequence differences To correct for multiple substitutions it is common to use distances corrected by a model of molecular evolution such as the Jukes Cantor model Jukes and Cantor 1969 UPGMA A simple but popular clustering algorithm for distance data is Unweighted Pair Group Method using Arithmetic averages UPGMA Michener and Sokal 1957 Sneath and Sokal 19 3 This method works by initially having all sequences in separate clusters and continuously joining these The tree is constructed by considering all initial clusters as leaf nodes in the tree and each time two clusters are joined a node is added to the tree as the parent of the two chosen nodes The clusters to be joined are chosen as those with minimal pairwise distance The branch lengths are set corresponding to the distance between clusters which is calculated CHAPTER 19 PHYLOGENETIC TREES 308 as the average distance between pairs of sequences in each cluster
100. of whole sequence 4 Reading frame 1 Reading frame 2 Reading frame 3 Reading frame 1 Reading frame 2 Reading frame 3 Translation of coding regions Y Translate CDS Translate ORF Genetic code translation table 1 Standard 2 JS tere Pe isd rit crea Figure 15 6 Choosing 1 and 3 reading frames and the standard translation table Here you have the following options Reading frames If you wish to translate the whole sequence you must specify the reading frame for the translation If you select e g two reading frames two protein sequences are generated Translate coding regions You can choose to translate regions marked by and CDS or ORF annotation This will generate a protein Sequence for each CDS or ORF annotation on the sequence Genetic code translation table Lets you specify the genetic code for the translation The translation tables are occasionally updated from NCBI The tables are not available in this CHAPTER 15 NUCLEOTIDE ANALYSES 230 printable version of the user manual Instead the tables are included in the Help menu in the Menu Bar in the appendix Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The newly created protein is shown but is not saved automatically To save a protein sequence drag it into the Navigation Area or press Ctrl S 6 S on Mac to activate a save dialog 15 5 1
101. of zooming in 3 3 2 Zoom Out It is possible to zoom out step by step on a sequence Click Zoom Out 45 in the toolbar click in the view until you reach a satisfying zoomlevel or Press on your keyboard The last option for zooming out is only available if you have a mouse with a scroll wheel or Press and hold Ctrl 38 on Mac Move the scroll wheel on your mouse backwards CHAPTER 3 USER INTERFACE 1 When you choose the Zoom Out mode the mouse pointer changes to a magnifying glass to reflect the mouse mode Note You might have to click in the view before you can use the keyboard or the scroll wheel to zoom If you want to get a quick overview of a sequence or a tree use the Fit Width function instead of the Zoom Out function If you press Shift while clicking in a View the zoom function is reversed Hence clicking on a sequence in this way while the Zoom Out mode toolbar item is selected zooms in instead of zooming out 3 3 3 Fit Width The Fit Width 4 function adjusts the content of the View so that both ends of the sequence alignment or tree is visible in the View in question This function does not change the mode of the mouse pointer 3 3 4 Zoom to 100 The Zoom to 100 function zooms the content of the View so that it is displayed with the highest degree of detail This function does not change the mode of the mouse pointer 3 3 5 Move The Move mode allows you to drag the content
102. option to search for motifs with a user specified similarity to the target sequence and furthermore the motifs found can be displayed in an overview table This is particularly useful when searching for motifs on many sequences To start the Toolbox motif search Toolbox General Sequence Analyses 171 Motif Search 4 Use the arrows to add or remove sequences or sequence lists from the selected elements You can perform the analysis on several DNA or several protein sequences at a time If the analysis is performed on several sequences at a time the method will search for patterns in the sequences and create an overview table of the motifs found in all sequences Click Next to adjust parameters see figure 14 26 G Motif Search eS 1 Select one or more sequences of same type 2 Set parameters Motif parameters Simple motif Java regular expression site regular expression Use motif list O Motif tataaa Accuracy 80 w Search for the reverse motif Include negative strand Exclude unknown regions Exclude matches in N regions For simple motifs g E Ceen Se JL gema Kona Figure 14 26 Setting parameters for the motif search The options for the motif search are e Motif types Choose what kind of motif to be used Simple motif Choosing this option means that you enter a simple motif e g ATGATGNNATG Java regular expression See section 14 7 3 Prosite regul
103. region as part of a signal peptide In the output of the BLAST search low complexity regions will be marked in lowercase gray characters default setting The low complexity region cannot be thought of as a significant match thus disabling the low complexity filter is likely to generate more hits to sequences which are not truly related Word size Change of the word size has a great impact on the seeded sequence space as described above But one can change the word size to find sequence matches which would otherwise not be found using the default parameters For instance the word size can be decreased when searching for primers or short nucleotides For blastn a suitable setting would be to decrease the default word size of 11 to 7 increase the E value significantly 1000 and turn off the complexity filtering For blastp a similar approach can be used Decrease the word size to 2 increase the E value and use a more stringent substitution matrix e g a PAM3O0 matrix Fortunately the optimal search options for finding short nearly exact matches can already be found on the BLAST web pages http www ncbi nlm nih gov BLAST Substitution matrix For protein BLAST searches a default substitution matrix is provided If you are looking at distantly related proteins you should either choose a high numbered PAM matrix or a low numbered BLOSUM matrix See Bioinformatics Explained on scoring matrices on http www clcbio com be The default
104. sequence e Comparative statistics layout If more sequences were selected in Step 1 this function generates statistics with comparisons between the sequences CHAPTER 14 GENERAL SEQUENCE ANALYSES 209 a G Create Sequence Statistics 8 1 Select sequences of same SEE pa amete type 2 Set parameters Layout Individual statistics layout Comparative statistics layout Background distribution For proteins Include background distribution of amino acids Based on Homo Sapiens human JCS previous pue JU Seh Xena Figure 14 15 Setting parameters for the sequence statistics You can also choose to include Background distribution of amino acids If this box is ticked an extra column with amino acid distribution of the chosen species is included in the table output The distributions are calculated from UniProt www uniprot org version 6 0 dated September 13 2005 Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish An example of protein sequence statistics is shown in figure 14 16 1 Protein statistics 1 1 Sequence information haemoglobin beta h0 chain Mus musculus 1 2 Half life Half life mammals Half life yeast Half life E Coli Figure 14 16 Comparative sequence statistics Nucleotide sequence statistics are generated using the same dialog as used for protein Sequence statistics However the output of Nucleotide s
105. sequences located in the Navigation Area can be used to create BLAST databases from Any given BLAST database can only include one molecule type If you wish to use a pre formatted BLAST database instead see section 12 3 1 To create a BLAST database go to Toolbox BLAST Create BLAST Database 24 This opens the dialog seen in figure 12 12 E E Create BLAST Database us 1 Choose where to run E a AMA AA A rhs Navigation rea Selected Elements 6 2 Select sequences of same J e CLC Data we 094296 3 Example Data Pu P39524 Cloning Pue P57792 Primers Pe Q29449 protein Pe Q9NTIZ Protein analyses f Q95x33 5 Protein orthologs su su N ES A EB as Par RNA secondary str Sequencing data She ATPBal X ATPSal genomic se 20 ATPBal mRNA A F coli Turina E Q enter search term gt AN Previous gt Next Finis XX Cancel Figure 12 12 Add sequences for the BLAST database Select sequences or sequence lists you wish to include in your database and click Next In the next dialog shown in figure 12 13 you provide the following information e Name The name of the BLAST database This name will be used when running BLAST searches and also as the base file name for the BLAST database files e Description You can add more details to describe the contents of the database e Location You can select the location to save the BLAST database files to You
106. server Check this option if you wish to use the license server e Automatically detect license server By checking this option you do not have to enter more information to connect to the server e Manually specify license server There can be technical limitations which mean that the license server cannot be detected automatically and in this case you need to specify more options manually CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 25 D CLC Protein Workbench Configure License Server connection Please choose how you would like to connect to your CLC License server V Enable license server connection Automatically detect license server Manually specify license server Port Disable license borrowing IF you choose this option users of this computer will not be able to borrow licenses From the License Server If you experience any problems please contact The CLC Support Team Proxy Settings Previous Finish Cancel Figure 1 19 Connecting to a license server Host name Enter the address for the licenser server Port Specify which port to use e Disable license borrowing on this computer If you do not want users of the computer to borrow a license see section 1 4 5 you can check this option Borrow a license A floating license can only be used when you are connected to the license server If you wish to use the CLC Protein Workbench when you are not connected to
107. several sequences at a time the method will search for patterns which is common between all the sequences Annotations will be added to all the sequences and a view is opened for each sequence Click Next to adjust parameters see figure 14 20 E q Pattern Discovery LES 1 Select one or more sequences of same type 2 Set parameters Define model Create and search with new model Use existing model O Set motif parameters Pattern length Min 4 Max 9 Noise 1 v Number of patterns to predict 1 w 2 A Previous gt Next Enshi 2 Cancel Figure 14 20 Setting parameters for the pattern discovery See text for details In order to search unknown sequences with an already existing model Select to use an already existing model which is seen in figure 14 20 Models are represented with the following icon in the navigation area HAR 14 6 1 Pattern discovery search parameters Various parameters can be set prior to the pattern discovery The parameters are listed below and a screen shot of the parameter settings can be seen in figure 14 20 e Create and search with new model This will create a new HMM model based on the selected sequences The found model will be opened after the run and presented in a table view It can be saved and used later if desired e Use existing model It is possible to use already created models to search for the same pattern in new sequences
108. such sequences regardless of whether they are highly variable or highly conserved at specific sites it is very difficult to generate a consensus sequence which covers the actual variability of a given position In order to better understand the information content or significance of certain positions a sequence logo can be used The sequence logo displays the information content of all positions in an alignment as residues or nucleotides stacked on top of each other see figure 18 8 The sequence logo provides a far more detailed view of the entire alignment than a simple consensus sequence Sequence logos can aid to identify protein binding sites on DNA sequences and can also aid to identify conserved residues in aligned domains of protein sequences and a wide range of other applications Each position of the alignment and consequently the sequence logo shows the sequence information in a computed score based on Shannon entropy Schneider and Stephens 1990 The height of the individual letters represent the sequence information content in that particular position of the alignment A sequence logo is a much better visualization tool than a simple consensus sequence An example hereof is an alignment where in one position a particular residue is found in 70 of the sequences If a consensus sequence is used it typically only displays the single residue with TO coverage In figure 18 8 an un gapped alignment of 11 E coli start codons including flan
109. than 10 000 nucleotides Note that a search can be saved 5 for later use You do not save the search results only the search parameters This means that you can easily conduct the same search later on when your data has changed 4 4 Search index This section has a technical focus and is not relevant if your search works fine However if you experience problems with your search results if you do not get the hits you expect it might be because of an index error The CLC Protein Workbench automatically maintains an index of all data in all locations in the Navigation Area If this index becomes out of sync with the data you will experience problems with strange results In this case you can rebuild the index Right click the relevant location Location Rebuild Index This will take a while depending on the size of your data At any time the process can be stopped in the process area see section 3 4 1 Chapter 5 User preferences and settings Contents 5 1 General preferences 000 ee eee ee 1 89 5 2 Default view preferences 0 08 ee eee eee 90 5 2 1 Number formatting in tables 91 5 2 2 Import and export Side Panel settings 2 08005 92 5 3 Advanced preferences lt lt lt lt 0 4 93 5 3 1 Default data location ae we aaa we ew a a 93 usas MODIBLA T oir nic iras 94 5 4 Export
110. the coding sequence of bovine myoglobin with the full mRNA of human gamma globin The top alignment is made with free end gaps while the bottom alignment is made with end gaps treated as any other The yellow annotation is the coding sequence in both sequences It is evident that free end gaps are ideal in this situation as the start codons are aligned correctly in the top alignment Treating end gaps as any other gaps in the case of aligning distant homologs where one sequence is partial leads to a spreading out of the short sequence as in the bottom alignment Both algorithms use progressive alignment The faster algorithm builds the initial tree by doing more approximate pairwise alignments than the slower option 18 1 3 Aligning alignments If you have selected an existing alignment in the first step 18 1 you have to decide how this alignment should be treated e Redo alignment The original alignment will be realigned if this checkbox is checked Otherwise the original alignment is kept in its original form except for possible extra equally sized gaps in all sequences of the original alignment This is visualized in figure 18 5 P68873 Q6WN20 P68231 Q6H1U7 P68945 Consensus Sequence Logo Conservation CHAPTER 18 SEQUENCE ALIGNMENT MVHLTPEEKS MVHLTGEEKA MVHLSGDEKN MVHLTAEEKN VHWTAEEKQ MVHLTAEEKN MVHCTSEEKs 20 AVTALWGKVN AVTALWGKVN AVHGLWSKVK AITSLWGKVA LI TGLWGKVN AVTALWGKVN Av TaLWeKVa VDEVGG
111. the criteria for discrimination of secretory and non secretory proteins The D score is introduced in SignalP version 3 0 and is a simple average of the S mean and Y max score The score shows superior discrimination performance of secretory and non secretory proteins to that of the S mean score which was used in SignalP version 1 and 2 For non secretory proteins all the scores represented in the SignalP3 NN output should ideally be very low The hidden Markov model calculates the probability of whether the submitted sequence contains a signal peptide or not The eukaryotic HMM model also reports the probability of a signal anchor previously named uncleaved signal peptides Furthermore the cleavage site is assigned by a probability score together with scores for the n region h region and c region of the signal peptide if it is found Other useful resources http www cbs dtu dk services SignalP Pubmed entries for some of the original papers CHAPTER 16 PROTEIN ANALYSES 240 http www ncbi nim nih gov entrez query fcgi db pubmed amp acmd Retrieve amp dopt AbstractPlusslist uids 9051 28 amp query hl 1i amp itool pubmed docsum http www ncbi nlm nih gov entrez query fcgi cmd Retrieve amp db PubMed amp list_ ulds 15223320 amp dopt Citation Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display
112. this dialog you can e Select which part of the view you want to print e Adjust Page Setup e See a print Preview window These three options are described in the three following sections 98 CHAPTER 6 PRINTING 99 a q Print Graphics zs Page Setup Parameters Orientation Portrait Paper Size A4 Horizontal Pagecount Not Applicable Vertical Pagecount Not Applicable Header Text Footer Text Show Pagenumber Yes Output Options Print visible area Print whole view X Cancel Help 23 Preview ED Page Setup Figure 6 1 The Print dialog 6 1 Selecting which part of the view to print In the print dialog you can choose to e Print visible area or e Print whole view These options are available for all views that can be zoomed in and out In figure 6 2 is a view of a circular sequence which is zoomed in so that you can only see a part of it pcDNA3 atp8a1 9118 bp e O E El 11 Eo HY Figure 6 2 A circular sequence as it looks on the screen When selecting Print visible area your print will reflect the part of the sequence that is visible in the view The result from printing the view from figure 6 2 and choosing Print visible area can be seen in figure 6 3 3 MV promoter T7 Promoter tp8a1 pcDNA3 atp8a1 9118 bp Figure 6 3 A print of the sequence selecting Print visible area On the other hand if you select Print whole view you will ge
113. to be shown e Accession sequences downloaded from databases like GenBank have an accession number e Latin name e Latin name accession e Common name e Common name accession Whether sequences can be displayed with this information depends on their origin Sequences that you have created yourself or imported might not include this information and you will only be able to see them represented by their name However sequences downloaded from databases like GenBank will include this information To change how sequences are displayed right click any element or folder in the Navigation Area Sequence Representation select format This will only affect sequence elements and the display of other types of elements e g alignments trees and external files will be not be changed If a sequence does not have this information there will be no text next to the sequence icon CHAPTER 3 USER INTERFACE 68 Rename element Renaming a folder or an element in the Navigation Area can be done in three different ways select the element Edit in the Menu Bar Rename or select the element F2 click the element once wait one second click the element again When you can rename the element you can see that the text is selected and you can move the cursor back and forth in the text When the editing of the name has finished press Enter or select another element in the Navigation Area If you want to discard the changes instead press
114. to try out the application For 30 days Please note that only a single 30 day evaluation license will be allowed for each computer Download a License Choose this option if you have a License Order ID and would like to download a License Import a License from a File Choose this option if you have a License File on your computer and would like to import it Upgrade a license from an older Workbench Choose this option if you have an older version of this workbench with a commercial license and would like to upgrade your license Configure License Server Connection Choose this option if your company or institution is using a central CLC License Server This option also enables you to disable a license server connection if you experience any problems please contact The CLC Support Team Proxy Settings _ o Limited Mode Previous Quit Figure 1 1 The license assistant showing you the options for getting started e Configure license server connection If your organization has a license server select this option to connect to the server Select an appropriate option and click Next If for some reason you don t have access to getting a license you can click the Limited Mode button see section 1 4 6 1 4 1 Request an evaluation license We offer a fully functional demo version of CLC Protein Workbench to all users free of charge Each user is entitled to 30 days demo of CLC Protein Workbench If
115. top see 10 5 You can also show a circular view of a sequence without opening the sequence first Select the sequence in the Navigation Area Show 45 As Circular Q 3 2 3 Close views When a view is closed the View Area remains open as long as there is at least one open view A view is closed by right click the tab of the View Close or select the view Ctrl W or hold down the Ctrl button Click the tab of the view while the button is pressed CHAPTER 3 USER INTERFACE 2 By right clicking a tab the following close options exist See figure 3 10 act P68046 O pop P68053Q pop Pos063 0 mer P File k k view k k k HBE Toolbox Show PF68225 MVHLTPEEKNAVTTLWG D Close erly Y Close Tab Area HBB TE Close all views Ctrl 5hift w E Reid Pee225 ESFGDLSSPDAVMGNPK ILDNL E save as Ctrl Shift 5 Figure 3 10 By right clicking a tab several close options are available e Close See above e Close Tab Area Closes all tabs in the tab area Close All Views Closes all tabs in all tab areas Leaves an empty workspace Close Other Tabs Closes all other tabs in all tab areas except the one that is selected 3 2 4 Save changes in a view When changes are made in a view the text on the tab appears bold and italic on Mac it is indicated by an before the name of the tab This indicates that the changes are not saved The Save function may be activated in two ways Click the tab of the vie
116. v H Download and Open Y Download and Save 2 ca Total number of hits 245 Figure 11 1 The GenBank search view 11 1 1 GenBank search options 149 Conducting a search in the NCBI Database from CLC Protein Workbench corresponds to conducting the search on NCBI s website When conducting the search from CLC Protein Workbench the results are available and ready to work with straight away You can choose whether you want to search for nucleotide sequences or protein sequences As default CLC Protein Workbench offers one text field where the search parameters can be entered Click Add search parameters to add more parameters to your search Note The search is a and search meaning that when adding search parameters to your search you search for both or all text strings rather than any of the text strings You can append a wildcard character by checking the checkbox at the bottom This means that you only have to enter the first part of the search text e g searching for genom will find both genomic and genome The following parameters can be added to the search e All fields Text searches in all parameters in the NCBI database at the same time Organism Text Description Text Modified Since Between 30 days and 10 years Molecule Genomic DNA RNA mRNA or rRNA Gene Location Genomic DNA RNA Mitochondrion or Chloroplast Sequence Length Number for maximum or minimum length of the seq
117. was selected before choosing the Toolbox action the sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements You can perform the analysis on several protein sequences at a time This will result in one output graph showing protein charge graphs for the individual proteins Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish CHAPTER 16 PROTEIN ANALYSES 241 E q Create Protein Charge Plot ES 1 Select a protein Selecta protein Projects Selected Elements 2 3 CLC_Data Ss ATPSal Example Data e 094296 Sw ATP8al Cloning Primers Protein analyses Protein orthologs su SN P39524 SN P57792 Sw Q29449 At QONTI2 FAs Q95X33 s RNA secondary struc 3 Sequencing data 4 mI Qy lt enter search term gt A Pees pe la Xen Figure 16 6 Choosing protein sequences to calculate protein charge 16 2 1 Modifying the layout Figure 16 shows the electrical charges for three proteins In the Side Panel to the right you can modify the layout of the graph Protein charge 100 0 m lt Q 100 ATP8a1 200 094296 0 2 4 6 8 10 12 14 pH Figure 16 7 View of the protein charge See section B in the appendix for information about the graph view 16 3 Transmembrane helix prediction Many p
118. will be displaced compared to the other sequences in the alignment 18 3 3 Delete residues and gaps Residues or gaps can be deleted for individual sequences or for the whole alignment For individual sequences CHAPTER 18 SEQUENCE ALIGNMENT 293 select the part of the sequence you want to delete right click the selection Edit Selection Delete the text in the dialog Replace The selection shown in the dialog will be replaced by the text you enter If you delete the text the selection will be replaced by an empty text i e deleted To delete entire columns select the part of the alignment you want to delete right click the selection Delete columns The selection may cover one or more sequences but the Delete columns function will always apply to the entire alignment 18 3 4 Copy annotations to other sequences Annotations on one sequence can be transferred to other sequences in the alignment right click the annotation Copy Annotation to other Sequences This will display a dialog listing all the sequences in the alignment Next to each sequence is a checkbox which is used for selecting which sequences the annotation should be copied to Click Copy to copy the annotation If you wish to copy all annotations on the sequence click the Copy All Annotations to other Sequences 18 3 5 Move sequences up and down Sequences can be moved up and down in the alignment drag the name of the sequence up or down When yo
119. window of the dialog Use the arrows to add or remove elements from the Navigation Area Click Next to adjust parameters 18 5 1 Pairwise comparison on alignment selection A pairwise comparison can also be performed for a selected part of an alignment right click on an alignment selection Pairwise Comparison HE This leads directly to the dialog described in the next section CHAPTER 18 SEQUENCE ALIGNMENT 297 a BN Create Pairwise Comparison EA 1 Select alignments of Select alignments of same typ same type Projects Selected Elements 1 Data EE ATP8al ortholog alignment ixample Data 3 Cloning Primers P Protein analyses gt Protein orthologs RNA secondary structure Fj Sequencing data Th Qr zenter search term gt Figure 18 13 Creating a pairwise comparison table 18 5 2 Pairwise comparison parameters There are four kinds of comparison that can be made between the sequences in the alignment as shown in figure 18 14 a I Create Pairwise Comparison EJ 1 Select alignments of select comparisons to p TL same type 2 Select comparisons to perform Select comparisons Y Gaps 4 Differences Distance 4 Similarity Y Identities mafia Dao previous pu J Jin J cama Figure 18 14 Adjusting parameters for pairwise comparison e Gaps Calculates the number of alignment positions where one se
120. 0 lt 0 n lt b me 223 CLC Protein Workbench offers different kinds of sequence analyses which apply to both protein and DNA The analyses are described in this chapter 14 1 Shuffle sequence In some cases it is beneficial to shuffle a sequence This is an option in the Toolbox menu under General Sequence Analyses It is normally used for statistical analyses e g when comparing an alignment score with the distribution of scores of shuffled sequences Shuffling a sequence removes all annotations that relate to the residues select sequence Toolbox in the Menu Bar General Sequence Analyses A Shuffle Sequence 2 195 CHAPTER 14 GENERAL SEQUENCE ANALYSES 196 or right click a sequence Toolbox General Sequence Analyses A Shuffle Sequence x This opens the dialog displayed in figure 14 1 a E Shuffle Sequence LES E Select one or more Selled one or more sequences of same type SCS sequences of same type Projects Selected Elements 1 p CLC Data XC ATP8al mRNA B b Example Data 7c ATP8al genomic xx Sw ATP8al H H Cloning H Primers Protein analyses H H Protein ortholog RNA secondary __ Sequencing data gt 4 p p Qy lt enter search term gt 4 Previous gt Next Finish X Cancel Figure 14 1 Choosing sequence for shuffling If a sequence was selected before choosing the Toolbox action this sequence is now listed in t
121. 000 Direct Download The workbench will attempt to contact the CLC Licenses Service and download the license directly This method requires internet access from the workbench Go to License Download web page Th rkbench will open a We o owser with the License Download web page From there you wil be able to download your license as a file re in the next step If you experience any problems please contact The CLC Support Team _ Proxy Settings Previous Previous Next Quit workbench Quit Workbench Figure 1 7 Entering a license ID provided by CLC bio the license ID in this example is artificial In this dialog there are two options e Direct download The workbench will attempt to contact the online CLC Licenses Service and download the license directly This method requires internet access from the workbench e Go to license download web page The workbench will open a Web Browser with the License Download web page when you click Next From there you will be able to download your license as a file and import it This option allows you to get a license even though the Workbench does not have direct access to the CLC Licenses Service If you select the first option and it turns out that you do not have internet access from the Workbench because of a firewall proxy server etc you will be able to click Previous and use the other option instead Direct download Selecting the first option takes you to the
122. 1 3 1 Which part of the view to export In this dialog you can choose to e Export visible area or e Export whole view These options are available for all views that can be zoomed in and out In figure 7 8 is a view of a circular sequence which is zoomed in so that you can only see a part of it O AY738515 0 A HBD HBB lt gt Figure 7 8 A circular sequence as it looks on the screen When selecting Export visible area the exported file will only contain the part of the sequence that is visible in the view The result from exporting the view from figure 7 8 and choosing Export visible area can be seen in figure 7 9 Figure 7 9 The exported graphics file when selecting Export visible area On the other hand if you select Export whole view you will get a result that looks like figure 7 10 This means that the graphics file will also include the part of the sequence which is not visible when you have zoomed in For 3D structures this first step is omitted and you will always export what is shown in the view equivalent to selecting Export visible area Click Next when you have chosen which part of the view to export 7 3 2 Save location and file formats In this step you can choose name and save location for the graphics file see figure 7 11 CLC Protein Workbench supports the following file formats for graphics export CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS AY738615 180 bp 111 Figure
123. 10 7 Sequence Lists The Sequence List shows a number of sequences in a tabular format or it can show the sequences together in a normal sequence view Having sequences in a sequence list can help organizing sequence data The sequence list may originate from an NCBI search chapter 11 1 Moreover if a multiple sequence fasta file is imported it is possible to store the data in a sequences list A Sequence List can also be generated using a dialog which is described here select two or more sequences right click the elements New Sequence List This action opens a Sequence List dialog CHAPTER 10 VIEWING AND EDITING SEQUENCES F G Create Sequence List m 1 Select sequences of same Mi E cds do Projects Selected Elements 6 E CLC Data 3 3 Example Data XxX ATP8al genomit 2 ATP8al mRNA Sw ATP8al 2222 Protein analyse Protein ortholog 233322 RNA secondary Sequencing dat j Qx lt enter search term gt 4 Figure 10 15 A Sequence List dialog 094296 P39524 P57792 Q29449 QONTI2 Q95X33 E 144 The dialog allows you to select more sequences to include in the list or to remove already chosen sequences from the list Clicking Finish opens the sequence list It can be saved by clicking Save or by dragging the tab of the view into the Navigation Area Opening a Sequence list is done by right click the sequence list in the Navigation
124. 14 GENERAL SEQUENCE ANALYSES 222 having to list all elements The simplest form of a regular expression is a literal string The syntax used for the regular expressions is the Java regular expression syntax see http java sun com docs books tutorial essential regex index html Below is listed some of the most important syntax rules which are also shown in the help pop up when you press Shift F1 A Z will match the characters A through Z Range You can also put single characters between the brackets The expression AGT matches the characters A G or T A D M P will match the characters A through D and M through P Union You can also put single characters between the brackets The expression AG M P matches the characters A G and M through P A M amp amp H P will match the characters between A and M lying between H and P Intersection You can also put single characters between the brackets The expression AM amp amp HGTDA matches the characters A through M which is H G T Dora A M will match any character except those between A and M Excluding You can also put single characters between the brackets The expression AG matches any character except A and G A Z amp amp M P will match any character A through Z except those between M and P Subtraction You can also put single characters between the brackets The expression A P amp amp CG matches any character between A and P except C and G
125. 4 Use tree from file e Select substitution model CLC Protein Workbench allows maximum likelihood tree estima tion to be performed under the assumption of one of four substitution models the Jukes Cantor Jukes and Cantor 1969 the Kimura 80 Kimura 1980 the HKY Hasegawa et al 1985 and the GTR also known as the REV model Yang 1994a models All models are time reversible The JC and K80 models assume equal base frequencies and the HKY and GTR models allow the frequencies of the four bases to differ they will be estimated by the observed frequencies of the bases in the alignment In the JC model all substitutions are assumed to occur at equal rates in the K80 and HKY models transition and transversion rates are allowed to differ The GIR model is the general time reversible model and allows all substitutions to occur at different rates In case of the K8O and HKY models the user may set a transtion transversion ratio value which will be used as starting value or fixed depending on the level of estimation chosen by the user see below For the substitution rate matrices describing the substitution models we use the parametrization of Yang Yang 1994a e Rate variation in CLC Protein Workbench substitution rates may be allowed to differ among the individual nucleotide sites in the alignment by selecting the include rate variation box When selected the discrete gamma model of Yang Yang 1994b is used to model rate variation
126. 4 An old license is detected When you click Next the Workbench checks on CLC bio s web server to see if you are entitled to upgrade your license Note If you should be entitled to get an upgrade and you do not get one automatically in this process please contact support clcbio com In this dialog there are two options e Direct download The workbench will attempt to contact the online CLC Licenses Service and download the license directly This method requires internet access from the workbench e Go to license download web page The workbench will open a Web Browser with the License Download web page when you click Next From there you will be able to download your license as a file and import it This option allows you to get a license even though the Workbench does not have direct access to the CLC Licenses Service If you select the first option and it turns out that you do not have internet access from the CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 23 Workbench because of a firewall proxy server etc you will be able to click Previous and use the other option instead Direct download Selecting the first option takes you to the dialog shown in figure 1 15 License Wizard eS a CLC Protein Workbench Requesting a license with id CLC LICENSE SRENMNSTED 0D43CA9 Requesting and downloading a license by establishing a direct connection to the CLC bio License Web Service Your License was successfu
127. 4 Element informati0N lt lt 141 LOS VIEWS TON orinar AA 142 10 6 Creating a new sequence 2 0 eee eee ee 142 10 7 Sequence Lists ccoo AA we we 143 10 7 1 Graphical view of sequence lists 2 0 ee ee ee es 144 10 7 2 Sequence list table a 144 10 7 3 Extract sequences aaoo a a a E ew ww a 145 CLC Protein Workbench offers five different ways of viewing and editing single sequences as described in the first five sections of this chapter Furthermore this chapter also explains how to create a new sequence and how to gather several sequences in a sequence list 10 1 View sequence When you double click a sequence in the Navigation Area the sequence will open automatically and you will see the nucleotides or amino acids The zoom options described in section 3 3 allow 122 CHAPTER 10 VIEWING AND EDITING SEQUENCES 123 you to e g zoom out in order to see more of the sequence in one view There are a number of options for viewing and editing the sequence which are all described in this section All the options described in this section also apply to alignments further described in section 18 2 10 1 1 Sequence settings in Side Panel Each view of a sequence has a Side Panel located at the right side of the view see figure 10 1 MOS et Fit Width 100 Pan CSS Zoom In Zoom Cut k Sequence layout k Annotation layout
128. 524 BBTTSHSGSR SKMTNSHANG WilPPsHZEP EETMDEDADO s 094296 BEREDMECSE SQMMSSSGON STNP EEAD 6 ED soa qui ae Ha CRS ES ARA A a GSN Ss sa ae aa aaa a 11 Consensus 0000 du ee eee ee eee ee ee ee Be ee ee eee 100 A Conservation 0 Docoooa Mo Dee ee ee at E y Op 41 A El 5S w Sequence layout Spacing Every 10 residues Y No wrap Auto wrap Fixed wrap m 60 residues 4 Numbers on sequences Relative to 1 Lock numbers Hide labels 4 Lock labels Sequence label Name v F Show selection boxes Figure 2 5 The protein alignment as it looks when you open it with background color according to the Rasmol color scheme and automatically wrapped First select No wrap in the Sequence Layout This means that each sequence in the alignment is kept on the same line To see more of the alignment you now have to scroll horizontally Next expand the Annotation Layout group and select Show Annotations Set the Offset to More offset and set the Label to Stacked Expand the Annotation Types group Here you will see a list of the types annotation that are carried by the sequences in the alignment see figure 2 6 Annotation layout Show annotations Position Offset a Show arrows Use gradients Annotation types B active site MN 4 Gene E DD Metal binding site ES Modified site ES tWP binding E Protein DD Region Doe Figure 2 6 T
129. 53 Sequence layout P68225 P Spacing P68873 F A v Every 10 residues P68228 A WGEVccEagc 30 O No wrap P68231 MMHBSCBEKN A DENccEAEG 30 Pos063 MHWTABEKO WADCGABABA 29 Auto wrap P68945 MHWTABEKO BETCBWcKYN MaDccaBadia 29 O Fixed wrap Consensus MVHLTXEEKN AVTGLWGKVN VDEVGGEALG ol oo Th Te e saene MYNE EREA AUTOLNGAVA VOEOGGEALG ame a Follow selection e qn pescas REENNNPWTO REEDSEcDES SPDANMeNPK 59 P68053 REENNNPWTO REEDSEcDES sPDANMeNPK 59 Hide labels G PDANMGNP P68225 Mi melo abro Figure 3 15 A maximized view The function hides the Navigation Area and the Toolbox 3 2 7 Side Panel The Side Panel allows you to change the way the contents of a view are displayed The options in the Side Panel depend on the kind of data in the view and they are described in the relevant sections about sequences alignments trees etc Side Panel are activated in this way select the view Ctrl U 38 U on Mac or right click the tab of the view View Show Hide Side Panel Note Changes made to the Side Panel will not be saved when you save the view See how to save the changes in the Side Panel in chapter 5 The Side Panel consists of a number of groups of preferences depending on the kind of data CHAPTER 3 USER INTERFACE 16 being viewed which can be expanded and collapsed by clicking the header of the group You can also expand or collapse all the groups by clicking the icons
130. 63 EQ gt HHT P66063 LLIVYPWTQRFFASFGNLSSPTAIIGNPMV 4 art P6B225 ED P66225 RLLVWWYPWTORFFESFGDLSSPDAVMGNP K Figure 3 13 A horizontal split screen The two views split the View Area Maximize Restore size of view The Maximize Restore View function allows you to see a view in maximized mode meaning a mode where no other views nor the Navigation Area is shown Maximizing a view can be done in the following ways select view Ctrl M or select view View Maximize restore View or select view right click the tab View Maximize restore View 1 or double click the tab of view The following restores the size of the view Ctrl M or View Maximize restore View or double click title of view CHAPTER 3 USER INTERFACE 19 art PERDAS O ser 68225 O ar P6s053 O act P68046 P68225 VDEVGGEALI P68046 DEVGGEALGF PF68225 RLLVWYPWT P656046 LLVVYPWT OF P68225 REFESFGDL P68046 FFDSFGDLS mA EE i yt Figure 3 14 A vertical split screen CLC Protein Workbench 3 0 Current workspace Default File Edit Search View Toolbox Workspace Help 15 a o Bi 5 PP fl pens l EL e vi uy TENTE xum E Es TERE X Show New Import Export Graphics Print Copy Workspace Search Fit Width 10096 Selection Zoom In Zoom Out HEE protein align T P68046 MABTABEKA GDENccEATc 29 P680
131. 7 10 The exported graphics file when selecting Export whole view The whole sequence is shown even though the view is zoomed in on a part of the sequence g Export Graphics e m 1 Output options m pe ME 2 Save in file Lookin EE Desktop 2 BE ds Recent Items Desktop Documents A Computer Ae Network Files of type Portable Document Format pdf v Directory C Users smoensted Desktop Name ATP8al pdf Previous gt Next XX Cancel Figure 7 11 Location and name for the graphics file Format Suffix Type Portable Network Graphics png bitmap JPEG Jpg bitmap Tagged Image File tif bitmap PostScript ps vector graphics Encapsulated PostScript eps vector graphics Portable Document Format pdf vector graphics Scalable Vector Graphics SVg vector graphics These formats can be divided into bitmap and vector graphics The difference between these two categories is described below Bitmap images In a bitmap image each dot in the image has a specified color This implies that if you zoom in on the image there will not be enough dots and if you zoom out there will be too many In these cases the image viewer has to interpolate the colors to fit what is actually looked at A bitmap image needs to have a high resolution if you want to zoom in This format is a good choice for storing images without large shapes e g dot plots It is also appropriate if you don t have the need for resizing and
132. 8 15 5 Translation of DNA or RNA to protein 0 0 80 eee een eee 228 15 5 1 Translate part of a nucleotide sequence o 230 15 6 Find open reading frames 2 0 eee et ee 4 230 15 6 1 Open reading frame parameters ee 230 CLC Protein Workbench offers different kinds of sequence analyses which only apply to DNA and RNA 15 1 Convert DNA to RNA CLC Protein Workbench lets you convert a DNA sequence into RNA substituting the T residues Thymine for U residues Urasil select a DNA sequence in the Navigation Area Toolbox in the Menu Bar Nucleotide Analyses Convert DNA to RNA 2 or right click a sequence in Navigation Area Toolbox Nucleotide Analyses ZA Convert DNA to RNA kg This opens the dialog displayed in figure 15 1 lf a sequence was selected before choosing the Toolbox action this sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish Note You can select multiple DNA sequences and sequence lists at a time If the sequence list contains RNA sequences as well they will not be converted 225 CHAPTER 15 NUCLEOTIDE ANALYSES 226 a EB Convert DNA to RNA ba 1 Select DNA sequences Select DNA seo
133. 8 329 341 CLC Standard Settings 96 CLC Workbenches 27 CLC file format 327 329 associating with CLC Protein Workbench 13 Cleavage 259 the Peptidase Database 263 Clone Manager file format 327 Cloning 312 315 Close view 1 Clustal file format 328 Coding sequence translate to protein 129 Codon frequency tables reverse translation 257 usage 258 col file format 329 Color residues 289 Comments 141 Common name batch edit 69 Compare workbenches 311 Complexity plot 207 Configure network 33 Conflicting enzymes 2 4 Consensus sequence 288 313 open 288 Conservation 288 graphs 313 Contact information 12 Contig 311 Copy 115 annotations in alignments 293 elements in Navigation Area 65 into sequence 130 search results GenBank 151 search results structure search 157 search results UniProt 154 sequence 142 143 sequence selection 227 text selection 142 cpf file format 94 chp file format 329 Create alignment 283 dot plots 197 enzyme list 278 local BLAST database 1 4 new folder 65 workspace 9 INDEX Create index file BLAST database 1 4 CSV export graph data points 113 formatting of decimal numbers 10 csv file format 329 CSV file format 327 329 ct file format 329 Custom annotation types 137 Data storage location 63 Data formats bioinformatic 326 graphics 329 Data sharing 63 Data structure 63 Database GenBank 148 local 63 NCBI 173 nucleo
134. AA ee O m E mu A i as a Ee a RR OO 1 SSS a EEE RR OO a ERES o ee E E REA ps EEE EEE EEE EEE A E EE eh E al IE a EEE A TE EE TT A TE Figure 12 19 BLAST graphical view A simple graphical overview of the hits found aligned to the query sequence The alignments are color coded ranging from black to red as indicated in the color label at the top Sequences producing significant alignments k headers to sort columns NM 174886 1 Homo sapiens TGFB induced factor TALE family homeobox TGIF 339 563 85 le 90 100 U E GM NM 173210 1 Homo sa piens TGFB induced factor TALE family homeobox TGIF 339 563 85 1e 90 100 MEO NM 173209 1 Homo sapiens TGFB induced factor TALE family homeobox TGIF 339 563 85 1e 90 100 UEGM NM 173211 1 Homo sa piens TGFB induced factor TALE family homeobox TGIF 339 563 85 1e 90 100 Ed NM_173207 1 Homo sapiens TGFB induced factor TALE family homeobox TGIF 339 563 85 1e 90 100 UEGM NM 173208 1 Homo sapiens TGFB induced factor TALE family homeobox TGIF 339 563 85 1e 90 100 U E GM NM 170695 2 Homo sa piens TGFB induced factor TALE family homeobox TGIF 339 563 85 1e 90 100 UE GM NM 003244 2 Homo sapiens TGFB induced factor TALE family homeobox TGIF 339 563 85 1e 90 100 UEGM NM 003246 2 Homo sapiens thrombospondin 1 THBS1 mRNA 38 2 38 2 4 7 2 100
135. AA24102 0 38 0 96 0 54 0 92 0 50 0 88 0 86 0 84 0 82 0 80 0 78 Complexity Local complexity 5 10 15 20 25 30 35 40 45 Position Figure 14 14 An example of a local complexity plot Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The values of the complexity plot approaches 1 0 as the distribution of amino acids become more complex See section B in the appendix for information about the graph view 14 4 Sequence statistics CLC Protein Workbench can produce an output with many relevant statistics for protein sequences Some of the statistics are also relevant to produce for DNA sequences Therefore this section deals with both types of statistics The required steps for producing the statistics are the same To create a statistic for the sequence do the following select sequence s Toolbox in the Menu Bar General Sequence Analyses 2 Create Sequence Statistics This opens a dialog where you can alter your choice of sequences which you want to create Statistics for You can also add sequence lists Note You cannot create statistics for DNA and protein sequences at the same time When the sequences are selected click Next This opens the dialog displayed in figure 14 15 The dialog offers to adjust the following parameters e Individual statistics layout If more sequences were selected in Step 1 this function generates separate statistics for each
136. AATGGGCGGTAGGCGTGTACGGTGGGAGG Imotif CGCAAATG GGCGGTAGGCGTG list index 5 CMV Aype Simple Idescriptior CMY promoter primer Figure 14 24 Showing dynamic motifs on the sequence To add Labels to the motif select the Flag or Stacked option They will put the name of the motif as a flag above the sequence The stacked option will stack the labels when there is more than one motif so that all labels are shown Below the labels option there are two options for controlling the way the sequence should be searched for motifs e Include reverse motifs This will also find motifs on the negative strand only available for nucleotide sequences e Exclude matches in N regions for simple motifs The motif search handles ambiguous characters in the way that two residues are different if they do not have any residues in common For example For nucleotides N matches any character and R matches A G For proteins X matches any character and Z matches E Q Genome sequence often have large regions with unknown sequence These regions are very often padded with N s Ticking this checkbox will not display hits found in N regions and if a one residue in a motif matches to an N it will be treated as a mismatch The list of motifs shown in figure 14 22 is a pre defined list that is included with the CLC Protein Workbench You can define your own set of motifs to use instead In order to do this you first need to create a Motif list E This will bring
137. ASI LIL cL Moved aligned region Wed Jan 21 10 40 45 CET 2009 User smoensted Parameters Read name Fuda 01d aliqned region 139 955 New aligned region 37 9585 Comments Edik gt Wo Comment Deleted selection Wed Jan 21 10 39 57 CET 2009 User smoensted Parameters Region 977 Modified element Rews Comments Edit No Comment Assembled sequences to reference Wed Jan 21 10 38 50 CET 2009 Figure 8 1 An element s history to your locale settings see section 5 1 e User The user who performed the operation If you import some data created by another person in a CLC Workbench that persons name will be shown e Parameters Details about the action performed This could be the parameters that was chosen for an analysis e Origins from This information is usually shown at the bottom of an element s history Here you can see which elements the current element origins from If you have e g created an alignment of three sequences the three sequences are shown here Clicking the element selects it in the Navigation Area and clicking the history link opens the element s own history e Comments By clicking Edit you can enter your own comments regarding this entry in the history These comments are saved 8 1 1 Sharing data with history The history of an element is attached to that element which means that exporting an element in CLC fo
138. All start codons in genetic code Other Here you can specify a number of start codons separated by commas e Both strands Finds reading frames on both strands e Open ended Sequence Allows the ORF to start or end outside the sequence If the sequence studied is a part of a larger sequence it may be advantageous to allow the ORF to start or end outside the sequence e Genetic code translation table e Include stop codon in result The ORFs will be shown as annotations which can include the stop codon if this option is checked The translation tables are occasionally updated from NCBI The tables are not available in this printable version of the user manual Instead the tables are included in the Help menu in the Menu Bar in the appendix CHAPTER 15 NUCLEOTIDE ANALYSES 232 e Minimum Length Specifies the minimum length for the ORFs to be found The length is specified as number of codons Using open reading frames for gene finding is a fairly simple approach which is likely to predict genes which are not real Setting a relatively high minimum length of the ORFs will reduce the number of false positive predictions but at the same time short genes may be missed see figure 15 9 th 0 J N NC 000913 selection ORF 8000 ORF aax ORF on e mp E ORF yaal Figure 15 9 The first 12 000 positions of the E coli sequence NC 000913 downloaded from GenBank The blue dark annotations are the genes while the yellow b
139. Area Show 42 Graphical Sequence List OR Table H The two different views of the same sequence list are shown in split screen in figure 10 16 10 7 1 Graphical view of sequence lists The graphical view of sequence lists is almost identical to the view of single sequences see section 10 1 The main difference is that you now can see more than one sequence in the same view However you also have a few extra options for sorting deleting and adding sequences e To add extra sequences to the list right click an empty white space in the view and select Add Sequences e To delete a sequence from the list right click the sequence s name and select Delete Sequence e To sort the sequences in the list right click the name of one of the sequences and select Sort Sequence List by Name or Sort Sequence List by Length e To rename a sequence right click the name of the sequence and select Rename Sequence 10 7 2 Sequence list table Each sequence in the table sequence list is displayed with CHAPTER 10 VIEWING AND EDITING SEQUENCES 145 PETIT sequence list E 50 E mM PERHIBA 50 100 PERHIBB DDD IT 50 100 a PERH2BA 30 100 v AB ES E FE 4 sequence list E Accession Definition Modification Date Length P maniculatus dee 27 APR 1953 110 PERHIBE M15289 P maniculatus dee 27 APR 1993 PERHZBA M15293 P maniculatus dee 27 APR 1993 PERH2BB M15290 P maniculatus d
140. Asn N 1 4 hours 3 min gt 10 hours Pro P gt 20 hours gt 20 hours e Gin Q 0 8 hour 10 min gt 10 hours Arg R 1 hour 2 min 2 min Ser S 1 9 hours gt 20 hours gt 10 hours Thr T 7 2 hours gt 20 hours gt 10 hours Val V 100 hours gt 20 hours gt 10 hours Trp W 2 8 hours 3 min 2 min Tyr Y 2 8 hours 10 min 2 min Table 14 2 Estimated half life Half life of proteins where the N terminal residue is listed in the first column and the half life in the subsequent columns for mammals yeast and E coli X Ala X Val X lle and X Leu are the amino acid compositional fractions The constants a and b are the relative volume of valine a 2 9 and leucine isoleucine b 3 9 side chains compared to the side chain of alanine Ikai 1980 Estimated half life The half life of a protein is the time it takes for the protein pool of that particular protein to be reduced to the half The half life of proteins is highly dependent on the presence of the N terminal amino acid thus overall protein stability Bachmair et al 1986 Gonda et al 1989 Tobias et al 1991 The importance of the N terminal residues is generally known as the N end rule The N end rule and consequently the N terminal amino acid simply determines the half life of proteins The estimated half life of proteins have been investigated in mammals yeast and E coli see Table 14 2 If leucine is found N terminally in mammalian proteins the estimated half life is
141. BLAST Table button f 3 at the bottom of the view Figure 12 10 is an example of a BLAST Table ES CAA26204 BLAST Rows 103 Summary oF hits From query 04426204 Per Description Bit score 1COH B Chain B Alpha Ferrous Carbonmonoxy Beta Cobaltou 3 36E 66 244 973 1 85 B Chain 6 T To T Highj Quaternary Transitions In Human 3 36E 66 244 073 JKE MAIN F BIT Pranto AULA AFRO ono Ne CC QFFEGB 243 432 Download and Open Download and Save Open at MEBI Open Structure Figure 12 10 Display of the output of a BLAST search in the tabular view The hits can be sorted by the different columns simply by clicking the column heading The BLAST Table includes the following information Query sequence The sequence which was used for the search Hit The Name of the sequences found in the BLAST search Id GenBank ID Description Text from NCBI describing the sequence E value Measure of quality of the match Higher E values indicate that BLAST found a less homologous sequence Score This shows the score of the local alignment generated through the BLAST search Bit score This shows the bit score of the local alignment generated through the BLAST search Bit scores are normalized which means that the bit scores from different alignments can be compared even if different scoring matrices have been used Hit start Shows the start position in the hit sequence Hit end Shows the end position in the hit
142. C Figure 10 3 Region 1 A single residue Region 2 A range of residues including both endpoints Region 3 A range of residues starting somewhere before 30 and continuing up to and including 40 Region 4 A single residue somewhere between 50 and 60 inclusive Region 5 A range of residues beginning somewhere between 70 and 80 inclusive and ending at 90 inclusive Region 6 A range of residues beginning somewhere between 100 and 110 inclusive and ending somewhere between 120 and 130 inclusive Region 7 A site between residues 140 and 141 Region 8 A site between two residues somewhere between 150 and 160 inclusive Region 9 A region that covers ranges from 170 to 180 inclusive and 190 to 200 inclusive Region 10 A region on negative strand that covers ranges from 210 to 220 inclusive Region 11 A region on negative strand that covers ranges from 230 to 240 inclusive and 250 to 260 inclusive bl et bl et 4000 1000 pBR322 4361 bp S protein Figure 10 4 A molecule shown in a circular view This view of the sequence shares some of the properties of the linear view of sequences as described in section 10 1 but there are some differences The similarities and differences are listed below e Similarities The editing options Options for adding editing and removing annotations Restriction Sites Annotation Types Find and Text Format preferences groups e Differences CHAPTE
143. Create alignment To add a fixpoint open the sequence or alignment and Select the region you want to use as a fixpoint right click the selection Set alignment fixpoint here This will add an annotation labeled Fixpoint to the sequence see figure 18 6 Use this procedure to add fixpoints to the other sequence s that should be forced to align to each other When you click Create alignment and go to Step 2 check Use fixpoints in order to force the alignment algorithm to align the fixpoints in the selected sequences to each other In figure 18 7 the result of an alignment using fixpoints is illustrated You can add multiple fixpoints e g adding two fixpoints to the sequences that are aligned will force their first fixpoints to be aligned to each other and their second fixpoints will also be CHAPTER 18 SEQUENCE ALIGNMENT 28 20 peso46 MHLTADENMA AvVTABWCREN HoEVEcCEADc 29 P68053 MACTGEBRA AVTABWGRIN PDEVECEADRSG 29 P6B225 PE rodas Es Mr o MMHLT Copy Selection gt Open Selection in New views PhBoTs PE 5 p MEGEEEERES AN 2 Edit Selection p6s228 MENLSGDERN AV Ei Add Annotation Add Gaps After P68231 MMALSG DERN AV Add Gaps Before 0 EY Delete Selection P68063 MHWTA E Efo EM Realign Selection 9 Set Alignment Fixpoink Here PERIJS MHWTA E Eko LI Set Numbers Relative to This Selection 9 Consensus MVHLTXEEKN AV EA create Pairwise Comparison Figure 18 6 Adding a fixpoint to a sequence in an
144. Description 10 5 View as text sequence can be viewed as text without any layout and text formatting This displays all the information about the sequence in the GenBank file format To view a sequence as text select a sequence in the Navigation Area Show in the Toolbar As text This way it is possible to see background information about e g the authors and the origin of DNA and protein sequences Selections or the entire text of the Sequence Text View can be copied and pasted into other programs Much of the information is also displayed in the Sequence info where it is easier to get an overview see section 10 4 In the Side Panel you find a search field for searching the text in the view 10 6 Creating a new sequence sequence can either be imported downloaded from an online database or created in the CLC Protein Workbench This section explains how to create a new sequence New 5 in the toolbar The Create Sequence dialog figure 10 14 reflects the information needed in the GenBank format but you are free to enter anything into the fields The following description is a guideline for entering information about a sequence e Name The name of the sequence This is used for saving the sequence e Common name common name for the species e Latin name The Latin name for the species CHAPTER 10 VIEWING AND EDITING SEQUENCES 143 q Create Sequence Es 1 Enter Sequence Data BESL Ae Name P70704 C
145. E E v act pcONA3 atp8al Ea 140 160 pcONA3 atpsal TTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAA CMV promoter a si em pcONn43 apoal GAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTA mi promoter pcONAs atpsal CGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATT fe O E ODE Figure 2 3 Sequence pcDNAS atp8a1 opened in a view This sequence is circular which is indicated by lt lt and gt gt at the beginning and the end of the sequence In the following we will show how the same sequence can be displayed in two different views one linear view and one circular view First Zoom in to see the residues again by using the Zoom In 540 or the 100 4 Then we make a split view by press and hold the Ctrl button on the keyboard 38 on Mac click Show as Circular at the bottom of the view CHAPTER 2 TUTORIALS 40 This opens an additional view of the vector with a circular display as can be seen in figure 2 4 act prONAS atp al E3 pcDNA3 atp8a1 GACGGAT CGGGAGATCTCCCGATCCCCTATGGICGACTCTCAGT 60 ab pcDNA3 atpsal ACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCC ber O E DE pcDNA3 atpsal Sal Ampicillin ORF ColE1 w F Sal dy peDNA3 atp al Ps th Sma Neomycin rate SV40 origin of replicatio SV40 promoter Aho Sal BHG Poly A 5p6 promoter O E 0 E Figure 2 4 The resulting two views which are split horizontally 9118 bp Make a selection o
146. EALG VXEVGGEALG VDEVGGEALG IEQTGGEALG VADCGAEALA VDEVGGEALG VsevGGEAL RLLVVYPW RLLVVYPW RLLVVYPW RLLIVYPW RLLIVYPW RLLVVYPW RLLYVY PW 20 P68873 MVHLTPEEKS AVTALWGKV NVDEVGG EALGRLLY Q6WN20 MVHLTGEEKA AVTALWGKV NVXEVGG EALGRLLV P68231 MVHLSGDEKN AVHGLWSKV KVDEVGG EALGRLLV Q6H1U7 MVHLTAEEKN AITSLWGKV AlEQTGG EALGRLLI P68945 VHWTAEEKQ LITGLWGKV NVADCGA EALARLLI P68873 MVHLTPEEKS AVTALWOKVX XXXNVDEWGG EALGRLLY Consensus MVHLTAEEKN AVTALWGKV NVDEVGG EALGRLLV Sequence Logo MVHCTsEEKe AvTaLWGKV AVsEvGG EALGRLLy 286 Conservation Figure 18 5 The top figures shows the original alignment In the bottom panel a single sequence with four inserted X s are aligned to the original alignment This introduces gaps in all sequences of the original alignment All other positions in the original alignment are fixed This feature is useful if you wish to add extra sequences to an existing alignment in which case you just select the alignment and the extra sequences and choose not to redo the alignment It is also useful if you have created an alignment where the gaps are not placed correctly In this case you can realign the alignment with different gap cost parameters 18 1 4 Fixpoints With fixpoints you can get full control over the alignment algorithm The fixpoints are points on the sequences that are forced to align to each other Fixpoints are added to sequences or alignments before clicking
147. EIVIEN a s 6b bob ww BG Ow EE Be ae we RS Se Ow RoR 2 6 17 4 Restriction enzyme lists lt 2 es 278 17 4 1 Create enzyme list 218 17 4 2 View and modify enzyme list 2 002 ee ee ee a 280 There are two ways of finding and showing restriction sites e In many cases the dynamic restriction sites found in the Side Panel of sequence views will be useful since it is a quick and easy way of showing restriction sites e In the Toolbox you will find the other way of doing restriction site analyses This way provides more control of the analysis and gives you more output options e g a table of restriction sites and you can perform the same restriction map analysis on several sequences in one step This chapter first describes the dynamic restriction sites followed by the toolbox way This section also includes an explanation of how to simulate a gel with the selected enzymes The 265 CHAPTER 17 RESTRICTION SITE ANALYSES 266 final section in this chapter focuses on enzyme lists which represent an easy way of managing restriction enzymes 17 1 Dynamic restriction sites If you open a sequence a sequence list etc you will find the Restriction Sites group in the Side Panel As shown in figure 17 1 you can display restriction sites as colored triangles and lines on the sequence The Restriction sites group in the side panel shows a list of enzymes represented
148. ER 16 PROTEIN ANALYSES 260 E BB Proteolytic Cleavage ES 1 Select protein sequences Selec protein sequences Projects Selected Elements 1 CLC_Data e ATP8al gt Example Data H Cloning Primers Protein analyses Protein ortholog RNA secondary Sequencing data gt 4 Ww Qr genter rs com gt A Figure 16 23 Choosing sequence CAA32220 for proteolytic cleavage CLC Protein Workbench allows you to detect proteolytic cleavages for several sequences at a time Correct the list of sequences by selecting a sequence and clicking the arrows pointing left and right Then click Next to go to Step 2 In Step 2 you can select proteolytic cleavage enzymes The list of available enzymes will be expanded continuously Presently the list contains the enzymes shown in figure 16 24 The full list of enzymes and their cleavage patterns can be seen in Appendix section E E g Proteolytic Cleavage 1 Select protein sequences p Set pal amerers 2 Select enzymes e 3 Set parameters Enzyme criteria Min number of cleavage sites 15 Max number of cleavage sites 1 Criteria For the table list of fragments Min fragment lenath 35 Max Fragment length 100 Min fragment mass Da 0 Max fragment mass Da 100 25 A Previous gt Next Y Erin X Cancel Figure 16 24 Setting parameters for proteolytic cleavage detecti
149. ERREERENORRONNDNE AD ONWRPERNWENNWONOKRWBNOUERD WONRORNWNHNOWWKRODOOWKOAONZ WOWKRRPORWBHWEKWERNOWOAKRNNDT PONBPBRwWBNHRWOKRKRWBWBAWOWWDWOO MD EPNRPORWOKNWONNOUWOOKKO MUNWRPORWNKWWONUNAKANOOHRM M WWNHNONWWNKAKRNANNWRONOD ONNNEBNEBNRWBWANDOOWRKRON IT WR WRNWORWNAWEAWWHRWWWK PRPENOBEBNWBONNANWBEKAWNHKRAWNEOC MNWBRORWKRUNWENKBKBRWBRONB SR RABAANOGANAENANORONHHAS PWKRNYONKHMAOWDDOORWBWWNWWWNHT NONRNBPANANVNAGONNARORNNHTO MNWRPARNRONNKRODOKROKBBKBE YD ONNORBENKBRBREBKBNNKBRKBBRBROBR O 4 PNNNNWBWENEPBRNWBNHKRENWNND lt RE WBONNBRKRNBRWWWNHNKRWWWOK lt lt lt S 70 UT2ZXFTIOMmMOOUzZzTI gt Table 14 1 The BLOSUM62 matrix A tabular view of the BLOSUM62 matrix containing all possible substitution scores Henikoff and Henikoff 1992 in a scoring matrix called BLOSUM62 In contrast to the PAM matrices the BLOSUM matrices are calculated from alignments without gaps emerging from the BLOCKS database http Fi DCCs Tiers org Sean Eddy recently wrote a paper reviewing the BLOSUM62 substitution matrix and how to calculate the scores Eddy 2004 Use of scoring matrices Deciding which scoring matrix you should use in order of obtain the best alignment results is a difficult task If you have no prior knowledge on the sequence the BLOSUM62 is probably the best choice This matrix has become the de facto standard for scoring matrices and is also used as the default matrix in BLAST searches The selection of a wrong scoring matrix will
150. Finish 16 1 2 Signal peptide prediction output After running the prediction as described above the protein sequence will show predicted signal peptide as annotations on the original sequence see figure 16 2 Signal Peptide ATPasel3a3 MDREERKT INQGQEDEME YGYNLSRWKLAIVSLGV Signal Peptide 60 ATPasel3a3 I CSGGFLLLLLYWMPEWRVKATCVRAA IKDCEVVLL a Ani Figure 16 2 N terminal signal peptide shown as annotation on the sequence Each annotation will carry a tooltip note saying that the corresponding annotation is predicted with SignalP version 3 0 Additional notes can be added through the Edit annotation 3 right click mouse menu See section 10 3 2 Undesired annotations can be removed through the Delete Annotation right click mouse menu See section 10 3 4 16 1 3 Bioinformatics explained Prediction of signal peptides Why the interest in signal peptides The importance of signal peptides was shown in 1999 when Gunter Blobel received the Nobel Prize in physiology or medicine for his discovery that proteins have intrinsic signals that govern CHAPTER 16 PROTEIN ANALYSES 236 their transport and localization in the cell Blobel 2000 He pointed out the importance of defined peptide motifs for targeting proteins to their site of function Performing a query to PubMed reveals that thousands of papers have been published regarding signal peptides secretion and subcellular localization including knowledge of using signa
151. GATGCCATGCGGAGGACAGTCGGAGATCCGCTCGCGCGCGGA Figure 5 2 Annotations added when the sequence is edited Deleted selection Editing of sequence selection 220 260 GAGATGCC GATCCGCTCGCGCGCGGAAGGTTAT Figure 5 3 Details of the editing 5 2 Default view preferences There are five groups of default View settings CHAPTER 5 USER PREFERENCES AND SETTINGS 91 Toolbar Side Panel Location LL 2 3 New View 4 View Format Di User Defined View Settings In general these are default settings for the user interface The Toolbar preferences let you choose the size of the toolbar icons and you can choose whether to display names below the icons The Side Panel Location setting lets you choose between Dock in views and Float in window When docked in view view preferences will be located in the right side of the view of e g an alignment When floating in window the side panel can be placed everywhere in your screen also outside the workspace e g on a different screen See section 5 5 for more about floating Side panels The New view setting allows you to choose whether the View preferences are to be shown automatically when opening a new view If this option is not chosen you can press Ctrl U 36 U on Mac to see the preferences panels of an open view The View Format allows you to change the way the elements appear in the Navigation Area The following text can be used to describe the element
152. Go to license download web page opens the license web page as shown in 1 4 Click the Request Evaluation License button and you will be able to save the license on your computer e g on the Desktop CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 18 Request an Evaluation License please cick the Dutton Delow Request Evaluation License ul a file containing the license will b d to To bi ur license you Choose License File button and locate the file on your c Figure 1 4 The license web page where you can download a license Back in the Workbench window you will now see the dialog shown in 1 5 License Wizard p CLC Protein Workbench Import a license from a file Please click the button below and locate the file containing your license No file selected Choose License File If you experience any problems please contact The CLC Support Team Proxy Settings Previous Ne Quit Workbench Figure 1 5 Importing the license downloaded from the web site Click the Choose License File button and browse to find the license file you saved before e g on your Desktop When you have selected the file click Next Accepting the license agreement Regardless of which option you chose above you will now see the dialog shown in figure 1 6 License Wizard p CLC Protein Workbench License Agreement Please read and accept the license agreement below to begin using you license
153. H 152 Even if you don t save the search the next time you open the search view it will remember the parameters from the last time you did a search 11 2 UniProt Swiss Prot TrEMBL search This section describes searches in UniProt and the handling of search results UniProt is a global database of protein sequences The UniProt search view figure 11 3 is opened in this way Search Search for Sequences in UniProt g UniProt search Es Choose database Swiss Prot TrEMBL All Fields v insulin E Created Since 30 Days v E Add search parameters a Start search C Append wildcard to search words Rows 13 Search results Filter Accession Name Description Organism Q32L79 INSL6 BOVIN Insulin like peptide INSL6 precursor Insulin like pep Bos taurus Bovine IQ3TDX8 Q3TIH3 Q3 NESR4_MOUSE Cytochrome b5 reductase 4 EC1 Musmusculus Mouse _ Q3UFB NTRK1_MOUSE High affinity nerve growth Factor receptor precurso Mus musculus Mouse QSNVK7 Q5R4U6 Q5 SPOP_PONPY Speckle type POZ protein Pongo pygmaeus Orangutan 063132 063130 Q6 ROS RAT Proto oncogene tyrosine protein kinase ROS precur Rattus norvegicus Rat 078DX7 Q60705 ROS MOUSE Proto oncogene tyrosine protein kinase ROS precur Mus musculus Mouse OSTL28 IULIL_METAC Ulilvsin precursor EC 3 Methanosarcina acetivorans H Download and Open Download and Save Total number of hi
154. H Annotation types k Restriction sites k Residue coloring Nucleotide info k Find k Text Format Figure 10 1 Overview of the Side Panel which is always shown to the right of a view When you make changes in the Side Panel the view of the sequence is instantly updated To show or hide the Side Panel select the View Ctrl U or Click the J at the top right corner of the Side Panel to hide Click the gray Side Panel button to the right to show Below each group of settings will be explained Some of the preferences are not the same for nucleotide and protein sequences but the differences will be explained for each group of settings Note When you make changes to the settings in the Side Panel they are not automatically saved when you save the sequence Click Save restore Settings i to save the settings see section 5 5 for more information Sequence Layout These preferences determine the overall layout of the sequence e Spacing Inserts a space at a specified interval No spacing The sequence is shown with no spaces Every 10 residues There is a space every 10 residues starting from the beginning of the sequence Every 3 residues frame 1 There is a space every 3 residues corresponding to the reading frame starting at the first residue CHAPTER 10 VIEWING AND EDITING SEQUENCES 124 Every 3 residues frame 2 There is a space every 3 residues corresponding to the reading frame startin
155. If you have a protein sequence but want to see the actual location on the chromosome this is easy to do using BLAST In this example we wish to map the protein sequence of the Human beta globin protein to a chromosome We know in advance that the beta globin is located somewhere on chromosome 11 Data used in this example can be downloaded from GenBank Search Search for Sequences at NCBI i Human chromosome 11 NC 000011 consists of 134452384 nucleotides and the beta globin AAA16334 protein has 147 amino acids BLAST configuration Next conduct a local BLAST search Toolbox BLAST Search E Local BLAST 4 Select the protein sequence as query sequence and click Next Since you wish to BLAST a protein sequence against a nucleotide sequence use tblastn which will automatically translate the nucleotide sequence selected as database As Target select NC 000011 that you downloaded If you are used to BLAST you will know that you usually have to create a BLAST database before BLASTing but the Workbench does this on the fly when you just select one or more sequences Click Next leave the parameters at their default click Next again and then Finish Inspect BLAST result When the BLAST result appears make a split view so that both the table and graphical view is visible see figure 2 16 This is done by pressing Ctrl on Mac while clicking the table view 33 at the bottom of the view In the table start out b
156. K Y Trypsin 140 150 11 1 368 50 5 58 R Trypsin NGAWEIVHWEK Vv Trypsin Z Length 151 160 10 1 069 30 7 07 K Trypsin VNVGDIVIIK G Trypsin J Mass 198 207 10 1 029 15 6 80 R Trypsin QGLPATSDIK D Trypsin Figure 16 26 The result of the proteolytic cleavage detection CHAPTER 16 PROTEIN ANALYSES 262 Depending on the settings in the program the output of the proteolytic cleavage site detection will display two views on the screen The top view shows the actual protein sequence with the predicted cleavage sites indicated by small arrows If no labels are found on the arrows they can be enabled by setting the labels in the annotation layout in the preference panel The bottom view shows a text output of the detection listing the individual fragments and information on these 16 10 2 Bioinformatics explained Proteolytic cleavage Proteolytic cleavage is basically the process of breaking the peptide bonds between amino acids in proteins ThiS process is carried out by enzymes called peptidases proteases or proteolytic cleavage enzymes Proteins often undergo proteolytic processing by specific proteolytic enzymes proteases peptidases before final maturation of the protein Proteins can also be cleaved as a result of intracellular processing of for example misfolded proteins Another example of proteolytic processing of proteins is secretory proteins or proteins targeted to organelles which have their signal peptide removed by specifi
157. L 2001 Predicting transmembrane protein topology with a hidden Markov model application to complete genomes J Mol Biol 305 3 56 7 580 Kyte and Doolittle 1982 Kyte J and Doolittle R F 1982 A simple method for displaying the hydropathic character of a protein J Mol Biol 157 1 105 132 Larget and Simon 1999 Larget B and Simon D 1999 Markov chain monte carlo algorithms for the bayesian analysis of phylogenetic trees Mol Biol Evol 16 750 759 Leitner and Albert 1999 Leitner T and Albert J 1999 The molecular clock of HIV 1 unveiled through analysis of a known transmission history Proc Natl Acad Sci USA 96 19 10752 10757 Maizel and Lenk 1981 Maizel J V and Lenk R P 1981 Enhanced graphic matrix analysis of nucleic acid and protein sequences Proc Natl Acad Sci US A 78 12 7665 7669 McGinnis and Madden 2004 McGinnis S and Madden T L 2004 BLAST at the core of a powerful and diverse set of sequence analysis tools Nucleic Acids Res 32 Web Server issue W20 W25 BIBLIOGRAPHY 337 Menne et al 2000 Menne K M Hermjakob H and Apweiler R 2000 A comparison of signal sequence prediction methods using a test set of signal peptides Bioinformatics 16 8 741 42 Michener and Sokal 1957 Michener C and Sokal R 1957 A quantitative approach to a problem in classification Evolution 11 130 162 Nielsen et al 1997 Nielsen H Engelbrecht J Brunak S a
158. LYSES 250 In CLC Protein Workbench we have implemented our own HMM algorithm for prediction of the Pfam domains Thus we do not use the original HMM implementation HMMER http hmmer wustl edu for domain prediction We find the most probable state path alignment through each profile HMM by the Viterbi algorithm and based on that we derive a new null model by averaging over the emission distributions of all M and states that appear in the state path M is a match state and is an insert state From that model we now arrive at an additive correction to the original bit score like it is done in the original HMMER algorithm In order to conduct the Pfam search Select a protein sequence Toolbox in the Menu Bar Protein Analyses a Pfam Domain Search gt or right click a protein sequence Toolbox Protein Analyses ia Pfam Domain Search a q Pfam Domain Search eS a 1 Select protein sequences WU 2 Set parameters Database and search type Search Full domains and fragments Search Full domains only Search Fragments only Database 100 most common domains w Additional databases Open Plug in and Resource manager Significance cutoff Max E value 1 A Previous Next Y Erin XX Cancel Figure 16 16 Setting parameters for Pfam domain search lf a sequence was selected before choosing the Toolbox action this sequence is now listed in the Selected Elem
159. NCBI http www ncbi nlm nih gov BLAST Download pages for the BLAST programs http www ncbi nlm nih gov BLAST download shtml Download pages for pre formatted BLAST databases Ep TEN eo mim nih oer blast ab O Reilly book on BLAST http www oreilly com catalog blast Explanation of scoring substitution matrices and more http www clcbio com be CHAPTER 12 BLAST SEARCH 185 Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents Chapter 13 3D molecule viewing Contents 13 1 Importing structure files 1 eee te 186 13 2 Viewing structure files ee tt ee 187 13 2 1 Moving and rotang 2462026 orcas 187 13 3 Selections and display of the 3D structure lt 188 13 3 1 Coloring of the 3D structure a a a aoao Bee a a a E RE e A IT 188 13 3 2 Hierarchical view changing how selections of the structure
160. NC_000003 iACCATTCGATGATTGCATTCAATTCATTCGATGACGATTCCATTCAATTCCGTTCAATGATTCCATTAGATTC Consensus iACCATTCGATGATTGCATTCAATTCATTCGATGACGATTCCATTCAATTCCGTTCAATGATTCCATTAGATTC 3388 Coverage O AN awe A GA LU A ONAT aes AA 8 1205 1326 1 TGACGATTCCATTCAATTCCGTTCAATGATTCCATTHEGATTC 1 2 413 1273 2 TGACGATTCCATTCAATTCCGTTCAATGATTCCATTEGATTC 98 1139 847 1 GACGATTCCATTCAATTCCGTTCAATGATTCCATTHEGATTC 2 90 40 189 2 GACGATTCCATTCAATTCCGTTCAATGATTCCATTHEGATTC 86 627 1969 1 GACGATTCCATTCAATTCCGTTCAATGATTCCATTHEGATTC 2 85 523 514 2 GACGATTCCATGCAATTCCGTTCAATGATTCCATTAGATTC 4 1256 1139 1 GACCATTCCATTCAATTCCGTTCAATGATTCCATTAGATTC 78 1008 834 2 GACGATTCCATTCAATTCCGTTCAATGATTCCATTAGATTC 64 294 1084 2 GACGATTCCATTCABTTCCGTTCAATGATTCCATTHEGATTC 8 722 1303 2 GACGATTCCATTCAATTCCGTTCAATGATTCCATTAGATTC Figure 7 14 A graph displayed along the mapped reads Right click the graph to export the data points to a file will be shown If the graph is covering a set of aligned sequences with a main sequence such as read mappings and BLAST results the dialog shown in figure 7 15 will be displayed These kinds of graphs are located under Alignment info in the Side Panel In all other cases a normal file dialog will be shown letting you specify name and location for the file g Export Graphics 1 Output options Export options Export excluding gaps Figure 7 15 Choosing to include data points with gaps In this dialog select whether you wish to inclu
161. ND EXPORT F 1 1 Sequence data formats File type FASTA AB1 ABI CLC Clone Manager CSV export CSV import DNAstrider DS Gene Embl GCG sequence GenBank Gene Construction Kit Lasergene Nexus Phred PIR NBRF Raw sequence SCF2 SCF3 Staden Swiss Prot Tab delimited text Vector NTI archives Vector NTI Database Zip export Zip import Suffix fsa fasta abt abi clc cmo CSV CSV str strider bsml embl geg bk gb gp gck pro seq NXS NeEXUS phd pir any SCf SCf sdn SWP txt 327 Import Export Description X X X X X gt lt X K K X XK X X X X X X X X X X ma4 pa4 0a4 X Zip Zip gzip tar X X X Simple format name amp description Including chromatograms Including chromatograms Rich format including all information Annotations in csv format One sequence per line name de scription optional sequence Only nucleotide sequence Rich information incl annotations Rich information incl annotations Including chromatograms Simple format name amp description Only sequence no name Including chromatograms Including chromatograms Rich information only proteins Annotations in tab delimited text for mat Archives in rich format Special import full database Selected files in CLC format Contained files folder structure APPENDIX F FORMATS FOR IMPORT AND EXPORT 328 F 1 2 Contig formats File type
162. Navigation Area Ctrl O 38 B on Mac Opening a view while another view is already open will show the new view in front of the other view The view that was already open can be brought to front by clicking its tab Note If you right click an open tab of any element click Show and then choose a different view of the same element this new view is automatically opened in a split view allowing you to see both views See section 3 1 5 for instructions on how to open a view using drag and drop 3 2 2 Show element in another view Each element can be shown in different ways A sequence for example can be shown as linear circular text etc In the following example you want to see a sequence in a circular view If the sequence is already open in a view you can change the view to a circular view Click Show As Circular at the lower left part of the view The buttons used for switching views are shown in figure 3 9 ha O E WE Figure 3 9 The buttons shown at the bottom of a view of a nucleotide sequence You can click the buttons to change the view to e g a circular view or a history view If the sequence is already open in a linear view ar and you wish to see both a circular and a linear view you can split the views very easily Press Ctrl 38 on Mac while you Click Show As Circular at the lower left part of the view This will open a split view with a linear view at the bottom and a circular view at the
163. R 1983 A computer program for predicting protein antigenic determinants Mol Immunol 20 4 483 489 Ikai 1980 Ikai A 1980 Thermostability and aliphatic index of globular proteins J Biochem Tokyo 88 6 1895 1898 Janin 1979 Janin J 1979 Surface and inside volumes in globular proteins Nature 2 1 5696 491 492 Jukes and Cantor 1969 Jukes T and Cantor C 1969 Mammalian Protein Metabolism chapter Evolution of protein molecules pages 21 32 New York Academic Press Karplus and Schulz 1985 Karplus P A and Schulz G E 1985 Prediction of chain flexibility in proteins Naturwissenschaften 2 212 213 Kimura 1980 Kimura M 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J Mol Evol 16 2 111 120 Klee and Ellis 2005 Klee E W and Ellis L B M 2005 Evaluating eukaryotic secreted protein prediction BMC Bioinformatics 6 256 Knudsen and Miyamoto 2001 Knudsen B and Miyamoto M M 2001 A likelihood ratio test for evolutionary rate shifts and functional divergence among proteins Proc Natl Acad Sci USA 98 25 14512 14517 Kolaskar and Tongaonkar 1990 Kolaskar A S and Tongaonkar P C 1990 A semi empirical method for prediction of antigenic determinants on protein antigens FEBS Lett 2 0 1 2 1 2 174 Krogh et al 2001 Krogh A Larsson B von Heijne G and Sonnhammer E
164. R 10 VIEWING AND EDITING SEQUENCES 132 In the Sequence Layout preferences only the following options are available in the circular view Numbers on plus strand Numbers on sequence and Sequence label You cannot zoom in to see the residues in the circular molecule If you wish to see these details split the view with a linear view of the sequence In the Annotation Layout you also have the option of showing the labels as Stacked This means that there are no overlapping labels and that all labels of both annotations and restriction sites are adjusted along the left and right edges of the view 10 2 1 Using split views to see details of the circular molecule In order to see the nucleotides of a circular molecule you can open a new view displaying a circular view of the molecule Press and hold the Ctrl button 38 on Mac click Show Sequence ri at the bottom of the view This will open a linear view of the sequence below the circular view When you zoom in on the linear view you can see the residues as shown in figure 10 5 O pBR322 bla bl pBR322 1000 4361 bp v a 9 E OB El 11 9 Ey Aer pBR322 AOS 2 40 l NEE cere tet et 60 80 pBR322 AGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTA ka O S m El me E v Figure 10 5 Two views showing the same sequence The bottom view is zoomed in Note If you make a selection in one of the views the other view will also make the
165. ROTLOEDL POG 18 POG 15 PEG 14 Neighborhood PRE 14 Scores from Words EIS BLOSUM62 matirx PNG 13 PDG 13 PHG 13 PMG 13 PSQ 13 PQA 12 PON 12 Threshold for Bree es neighborhood words T 13 Figure 12 16 Neighborhood BLAST words based on the BLOSUM62 matrix Only words where the threshold T exceeds 13 are included in the initial seeding After initial finding of words seeding the BLAST algorithm will extend the only 3 residues long alignment in both directions see figure 12 17 Each time the alignment is extended an alignment score is increases decreased When the alignment score drops below a predefined threshold the extension of the alignment stops This ensures that the alignment is not extended to regions where only very poor alignment between the query and hit sequence is possible If the obtained alignment receives a score above a certain threshold it will be included in the final BLAST result By tweaking the word size W and the neighborhood word threshold T it is possible to limit the search space E g by increasing T the number of neighboring words will drop and thus limit the search space as shown in figure 12 18 This will increase the speed of BLAST significantly but may result in loss of sensitivity Increasing the word size W will also increase the speed but again with a loss of sensitivity CHAPTER 12 BLAST SEARCH 1 9 NANAH_____ Query 325 SLAALLNKCKTPOGQRLVNQWIKOPLMDKNRIEERLNLVEA 365 LA L TP
166. TGGGAITIGCTAAAACAGCITCCIGITACIGAGAIGICITCAATIGGAATACA GICATTCCAAGAACTATAAACTTAAAGCTACTGTAGAAACAAAGGSTTITCITITITAAA PECTED GICATICCAAGAACTATABACTTASAGCTACTGIAGAAACAAAGEGTITICITITITASA IGITICITGGIAGATIATICATAATGTGAGATGGITCCCAATATCATGIGA POPU TEEPE eee TGTTICTTGGIAGATTATTCATAATGTIGAGATGGTICCCAATATCATGIGA 171 1163 1112 Score 224 bits 113 Expect 6e 56 Identities 161 161 100 Gaps 0 161 0 Strand Flus Flus Query Sbjct 213 GACTGIGCAATACITAGAGAACCIATAGCATCITCICATICCCATGIGGAACAGGATGCC PEP UTEP Eee GACTGIGCAATACTTAGAGAACCTATAGCATCTICICATICCCATGIGGAACAGGATGCC CACATACTGICTAATTAATAAATITICCACtrct ttt cCABACAAGTATGAATCTAGITGS AAA CACATACTGICTAAITAATAAATITICCATITITITICAARACAACGIATGAATCIAGITGG 1324 Query 273 Sbjct TIGATGCCttttttttCATGACATAATAAAGIAITITCIIT ILEANA NANA eee TIGATGCCTITITTTICATGACATAATAAAGTATIITCIIT Query 373 Sbjct 1365 Figure 12 21 Alignment view of BLAST results Individual alignments are represented together with BLAST scores and more 12 5 7 want to BLAST against my own sequence database is this possible It is possible to download the entire BLAST program package and use it on your own computer institution computer cluster or similar This is preferred if you want to search in proprietary sequences or sequences unavailable in the public databases stored at NCBI The downloadable BLAST package can either be installed as a web based tool or as a command line tool It is available for
167. The Advanced settings include the possibility to set up a proxy server This is described in section 1 8 5 3 1 Default data location If you have more than one location in the Navigation Area you can choose which location should be the default data location The default location is used when you e g import a file without selecting a folder or element in the Navigation Area first Then the imported element will be placed in the default location CHAPTER 5 USER PREFERENCES AND SETTINGS 94 Note The default location cannot be removed You have to select another location as default first 5 3 2 NCBI BLAST URL to use for BLAST It is possible to specify an alternate server URL to use for BLAST searches The standard URL for the BLAST server at NCBI is http blast ncbi nlim nih gov Blast cgi Note Be careful to specify a valid URL otherwise BLAST will not work 5 4 Export import of preferences The user preferences of the CLC Protein Workbench can be exported to other users of the program allowing other users to display data with the same preferences as yours You can also use the export import preferences function to backup your preferences To export preferences open the Preferences dialog Ctrl K on Mac and do the following Export Select the relevant preferences Export Choose location for the exported file Enter name of file Save Note The format of exported preferences is cpf This notation must be su
168. The annotations are placed above each other with a little space between This can take up a lot of Space on the screen e Label The name of the annotation can shown as a label Additional information about the sequence is shown if you place the mouse cursor on the annotation and keep it still No labels No labels are displayed On annotation The labels are displayed in the annotation s box Over annotation The labels are displayed above the annotations Before annotation The labels are placed just to the left of the annotation Flag The labels are displayed as flags at the beginning of the annotation Stacked The labels are offset so that the text of all labels is visible This means that there is varying distance between each sequence line to make room for the labels e Show arrows Displays the end of the annotation as an arrow This can be useful to see the orientation of the annotation for DNA sequences Annotations on the negative strand will have an arrow pointing to the left e Use gradients Fills the boxes with gradient color In the Annotation Types group you can choose which kinds of annotations that should be displayed This group lists all the types of annotations that are attached to the sequence s in the view For sequences with many annotations it can be easier to get an overview if you deselect the annotation types that are not relevant Unchecking the checkboxes in the Annotation Layout will not remo
169. The best way to go depends on how your data is currently stored in Vector NTI e Your data is stored in the Vector NTI Local Database which can be accessed through Vector NTI Explorer This is described in the first section below e Your data is stored as single files on your computer just like Word documents etc This is described in the second section below Import from the Vector NTI Local Database If your Vector NTI data are stored in a Vector NTI Local Database as the one shown in figure 7 2 you can import all the data in one step or you can import selected parts of it Importing the entire database in one step From the Workbench there is a direct import of the whole database see figure 3 CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 105 a al Exploring Local Vector NTI Database oO ss Table Edit View Analyses Align Database Assemble Tools Help Tl DNA RNA Molecules CNS BA ta SIE All Subsets All database DNA RNA Molecules E DNA RNA Molecules MAIN alll Invitrogen vectors Lu ADCY7 6196 Linear Basic NCBI Entrez NCBI Adeno2 35937 Linear Basic NCBI Entrez NCBI ADRA 1A 2306 Linear Basic NCBI Entrez NCBI ds BaculoDirect Linear DNA 139370 Linear Basic Invitrogen Invitr BaculoDirect Linear DNA Clonin 5770 Linear Construc Invitrogen Invitr 33 BPV1 7945 Circular Basic NCBI Entrez NCBI a BRAF 2510 Linear Basic NCBI Entrez NCBI se CDK2 2226 Linear Basic NCBI Entrez NCBI
170. The symbol matches any character X n will match a repetition of an element indicated by following that element with a numerical value or a numerical range between the curly brackets For example ACG 2 matches the string ACGG and ACG 2 matches ACGACG X n m will match a certain number of repetitions of an element indicated by following that element with two numerical values between the curly brackets The first number is a lower limit on the number of repetitions and the second number is an upper limit on the number of repetitions For example ACT 1 3 matches ACT ACTT and ACTTT X n represents a repetition of an element at least n times For example AC 2 matches all strings ACAC ACACAC ACACACAC The symbol restricts the search to the beginning of your sequence For example if you search through a sequence with the regular expression AC the algorithm will find a match if AC occurs in the beginning of the sequence The symbol restricts the search to the end of your sequence For example if you search through a sequence with the regular expression GT the algorithm will find a match if GT occurs in the end of the sequence Examples The expression ACG ACIG 2 matches all strings of length 4 where the first character is A C or G and the second is any character except A C and the third and fourth character is G The expression G A matches all strings of length 3 in the end of your sequence where the first
171. This is an overview of available resources on CLC bio s server To install a plug in click the Download Plug ins tab This will display an overview of the plug ins that are available for download and installation see figure 1 24 Manage Plug ins and Resources ha Ca o Cl Manage Plug ins Download Plug ins Manage Resources Download Resources oO Bookmark Navigator Version 1 03 E cen J Additional allignments With this extension you can bookmark elements in the Navigation Area Version 1 02 Description Perform alignments with many different programs from within the workbench ClustalW Windows Mac Linux Muscle Windows Mac Linux T Coffee Mac Linux Download and install e MAFFT Mac Linux Kalign Mac Linux Extract Annotations g version 1 02 Extracts annotations from one or more sequences The result is a More information is available on the sequence list containing sequences covered by the specified Additional alignments plugin website annotations Additional information E Usage g Annee MEN GET ME Located in Toolbox gt Alignments and Trees gt Additional Alignments Version 1 02 Using this plug in it is possible to annotate a sequence from list of annotations found in a GFF file Y E Additional Alignments Located in the Toolbox E EF Clustal Alignment Q SignalP EF Muscle Alignment Version 1 02 333 1 Clustal Alignment ht a ias Figure 1 24 The plug ins that are ava
172. Tx Translated DNA sequence against a Translated DNA database Automatic translation of your DNA query sequence and the DNA database in six frames The resulting peptide query sequences are used to search the resulting peptide database Note that this type of search is computationally intensive BLAST programs for protein query sequences e BLASTp Protein sequence against Protein database Used to look for peptide sequences with homologous regions to your peptide query sequence e tBLASTn Protein sequence against Translated DNA database Peptide query sequences are searched against an automatically translated in six frames DNA database If you search against the Protein Data Bank protein database homologous sequences are found to the query sequence these can be downloaded and opened with the 3D view Click Next This window see figure 12 4 allows you to choose parameters to tune your BLAST search to meet your requirements e BLAST at NCBI 1 Select sequences of same Set para AE type 2 Choose program 3 Set BLAST parame ters Choose parame ters Limit by entrez query all organisms x a fiten _ Filter low Complexity _ Mask lower case Expect 10 Word size E Matrix BLOSUM62 v Gap cost Existence 11 Extension 1 y Max number of hit sequences 1 oo 2 Ls Previous gt Next X Cancel Figure 12 4 Parameters that can be set before submitting a BLAST search When cho
173. UENCES Projects Selected Elements 1 J CLC Data xx ATP8al mRNA gt Example Data Xx ATP8al genomic s xx Cloning Primers Protein analyses 3 Protein orthologs RNA secondary st Sequencing data gt Q nter search term gt 4 poros tee Kena Figure 15 1 Translating DNA to RNA 15 2 Convert RNA to DNA CLC Protein Workbench lets you convert an RNA sequence into DNA substituting the U residues Urasil for T residues Thymine select an RNA sequence in the Navigation Area Toolbox in the Menu Bar Nucleotide Analyses A Convert RNA to DNA 3 or right click a sequence in Navigation Area Toolbox Nucleotide Analyses 7 Convert RNA to DNA 3 This opens the dialog displayed in figure 15 2 ra E Convert RNA to DNA 58 1 Select RNA sequences SelecP RNA sec puentes Projects Selected Elements 1 CLC Data xx ATP8al mRNA 3 UTR large gt Example Data Xc ATP8al genomic s XxX ATP8al mRNA Cloning Primers Protein analyses 4 7 Protein orthologs RNA secondary st 5200 20 ATP8al mRNA 5 Sequencing data EA 4 an p Qe zenter search term gt 4 aie EN gt Figure 15 2 Translating RNA to DNA If a sequence was selected before choosing the Toolbox action this sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists fro
174. Workbench supports the use of a HTTP proxy and an anonymous SOCKS proxy To configure your proxy settings open CLC Protein Workbench and go to the Advanced tab of the Preferences dialog figure 1 28 and enter the appropriate information The Preferences dialog is opened from the Edit menu CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 34 Use HTTP Proxy Server O u HTTP Proxy Port f HTTP Proxy Requires Login Passwor d Use SOCKS Proxy Server SOCKS Host Port be ILE You may have to restart the application For these changes to take effect Default Data Location Default Data Location CLC Data w m CSI BLAST URL to use when blasting http blast ncbi nlm nih gov Blast cgi Maximum number of simultaneous requests 10 Delay in ms between requests 3000 XM Cancel Help Export Import Figure 1 28 Adjusting proxy preferences You have the choice between a HTTP proxy and a SOCKS proxy CLC Protein Workbench only supports the use of a SOCKS proxy that does not require authorization Exclude hosts can be used if there are some hosts that should be contacted directly and not through the proxy server The value can be a list of hosts each separated by a and in addition a wildcard character can be used for matching For example foo com localhost If you have any problems with these settings you should contact your systems administrator 1 9 Th
175. a ena Figure 7 13 Page setup parameters for vector formats The settings for the page setup are shown and clicking the Page Setup button will display a dialog where these settings can ba adjusted This dialog is described in section 6 2 The page setup is only available if you have selected to export the whole view if you have chosen to export the visible area only the graphics file will be on one page with no headers or footers 7 3 4 Exporting protein reports It is possible to export a protein report using the normal Export function E which will generate a pdf file with a table of contents Click the report in the Navigation Area Export ES in the Toolbar select pdf You can also choose to export a protein report using the Export graphics function this way you will not get the table of contents but in 1 4 Export graph data points to a file Data points for graphs displayed along the sequence or along an alignment mapping or BLAST result can be exported to a semicolon separated text file csv format An example of such a graph is shown in figure 7 14 This graph shows the coverage of reads of a read mapping produced with CLC Genomics Workbench To export the data points for the graph right click the graph and choose Export Graph to Comma separated File Depending on what kind of graph you have selected different options CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 114 ES SGCG GS EEE
176. a wide range of different operating systems The BLAST package can be downloaded free of charge from the following location http www ncbi nlm nih gov BLAST download shtml Pre formatted databases are available from a dedicated BLAST ftp site ftp ftp ncbi nlm nih gov blast db Moreover it is possible to download programs scripts from the same site enabling automatic download of changed BLAST databases Thus it is possible to schedule a nightly update of changed databases and have the updated BLAST database stored locally or on a shared network drive at all times Most BLAST databases on the NCBI site are updated on a daily basis to include all recent sequence submissions to GenBank A few commercial software packages are available for searching your own data The advantage of using a commercial program is obvious when BLAST is integrated with the existing tools of these programs Furthermore they let you perform BLAST searches and retain annotations on the query sequence see figure 12 22 It is also much easier to batch download a selection of hit sequences for further inspection CHAPTER 12 BLAST SEARCH 184 Intron 2 intron 2 f CGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATS CGTGGATCCTGAGAACTTCAGGGTGAGTC TGTGGATCCTGAGAACTTCAAGGTGAGTC TGOTGGATCCTGAGAACTTCAAGGTGAGTC TGOTGGATCCTGAGAACTTCAAGGTGAGT CGTGGACCCTGAGAACTTCCTGGTGAGT Figure 12 22 Snippet of alignment view of BLAST results from CLC Main Workbench Individual alignments are r
177. about creating and modifying enzyme lists Below there are two panels e Tothe left you see all the enzymes that are in the list select above If you have not chosen to use an existing enzyme list this panel shows all the enzymes available e To the right there is a list of the enzymes that will be used Select enzymes in the left side panel and add them to the right panel by double clicking or clicking the Add button E If you e g wish to use EcoRV and BamHI select these two enzymes and add them to the right side panel If you wish to use all the enzymes in the list Click in the panel to the left press Ctrl A 38 A on Mac Add gt The enzymes can be sorted by clicking the column headings i e Name Overhang Methylation or Popularity This is particularly useful if you wish to use enzymes which produce e g a 3 overhang In this case you can sort the list by clicking the Overhang column heading and all the enzymes producing 3 overhangs will be listed together for easy selection When looking for a specific enzyme it is easier to use the Filter If you wish to find e g Hindlll sites simply type Hindlll into the filter and the list of enzymes will shrink automatically to only include the Hindlll enzyme This can also be used to only show enzymes producing e g a 3 overhang as shown in figure 17 20 If you need more detailed information and filtering of the enzymes either place your mouse cursor on an e
178. ackground of the letters using a gradient where the left side color is used for low coverage and the right side is used for maximum coverage Graph The coverage is displayed as a graph beneath the query sequence Learn how to export the data behind the graph in section 4 CHAPTER 12 BLAST SEARCH 170 x Height Specifies the height of the graph x Type The graph can be displayed as Line plot Bar plot or as a Color bar Color box For Line and Bar plots the color of the plot can be set by clicking the color box If a Color bar is chosen the color box is replaced by a gradient color box as described under Foreground color The remaining View preferences for BLAST Graphics are the same as those of alignments See section 18 2 Some of the information available in the tooltips is e Name of sequence Here is shown some additional information of the sequence which was found This line corresponds to the description line in GenBank if the search was conducted on the nr database e Score This shows the bit score of the local alignment generated through the BLAST search e Expect Also known as the E value A low value indicates a homologous sequence Higher E values indicate that BLAST found a less homologous sequence e Identities This number shows the number of identical residues or nucleotides in the obtained alignment e Gaps This number shows whether the alignment has gaps or not e Strand This is only valid for n
179. ailable CHAPTER 13 3D MOLECULE VIEWING 193 Selected model All models e None Will not display models e Color Clicking the color box allows you to select a color Opacity Determines the level of opacity 13 4 3D Output The output of the 3D view is rendered on the screen in real time and changes to the preferences are visible immediately From CLC Protein Workbench you can export the visible part of the 3D view to different graphic formats by pressing the Graphics button l l on the Menu bar This will allow you to export in the following formats Format Suffix Type Portable Network Graphics png bitmap JPEG Jpg bitmap Tagged Image File tif bitmap PostScript ps vector graphics Encapsulated PostScript eps vector graphics Portable Document Format pdf vector graphics Scalable Vector Graphics SVg vector graphics Printing is not fully implemented with the 3D editor Should you wish to print a 3D view this can be done by either exporting to a graphics format and printing that or use the scheme below Windows e Adjust your 3D view in CLC Protein Workbench e Press Print Screen on your keyboard or Alt Print Screen e Paste the result into an image editor e g Paint or GIMP http www gimp org e Crop edit the screen shot e Save in your preferred file format and or print e Set up your 3D view e Press amp shift 3 or shift 4 to take screen shot e Open the saved file pdf or png
180. al 1985 Welling et al used information on the relative occurrence of amino acids in antigenic regions to make a scale which is useful for prediction of antigenic regions This method is better than the Hopp Woods scale of hydrophobicity which is also used to identify antigenic regions e Kolaskar Tongaonkar A semi empirical method for prediction of antigenic regions has been developed Kolaskar and Tongaonkar 1990 This method also includes information of surface accessibility and flexibility and at the time of publication the method was able to predict antigenic determinants with an accuracy of 715 e Surface Probability Display of surface probability based on the algorithm by Emini et al 1985 This algorithm has been used to identify antigenic determinants on the surface of proteins e Chain Flexibility Display of backbone chain flexibility based on the algorithm by Karplus and Schulz 1985 It is known that chain flexibility is an indication of a putative antigenic determinant Find The Find function can also be invoked by pressing Ctrl Shift F 38 Shift F on Mac The Find function can be used for searching the sequence Clicking the find button will search for the first occurrence of the search term Clicking the find button again will find the next occurrence and so on If the search string is found the corresponding part of the sequence will be selected CHAPTER 10 VIEWING AND EDITING SEQUENCES 128 e Search te
181. ale Setting Style English United States Show all dialogs with Never show this dialog again Show Dialogs Help Y OK X Cancel Export Import Figure 5 1 Preferences include General preferences View preferences Colors preferences and Advanced settings e Audit Support If this option is checked all manual editing of sequences will be marked with an annotation on the sequence see figure 5 2 Placing the mouse on the annotation will reveal additional details about the change made to the sequence see figure 5 3 Note that no matter whether Audit Support is checked or not all changes are also recorded in the History LJ see section 8 e Number of hits The number of hits shown in CLC Protein Workbench when e g searching NCBI The sequences shown in the program are not downloaded until they are opened or dragged saved into the Navigation Area e Locale Setting Specify which country you are located in This determines how punctation is used in numbers all over the program e Show Dialogs A lot of information dialogs have a checkbox Never show this dialog again When you see a dialog and check this box in the dialog the dialog will not be shown again If you regret and wish to have the dialog displayed again click the button in the General Preferences Show Dialogs Then all the dialogs will be shown again Deleted selection Editing of sequence selection 220 0 260 GA
182. alignment score matrix come from Nat Biotechnol 22 8 1035 1036 Eisenberg et al 1984 Eisenberg D Schwarz E Komaromy M and Wall R 1984 Analysis of membrane and surface protein sequences with the hydrophobic moment plot J Mol Biol 1 9 1 125 142 Emini et al 1985 Emini E A Hughes J V Perlow D S and Boger J 1985 Induction of hepatitis a virus neutralizing antibody by a virus specific synthetic peptide J Virol 55 3 836 839 Engelman et al 1986 Engelman D M Steitz T A and Goldman A 1986 Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins Annu Rev Biophys Biophys Chem 15 321 353 Felsenstein 1981 Felsenstein J 1981 Evolutionary trees from DNA sequences a maximum likelinood approach J Mol Evol 17 6 368 376 Feng and Doolittle 1987 Feng D F and Doolittle R F 1987 Progressive sequence align ment as a prerequisite to correct phylogenetic trees J Mol Evol 25 4 351 360 Forsberg et al 2001 Forsberg R Oleksiewicz M B Petersen A M Hein J Botner A and Storgaard T 2001 A molecular clock dates the common ancestor of European type porcine reproductive and respiratory syndrome virus at more than 10 years before the emergence of disease Virology 289 2 1 74 179 Galperin and Koonin 1998 Galperin M Y and Koonin E V 1998 Sources of systematic error in functional annotation of genomes domain rearr
183. all sequences found in the list This can also be done for Alignments EE Contigs and read mappings gt e Read mapping tables f BLAST result 2 BLAST overview tables 2 e RNA Seq samples ZE and of course sequence lists For mappings and BLAST results the main sequences i e reference consensus and query sequence will not be extracted To extract the sequences Toolbox General Sequence Analyses A Extract Sequences This will allow you to select the elements that you want to extract sequences from see the list above Clicking Next displays the dialog shown in 10 17 Extract Sequences Please select a Set pe sequencelist Select destination Destination Extract bo single sequences Extract to new sequence list Number of sequences 12 sequences or paired end pairs found Figure 10 17 Choosing whether the extracted sequences should be placed in a new list or as single sequences Here you can choose whether the extracted sequences should be placed in a new list or extracted as single sequences For sequence lists only the last option makes sense but for alignments mappings and BLAST results it would make sense to place the sequences in a list CHAPTER 10 VIEWING AND EDITING SEQUENCES 147 Below these options you can see the number of sequences that will be extracted Click Next if you wish to adjust how to handle the results see section 9 1 If not click Fin
184. an search for e Name e Length e Organism See section 4 2 2 for more information on individual search terms For all other data you can only search for name lf you use Any field it will search all of the above plus the following e Description Keywords Common name Taxonomy name CHAPTER 4 SEARCHING YOUR DATA 88 To see this information for a sequence switch to the Element Info y view see section 10 4 For each search line you can choose if you want the exact term by selecting is equal to or if you only enter the start of the term you wish to find select begins with An example is shown in figure 4 7 dl Search Search in Location CLC Data we within Nucleotide Sequence Organism homo sapiens ES 4 Add Filter Search Description Length Path BCOS0969 Homo sapiens breast cancer 1 early onset mRNA fc 2050 WLS Datal omo sapiens breast cancer 1 early onset mRNA c 3273 ELO Datat omo sapiens breast cancer 1 early onset mRNA fc 1468 JLC Data omo sapiens breast cancer 1 early onset mANA fc 7790 CLE Daal omo sapiens breast cancer 1 early onset mRNA fc LC Data E 2062429 omo sapiens breast cancer 1 early onset mARNA fe 13922 CLC Data Showing 1 6 ap Figure 4 7 Searching for human sequences shorter than 10 000 nucleotides This example will find human nucleotide sequences organism is Homo sapiens and it will only find sequences shorter
185. angement non orthologous gene displacement and operon disruption In Silico Biol 1 1 55 67 Gill and von Hippel 1989 Gill S C and von Hippel P H 1989 Calculation of protein extinction coefficients from amino acid sequence data Anal Biochem 182 2 319 326 Gonda et al 1989 Gonda D K Bachmair A Wunning l Tobias J W Lane W S and Varshavsky A 1989 Universality and structure of the N end rule J Biol Chem 264 28 16700 16712 Guindon and Gascuel 2003 Guindon S and Gascuel O 2003 A Simple Fast and Accu rate Algorithm to Estimate Large Phylogenies by Maximum Likelihood Systematic Biology 52 5 696 704 Hasegawa et al 1985 Hasegawa M Kishino H and Yano T 1985 Dating of the human ape splitting by a molecular clock of mitochondrial DNA Journal of Molecular Evolution 22 2 160 174 Hein 2001 Hein J 2001 An algorithm for statistical alignment of sequences related by a binary tree In Pacific Symposium on Biocomputing page 179 Hein et al 2000 Hein J Wiuf C Knudsen B M ller M B and Wibling G 2000 Statistical alignment computational properties homology testing and goodness of fit J Mol Biol 302 1 265 279 BIBLIOGRAPHY 336 Henikoff and Henikoff 1992 Henikoff S and Henikoff J G 1992 Amino acid substitution matrices from protein blocks Proc Natl Acad Sci U S A 89 22 10915 10919 Hopp and Woods 1983 Hopp T P and Woods K
186. annotation types will be converted to unsure when exporting in GenBank format As long as you use the sequence in CLC format you own annotation type will be preserved CHAPTER 10 VIEWING AND EDITING SEQUENCES 139 describes the kind of information you wish to add If an appropriate qualifier is not present in the list you can type your own qualifier The pre defined qualifiers are derived from the GenBank format You can add as many qualifier key lines as you wish by clicking the button Redundant lines can be removed by clicking the delete icon 3 The information entered on these lines is shown in the annotation table See section 10 3 1 and in the yellow box which appears when you place the mouse cursor on the annotation If you write a hyperlink in the Key text field like e g www clcbio com it will be recognized as a hyperlink Clicking the link in the annotation table will open a web browser Click OK to add the annotation Note The annotation will be included if you export the sequence in GenBank Swiss Prot or CLC format When exporting in other formats annotations are not preserved in the exported file 10 3 3 Edit annotations To edit an existing annotation from within a sequence view right click the annotation Edit Annotation This will show the same dialog as in figure 10 10 with the exception that some of the fields are filled out depending on how much information the annotation contains There is another way o
187. ar expression For proteins you can enter different protein patterns from the PROSITE database protein patterns using regular expressions and describing specific amino acid sequences The PROSITE database contains a great number of patterns and have been used to identify related proteins see http www expasy ora egi b1n prosite lLstspL CHAPTER 14 GENERAL SEQUENCE ANALYSES 221 a zl Use motif list Clicking the small button acy will allow you to select a saved motif list see section 14 7 4 e Motif If you choose to search with a simple motif you should enter a literal string as your motif Ambiguous amino acids and nucleotides are allowed Example ATGATGNNATG If your motif type is Java regular expression you should enter a regular expression according to the syntax rules described in section 14 7 3 Press Shift F1 key for options For proteins you can search with a Prosite regular expression and you should enter a protein pattern from the PROSITE database e Accuracy If you search with a simple motif you can adjust the accuracy of the motif to the match on the sequence If you type in a simple motif and let the accuracy be 80 the motif search algorithm runs through the input sequence and finds all subsequences of the same length as the simple motif such that the fraction of identity between the subsequence and the simple motif is at least 80 A motif match is added to the sequence as an annotation with the exact f
188. arameters when creating the alignment see section 18 1 However gaps and residues can also be moved after the alignment is created select one or more gaps or residues in the alignment drag the selection to move This can be done both for single sequences but also for multiple sequences by making a selection covering more than one sequence When you have made the selection the mouse pointer turns into a horizontal arrow indicating that the selection can be moved see figure 18 9 Note Residues can only be moved when they are next to a gap AGG GAGTCAT AGG GAGTCAT AGG GAGTCAT AGG GAGTCAT AGG GAGCAGT AGG GAGCAGT AGG GTACAGT ATG GTGCACC ATG GTGCACC ATG GTGCATC ATG GTGCATC Figure 18 9 Moving a part of an alignment Notice the change of mouse pointer to a horizontal arrow 18 3 2 Insert gaps The placement of gaps in the alignment can be changed by modifying the parameters when creating the alignment However gaps can also be added manually after the alignment is created To insert extra gaps select a part of the alignment right click the selection Add gaps before after If you have made a selection covering e g five residues a gap of five will be inserted In this way you can easily control the number of gaps to insert Gaps will be inserted in the sequences that you selected If you make a selection in two sequences in an alignment gaps will be inserted into these two sequences This means that these two sequences
189. ately by mass spectrometry in a laboratory Isoelectric point The isoelectric point pl of a protein is the pH where the proteins has no net charge The plis calculated from the pKa values for 20 different amino acids At a pH below the pl the protein carries a positive charge whereas if the pH is above pl the proteins carry a negative charge In other words pl is high for basic proteins and low for acidic proteins This information can be used in the laboratory when running electrophoretic gels Here the proteins can be separated based on their isoelectric point Aliphatic index The aliphatic index of a protein is a measure of the relative volume occupied by aliphatic side chain of the following amino acids alanine valine leucine and isoleucine An increase in the aliphatic index increases the thermostability of globular proteins The index is calculated by the following formula Aliphaticindex X Ala ax X Val 6 X Leu 6 X Ile CHAPTER 14 GENERAL SEQUENCE ANALYSES 212 Amino acid Mammalian Yeast E coli Ala A 4 4 hour gt 20 hours gt 10 hours Cys C 1 2 hours gt 20 hours gt 10 hours Asp D 1 1 hours 3 min gt 10 hours Glu E 1 hour 30 min gt 10 hours Phe F 1 1 hours 3 min 2 min Gly G 30 hours gt 20 hours gt 10 hours His H 3 5 hours 10 min gt 10 hours lle 1 20 hours 30 min gt 10 hours Lys K 1 3 hours 3 min 2 min Leu L 5 5 hours 3 min 2 min Met M 30 hours gt 20 hours gt 10 hours
190. ating the Workbench to a new version this information is not preserved This means that you should keep this information in a separate place as back up The ability to change the tables is mainly aimed at centrally deployed installations of the Workbench 333 Bibliography Altschul and Gish 1996 Altschul S F and Gish W 1996 Local alignment statistics Methods Enzymol 266 460 480 Altschul et al 1990 Altschul S F Gish W Miller W Myers E W and Lipman D J 1990 Basic local alignment search tool J Mol Biol 215 3 403 410 Andrade et al 1998 Andrade M A O Donoghue S l and Rost B 1998 Adaptation of protein surfaces to subcellular location J Mol Biol 276 2 51 7 525 Bachmair et al 1986 Bachmair A Finley D and Varshavsky A 1986 In vivo half life of a protein is a function of its amino terminal residue Science 234 4773 179 186 Bateman et al 2004 Bateman A Coin L Durbin R Finn R D Hollich V Griffiths Jones S Khanna A Marshall M Moxon S Sonnhammer E L L Studholme D J Yeats C and Eddy S R 2004 The Pfam protein families database Nucleic Acids Res 32 Database issue D138 D141 Bendtsen et al 2004a Bendtsen J D Jensen L J Blom N Heijne G V and Brunak S 2004a Feature based prediction of non classical and leaderless protein secretion Protein Eng Des Sel 17 4 349 356 Bendtsen et al 2005 Bendtsen
191. ations on the protein sequence will be mapped to the resulting DNA sequence In the tooltip on the transferred annotations there is a note saying that the annotation derives from the original sequence CHAPTER 16 PROTEIN ANALYSES 257 The Codon Frequency Table is used to determine the frequencies of the codons Select a frequency table from the list that fits the organism you are working with A translation table of an organism is created on the basis of counting all the codons in the coding sequences Every codon in a Codon Frequency Table has its own count frequency per thousand and fraction which are calculated in accordance with the occurrences of the codon in the organism You can customize the list of codon frequency tables for your installation see section l Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The newly created nucleotide sequence is shown and if the analysis was performed on several protein sequences there will be a corresponding number of views of nucleotide sequences The new sequence is not saved automatically To save the sequence drag it into the Navigation Area or press Ctrl S 38 S on Mac to show the save dialog 16 9 2 Bioinformatics explained Reverse translation In all living cells containing hereditary material such as DNA a transcription to mRNA and subsequent a translation to proteins occur This is of course simplified but is in general what is happ
192. be found in the original paper by Schneider and Stephens 1990 Nevertheless the conservation of every position is defined as Rse which is the difference between the maximal entropy Smar and the observed entropy for the residue distribution S s N Rseg T ae Dobs logs i gt pn logo pn n 1 Pn is the observed frequency of a amino acid residue or nucleotide of symbol n at a particular position and N is the number of distinct symbols for the sequence alphabet either 20 for proteins or four for DNA RNA This means that the maximal sequence information content per position is log 4 2 bits for DNA RNA and logs 20 4 32 bits for proteins The original implementation by Schneider does not handle sequence gaps We have slightly modified the algorithm so an estimated logo is presented in areas with Sequence gaps lf amino acid residues or nucleotides of one sequence are found in an area containing gaps we have chosen to show the particular residue as the fraction of the sequences Example if one position in the alignment contain 9 gaps and only one alanine A the A represented in the logo has a hight of 0 1 Other useful resources The website of Tom Schneider http www Immb nci ferf gov Loms WebLogo CHAPTER 18 SEQUENCE ALIGNMENT 292 http weblogo berkeley edu Crooks et al 2004 18 3 Edit alignments 18 3 1 Move residues and gaps The placement of gaps in the alignment can be changed by modifying the p
193. blast databases from sequences within your CLC Protein Workbench section 12 3 3 CHAPTER 12 BLAST SEARCH 173 12 3 1 Make pre formatted BLAST databases available To use databases that have been downloaded or created outside the Workbench you can either e Put the database files in one of the locations defined in the BLAST database manager see section 12 4 e Add the location where your BLAST databases are stored using the BLAST database manager see section 12 4 See figure 12 14 12 3 2 Download NCBI pre formatted BLAST databases Many popular pre formatted databases are available for download from the NCBI You can download any of the databases available from the list at ftp ftp ncbi nih gov blast db from within your CLC Protein Workbench You must be connected to the internet to use this tool If you choose or Toolbox BLAST Download BLAST Databases 2 a window like the one in figure 12 11 pops up showing you the list of databases available for download e Download BLAST Databases ize o Select download location home joeuser blastdbs F X cancel Figure 12 11 Choose from pre formatted BLAST databases at the NCBI available for download In this window you can see the names of the databases the date they were made available for download on the NCBI site the size of the files associated with that database and a brief description of each database You can also see whether the
194. bmitted to the name of the exported file in order for the exported file to work Before exporting you are asked about which of the different settings you want to include in the exported file One of the items in the list is User Defined View Settings If you export this only the information about which of the settings is the default setting for each view is exported If you wish to export the Side Panel Settings themselves see section 5 2 2 The process of importing preferences is similar to exporting Press Ctrl K 38 on Mac to open Preferences Import Browse to and select the cpf file Import and apply preferences 5 4 1 The different options for export and importing To avoid confusion of the different import and export options here is an overview e Import and export of bioinformatics data such as sequences alignments etc described in section 1 1 e Graphics export of the views which creates image files in various formats described in section 3 e Import and export of Side Panel Settings as described in the next section e Import and export of all the Preferences except the Side Panel settings This is described above CHAPTER 5 USER PREFERENCES AND SETTINGS 95 5 5 View settings for the Side Panel The Side Panel is shown to the right of all views that are opened in CLC Protein Workbench By using the settings in the Side Panel you can specify how the layout and contents of the view Figure 5 8 is an exam
195. by different colors corresponding to the colors of the triangles on the sequence By selecting or deselecting the enzymes in the list you can specify which enzymes restriction sites should be displayed Restriction sites Y Show Labels Stacked Sorting Aa LI Po V Non cutters 4 Single cutters E 4 Bami O DME O E 7 corv 1 E 4 Hindi 1 O Mirmo O MA publi O COIE O Double cutters MN 7 sana O MA RI sma 2 O Multiple cutters a na E salt 3 Figure 17 1 Showing restriction sites of ten restriction enzymes ST TAGAGGGCCCGTTTAAACC The color of the restriction enzyme can be changed by clicking the colored box next to the enzyme s name The name of the enzyme can also be shown next to the restriction site by selecting Show name flags above the list of restriction enzymes There is also an option to specify how the Labels shown be shown e No labels This will just display the cut site with no information about the name of the enzyme Placing the mouse button on the cut site will reveal this information as a tool tip e Flag This will place a flag just above the sequence with the enzyme name see an example in figure 17 2 Note that this option will make it hard to see when several cut sites are located close to each other In the circular view this option is replaced by the Radial option CHAPTER 17 RESTRICTION SITE ANALYSES 267 e Radial This option is only available in the circula
196. c signal peptidases before release to the extracellular environment or specific organelle Below a few processes are listed where proteolytic enzymes act on a protein substrate e N terminal methionine residues are often removed after translation Signal peptides or targeting sequences are removed during translocation through a mem brane Viral proteins that were translated from a monocistronic MRNA are cleaved Proteins or peptides can be cleaved and used as nutrients e Precursor proteins are often processed to yield the mature protein Proteolytic cleavage of proteins has shown its importance in laboratory experiments where it is often useful to work with specific peptide fragments instead of entire proteins Proteases also have commercial applications As an example proteases can be used as detergents for cleavage of proteinaceous stains in clothing The general nomenclature of cleavage site positions of the substrate were formulated by Schechter and Berger 1967 68 Schechter and Berger 1967 Schechter and Berger 1968 They designate the cleavage site between P1 P1 incrementing the numbering in the N terminal direction of the cleaved peptide bond P2 P3 P4 etc On the carboxyl side of the cleavage site the numbering is incremented in the same way P1 P2 P3 etc This is visualized in figure 16 27 Proteases often have a specific recognition site where the peptide bond is cleaved As an example trypsin o
197. ce in the Navigation Area Show Annotation Table E or If the sequence is already open Click Show Annotation Table at the lower left part of the view This will open a view similar to the one in figure 10 9 ES NM 000044 annotation 13 rad Rows 28 E New Annotation Filter r 55 E Name Type Region Qualifiers Shown annotation types CDS forganismrHomo sapiens Gene mol_Eype mRNA Repeat region Source fdb xref taxon 9606 j i v Source fchromosome X Ji map Xq11 2 q12 Mists 7 Select all gene AR Deselect all 1023 1097 standard_name GDB 600694 db_xref UniSTS 99252 gene AR 836 958 Fstandard_name DX374098 fdb_xref UniSTS 38944 ace O B Oh El we amp Figure 10 9 A table showing annotations on the sequence In the Side Panel you can show or hide individual annotation types in the table E g if you only wish to see gene annotations de select the other annotation types so that only gene is selected Each row in the table is an annotation which is represented with the following information e Name Type Region Qualifiers The Name Type and Region for each annotation can be edited simply by double clicking typing the change directly and pressing Enter CHAPTER 10 VIEWING AND EDITING SEQUENCES 137 This information corresponds to the information in the dialog when you edit and add annotations see section 10 3 2 You can benef
198. cedure can be used for joining two alignments 18 3 7 Realign selection If you have created an alignment it is possible to realign a part of it leaving the rest of the alignment unchanged select a part of the alignment to realign right click the selection Realign selection This will open Step 2 in the Create alignment dialog allowing you to set the parameters for the realignment See section 18 1 It is possible for an alignment to become shorter or longer as a result of the realignment of a region This is because gaps may have to be inserted in or deleted from the sequences not selected for realignment This will only occur for entire columns of gaps in these sequences ensuring that their relative alignment is unchanged Realigning a selection is a very powerful tool for editing alignments in several situations e Removing changes If you change the alignment in a specific region by hand you may end up being unhappy with the result In this case you may of course undo your edits but another option is to select the region and realign it e Adjusting the number of gaps If you have a region in an alignment which has too many gaps in your opinion you can select the region and realign it By choosing a relatively high gap cost you will be able to reduce the number of gaps e Combine with fixpoints If you have an alignment where two residues are not aligned but you know that they should have been You can now set an alignment
199. cel Help Figure 10 10 The Add Annotation dialog The left hand part of the dialog lists a number of Annotation types When you have selected an annotation type it appears in Type to the right You can also select an annotation directly in this CHAPTER 10 VIEWING AND EDITING SEQUENCES 138 list Choosing an annotation type is mandatory If you wish to use an annotation type which is not present in the list simply enter this type into the Type field 2 The right hand part of the dialog contains the following text fields e Name The name of the annotation which can be shown on the label in the sequence views Whether the name is actually shown depends on the Annotation Layout preferences see section 10 3 1 e Type Reflects the left hand part of the dialog as described above You can also choose directly in this list or type your own annotation type e Region If you have already made a selection this field will show the positions of the selection You can modify the region further using the conventions of DDBJ EMBL and GenBank The following are examples of how to use the syntax based on http ur ncbi nlm fiih gov collab FPT 467 Points to a single residue in the presented sequence 340 565 Points to a continuous range of residues bounded by and including the starting and ending residues lt 345 500 Indicates that the exact lower boundary point of a region is unknown The location begins at some resi
200. ch License Agreement Please read and accept the license agreement below to begin using you license END USER LICENSE AGREEMENT FOR CLC BIO SOFTWARE 2 CLC Genomics Workbench 1 0 1 Recitals 1 1 This End User License Agreement EULA is a legal agreement between you either an individual person lora single legal entity who will be referred to in this EULA as You and CLC bio A S CVR no 28 30 50 87 for the software products that accompanies this EULA including any associated media printed materials and electronic documentation the Software Product I accept these terms If you experience any problems please contact The CLC Support Team Figure 1 13 Read the license agreement carefully If the Workbench succeeds to find an existing license the next dialog will look as shown in figure 1 14 License Wizard EJ p CLC Protein Workbench Upgrade a License The workbench will attempt to find a valid license for a previous version Tf a license can not be located or if you would like to upgade a different license please click the Choose a different License File button and locate it manually C Program Files CLC Combined Workbench 3 licenses workbench clccombinedwb key License Number CLCCOMBINEDWB3 Choose a different License File If you experience any problems please contact The CLC Support Team Proxy Settings Previous Quit Workbench Figure 1 1
201. ch NCBI Search UniProt Select All Selection Mode Show hide Side Panel Sort folder Split Horizontally Split Vertically Undo User Preferences Zoom In Mode Zoom In without clicking Zoom Out Mode Zoom Out without clicking Inverse zoom mode Windows Linux Shift arrow keys Ctrl tab Ctrl W Ctrl Shift W Ctrl C Ctrl X Delete Alt F4 Ctrl E Ctrl G Space or F1 Ctrl Ctrl M Ctrl arrow keys arrow keys Ctrl Shift N Ctrl N Ctrl O Ctrl V Ctrl P Ctrl Y F2 Ctrl S Ctrl F Ctrl Shift F Ctrl B Ctrl Shift U Ctrl A Ctrl 2 Ctrl U Ctrl Shift R Ctrl T Ctrl J Ctrl Z Ctrl K Ctrl plus plus Ctrl minus minus press and hold Shift 81 Mac OS X Shift arrow keys Ctrl Page Up Down ao W a6 Shift W C a X Delete or Backspace db Q ao E a G Space or F1 ab M db arrow keys arrow keys Shift N N 0 MV P Y S F Shift F B Shift U 2 U Shift R 7 J Z 3 plus d 4 SE 36 36 36 36 36 36 36 38 3 36 36 36 98 1 36 36 36 36 de de minus press and hold Shift Combinations of keys and mouse movements are listed below tOn Linux changing tabs is accomplished using Ctrl Page Up Page Down CHAPTER 3 USER INTERFACE 82 Action Windows Linux Mac OS X Mouse movement Maximize
202. character is C the second any character and the third any character except A CHAPTER 14 GENERAL SEQUENCE ANALYSES 223 14 7 4 Create motif list CLC Protein Workbench offers advanced and versatile options to create lists of sequence patterns or known motifs represented either by a literal string or a regular expression A motif list is created from the Toolbox Toolbox General Sequence Analyses Create Motif List f This will open an empty list where you can add motifs by clicking the Add button at the bottom of the view This will open a dialog shown in figure 14 28 Add motif o Simple E Java Prosite Name TATA box Press Shift Fi for options Description binding site of either transcription Factors or histones Ken Figure 14 28 Entering a new motif in the list In this dialog you can enter the following information e Name The name of the motif In the result of a motif search this name will appear as the name of the annotation and in the result table e Motif The actual motif See section 14 7 2 for more information about the syntax of motifs e Description You can enter a description of the motif In the result of a motif search the description will appear in the result table and added as a note to the annotation on the sequence visible in the Annotation table 3 or by placing the mouse cursor on the annotation bi N e Type You can enter three different types of motifs Si
203. cids 330 Undo limit 89 Undo Redo 2 UniProt 152 search 152 312 search sequence in 159 UPGMA algorithm 307 314 Urls Navigation Area 109 User defined view settings 91 User interface 62 Vector graphics export 111 VectorNTI file format 327 View 69 alignment 288 dot plots 199 GenBank format 142 preferences 5 save changes 2 sequence 122 349 sequence as text 142 View Area 69 illustration 62 View preferences 90 show automatically 91 style sheet 95 View settings user defined 91 Virtual gel 315 vsf file format for settings 92 Web page import sequence from 104 Wildcard append to search 149 152 155 Windows installation 12 Workspace 9 create 9 delete 80 save 9 select 9 Wrap sequences 123 Is file format 329 xIsx file format 329 xml file format 329 Zip file format 32 7 329 Zoom 6 tutorial 38 Zoom In 76 Zoom Out 6 Zoom to 100 7 7 Zoom 3D structure 187
204. conservation is shown on a color scale with blue residues being the least conserved and red residues being the most conserved It is therefore commonplace to either ignore this complication and assume sequences to be unrelated or to use heuristic corrections for shared ancestry The second challenge is to find the optimal alignment given a scoring function For pairs of sequences this can be done by dynamic programming algorithms but for more than three sequences this approach demands too much computer time and memory to be feasible A commonly used approach is therefore to do progressive alignment Feng and Doolittle 1987 where multiple alignments are built through the successive construction of pairwise alignments These algorithms provide a good compromise between time spent and the quality of the resulting alignment Presently the most exciting development in multiple alignment methodology is the construction of statistical alignment algorithms Hein 2001 Hein et al 2000 These algorithms employ a scoring function which incorporates the underlying phylogeny and use an explicit stochastic model of molecular evolution which makes it possible to compare different solutions in a statistically rigorous way The optimization step however still relies on dynamic programming and practical use of these algorithms thus awaits further developments Creative Commons License All CLC bio s scientific articles are licensed under a Creative C
205. ctions and display of the 3D structure 116 116 118 118 121 122 122 130 133 141 142 142 143 148 148 152 154 158 160 161 167 172 1 5 1 6 CONTENTS 134A MONDE va ee ae sd dd as 14 General sequence analyses 14 1 Shuffle sequence 14 2 DOLDIS rrei eee ede db Ed Ea 14 3 Local complexity plot 14 4 Sequence statistics 14 5 Join sequences 14 6 Pattern Discovery 14 7 MON SCOM a cs psss da E 15 Nucleotide analyses 15 1 Convert DNA to RNA 15 2 ConvertRNAtoDNA 15 3 Reverse complements of sequences 15 4 Reverse Sequence 0 15 5 Translation of DNA or RNA to protein 15 6 Find open reading frames 16 Protein analyses 16 1 Signal peptide prediction 16 2 Protem eharge o sure a dem 16 3 Transmembrane helix prediction 16 4 Antigenicity 16 5 Hydrophobicity 16 6 Pfam domain search 16 7 Secondary structure prediction 16 8 Protein repot cesa misas ale 16 9 Reverse translation from protein into DNA 16 10 Proteolytic cleavage detection 17 Restriction site analyses 17 1 Dynamic restriction sites 17 2 Restriction site analysis from the Toolbox 193 195 195 197 207 208 214 215 217 225 225 226 221 228 228 230 233 234 240 241 242 244 249 251 253 255 259 CONTENTS
206. ctures are not downloaded from the NCBI website but is downloaded from the RCSB Protein Data Bank http www rcsb org pdb home home do in mmCIF format Copy paste from structure search results When using copy paste to bring the search results into the Navigation Area the actual files are downloaded To copy paste files into the Navigation Area select one or more of the search results Ctrl C 36 C on Mac select location or folder in the Navigation Area Ctrl V Note Search results are downloaded before they are saved Downloading and saving several files may take some time However since the process runs in the background displayed in the Status bar it is possible to continue other tasks in the program Like the search process the download process can be stopped This is done in the Toolbox in the Processes tab 11 3 3 Save structure search parameters The search view can be saved either using dragging the search tab and and dropping it in the Navigation Area or by clicking Save E When saving the search only the parameters are saved CHAPTER 11 ONLINE DATABASE SEARCH 158 not the results of the search This is useful if you have a special search that you perform from time to time Even if you don t save the search the next time you open the search view it will remember the parameters from the last time you did a search 11 4 Sequence web info CLC Protein Workbench provides direct access to web based search i
207. cytosine Yes C Popularity Pyull cagctg Blunt GE Healthc N4 methylcytosine Yes Ml Select all Create New Enzyme List from Selection Add Remove Enzymes Deselect All Figure 17 22 An enzyme list and you can use the filter at the top right corner to search for specific enzymes recognition sequences etc If you wish to remove or add enzymes click the Add Remove Enzymes button at the bottom of the view This will present the same dialog as shown in figure 17 19 with the enzyme list shown to the right If you wish to extract a subset of an enzyme list CHAPTER 17 RESTRICTION SITE ANALYSES 281 open the list select the relevant enzymes right click Create New Enzyme List from Selection i If you combined this method with the filter located at the top of the view you can extract a very specific set of enzymes E g if you wish to create a list of enzymes sold by a particular distributor type the name of the distributor into the filter and select and create a new enzyme list from the selection Chapter 18 Sequence alignment Contents 18 1 Create an alignment 2 0 onua 283 ISLI CODADOO cau be risos Pewee ea ee oe ee eS 284 18 1 2 Fast or accurate alignment algorithm 050582086 284 18 1 3 Aligning alignments a 44644 2 eee eee eae eee eae ees 285 LE POMS gta eee Oka ea DAE ee ee ee ee E 286 18 2 View alignments 2 00 ee eee 288 18 2 1 Bioinformatics ex
208. d on http www google com 11 4 2 NCBI The NCBI search function searches in GenBank at NCBI http www ncbi nlm nih gov using an identification number when you view the sequence as text it is the GI number Therefore the sequence file must contain this number in order to look it up at NCBI All sequences downloaded from NCBI have this number CHAPTER 11 ONLINE DATABASE SEARCH 159 11 4 3 PubMed References The PubMed references search option lets you look up Pubmed articles based on references contained in the sequence file when you view the sequence as text it contains a number of PUBMED lines Not all sequence have these PubMed references but in this case you will se a dialog and the browser will not open 11 4 4 UniProt The UniProt search function searches in the UniProt database http www ebi uniprot org using the accession number Furthermore it checks whether the sequence was indeed downloaded from UniProt 11 4 5 Additional annotation information When sequences are downloaded from GenBank they often link to additional information on taxonomy conserved domains etc If such information is available for a sequence it is possible to access additional accurate online information If the db xref identifier line is found as part of the annotation information in the downloaded GenBank file it is possible to easily look up additional information on the NCBI web site To access this feature simply right click an ann
209. d put OR between them 4 2 3 Quick search history You can access the 10 most recent searches by clicking the icon GQ next to the search field see figure 4 5 Qe llenath 100 TO 150 Search length 100 TO 150 ckagina signal IN lt human name humhbb insulin abona Figure 4 5 Recent searches Clicking one of the recent searches will conduct the search again 4 3 Advanced search As a supplement to the Quick search described in the previous section you can use the more advanced search Search Local Search or Ctrl F 36 F on Mac This will open the search view as shown in figure 4 6 The first thing you can choose is which location should be searched All the active locations are shown in this list You can also choose to search all locations Read more about locations in section 3 1 1 Furthermore you can specify what kind of elements should be searched CHAPTER 4 SEARCHING YOUR DATA 87 do Search O Search in Location within Add Filter x Label Description Length L Figure 4 6 Advanced search e All sequences e Nucleotide sequences e Protein sequences e All data When searching for sequences you will also get alignments sequence lists etc as result if they contain a sequence which match the search criteria Below are the search criteria First select a relevant search filter in the Add filter list For sequences you c
210. d reading Rows 15 169 Find reading Frame output Filter Match any s Match all Length o rr Apply Start End Length Found ak strand Start codon 14 rate ovo negative ANT bd 3462 ao 426 negative CAC 414 556564 1851 negative CAC 24342 aboz 1663 negative ATA EA Cec STE mansabi TT Figure C 3 The advanced filter showing open reading frames larger than 400 that are placed on the negative strand Both for the simple and the advanced filter there is a counter at the upper left corner which tells you the number of rows that pass the filter 91 in figure C 2 and 15 in figure C 3 Appendix D BLAST databases Several databases are available at NCBI which can be selected to narrow down the possible BLAST hits D 1 Peptide sequence databases D 2 nr Non redundant GenBank CDS translations PDB SwissProt PIR PRF excluding those in env_nr refseq Protein sequences from NCBI Reference Sequence project http www ncbi nlm nih gov RefSeg swissprot Last major release of the SWISS PROT protein sequence database no incre mental updates pat Proteins from the Patent division of GenBank pdb Sequences derived from the 3 dimensional structure records from the Protein Data Bank http www rcsb org pdb env nr Non redundant CDS translations from env nt entries month All new or revised GenBank CDS translations PDB SwissProt PIR PRF released in the last 30 days N
211. d to have the same origin and are thus joined Consider the joining of alignments A and B If a sequence named in A and B is found in both A and B the spliced alignment will contain a sequence named in A and B which represents the characters from A and B joined in direct extension of each other If a sequence with the name in A not B is found in A but not in B the spliced alignment will contain a sequence named in A not B The first part of this sequence will contain the characters from A but since no sequence information is available from B a number of gap characters will be added to the end of the sequence corresponding to the number of residues in B Note that the function does not require that the individual alignments contain an equal number of sequences 18 5 Pairwise comparison For a given set of aligned sequences see chapter 18 it is possible make a pairwise comparison in which each pair of Sequences are compared to each other This provides an overview of the diversity among the sequences in the alignment In CLC Protein Workbench this is done by creating a comparison table Toolbox in the Menu Bar Alignments and Trees 2 Pairwise Comparison EB or right click alignment in Navigation Area Toolbox Alignments and Trees Pairwise Comparison FE This opens the dialog displayed in figure 18 13 If an alignment was selected before choosing the Toolbox action this alignment is now listed in the Selected Elements
212. database has any dependencies This aspect is described below You can also specify which of your database locations you would like to store the files in Please see the Manage BLAST Databases section for more on this section 12 4 There are two very important things to note if you wish to take advantage of this tool e Many of the databases listed are very large Please make sure you have room for them If you are working on a shared system we recommend you discuss your plans with your system administrator and fellow users e Some of the databases listed are dependent on others This will be listed in the Dependencies column of the Download BLAST Databases window This means that while CHAPTER 12 BLAST SEARCH 1 4 the database your are interested in may seem very small it may require that you also download a very big database on which it depends An example of the second item above is Swissprot To download a database from the NCBI that would allow you to search just Swissprot entries you need to download the whole nr database in addition to the entry for Swissprot 12 3 3 Create local BLAST databases In the CLC Protein Workbench you can create a local database that you can use for local BLAST searches You can specify a location on your computer to save the BLAST database files to The Workbench will list the BLAST databases found in these locations when you set up a local BLAST search see section 12 1 3 DNA RNA and protein
213. de DNA or protein Total size 1000 residues The number of residues in the database either bases or amino acid e Location The location of the database Below the list of BLAST databases there is a button to Remove Database This option will delete the database files belonging to the database selected 12 4 1 Migrating from a previous version of the Workbench In versions released before 2011 the BLAST database management was very different from this In order to migrate from the older versions please add the folders of the old BLAST databases as locations in the BLAST database manager see section 12 4 The old representations of the BLAST databases in the Navigation Area can be deleted If you have saved the BLAST databases in the default folder they will automatically appear because the default database location used in CLC Protein Workbench 5 8 is the same as the default folder specified for saving BLAST databases in the old version 12 5 Bioinformatics explained BLAST BLAST Basic Local Alignment Search Tool has become the defacto standard in search and alignment tools Altschul et al 1990 The BLAST algorithm is still actively being developed and is one of the most cited papers ever written in this field of biology Many researchers use BLAST as an initial screening of their sequence data from the laboratory and to get an idea of what they are working on BLAST is far from being basic as the name indicates it is a high
214. de and subcellular prediction methods will be described Most signal peptide prediction methods require the presence of the correct N terminal end of thttp www ncbi nlm nih gov entrez CHAPTER 16 PROTEIN ANALYSES 231 Sec signal peptide Cleavage site region a y hr pa TODO a Mature RIK A A 3 dl 3 AM Twin arginine signal peptide Cleavage site n regiom h region c region Mature sal Pp Sa E RK RAXFLK A A Lipoprotein signal peptide Cleavage site i regi n region E amp regis m Matire Prepillin like signal peptide Cleavage site nregion p h region a be a Mature Bacteriocin signal peptide Cleavage site region C region Mature i ata Non classical secreted protein d Mature Figure 16 3 Schematic representation of various signal peptides Red color indicates n region gray color indicates h region cyan indicates c region All white circles are part of the mature protein 1 indicates the first position of the mature protein The length of the signal peptides is not drawn to scale the preprotein for correct classification As large scale genome sequencing projects sometimes assign the 5 end of genes incorrectly many proteins are annotated without the correct N terminal Reinhardt and Hubbard 1998 leading to incorrect prediction of subcellular localization These erroneous predictions can be ascribed directly to poor gene finding Other methods for predictio
215. de positions where the main sequence the reference sequence for read mappings and the query sequence for BLAST results has gaps If you are exporting e g coverage information from a read mapping you would probably want to exclude gaps if you want the positions in the exported file to match the reference i e chromosome coordinates If you export including gaps the data points in the file no longer corresponds to the reference coordinates because each gap will shift the coordinates Clicking Next will present a file dialog letting you specify name and location for the file The output format of the file is like this CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 115 Position Valice LS MA corto AE 7 5 Copy paste view output The content of tables e g in reports folder lists and sequence lists can be copy pasted into different programs where it can be edited CLC Protein Workbench pastes the data in tabulator separated format which is useful if you use programs like Microsoft Word and Excel There is a huge number of programs in which the copy paste can be applied For simplicity we include one example of the copy paste function from a Folder Content view to Microsoft Excel First step is to select the desired elements in the view click a line in the Folder Content view hold Shift button press arrow down up key See figure 16 L3 Sequences Contents of Sequences Filter Name Description Length
216. default application for this file type e g Microsoft Word for doc files and Adobe Reader for pdf External files are imported and exported in the same way as bioinformatics files see sec tion 7 1 1 Bioinformatics files not recognized by CLC Protein Workbench are also treated as external files 1 3 Export graphics to files CLC Protein Workbench supports export of graphics into a number of formats This way the visible output of your work can easily be saved and used in presentations reports etc The Export Graphics function l is found in the Toolbar CLC Protein Workbench uses a WYSIWYG principle for graphics export What You See Is What You Get This means that you should use the options in the Side Panel to change how your data e g a sequence looks in the program When you export it the graphics file will look exactly the same way It is not possible to export graphics of elements directly from the Navigation Area They must first be opened in a view in order to be exported To export graphics of the contents of a view select tab of View Graphics This will display the dialog shown in figure 7 7 q Export Graphics ES reo 1 Output options RBS saias sales Export options O Export visible area Export whole area Figure 7 Selecting to export whole view or to export only the visible area Y Y next Finish PK Cancel CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 110
217. dialog shown in figure 1 8 A progress for getting the license is shown and when the license is downloaded you will be able to click Next Go to license download web page Selecting the second option Go to license download web page opens the license web page as shown in 1 9 Click the Request Evaluation License button and you will be able to save the license on your computer e g on the Desktop Back in the Workbench window you will now see the dialog shown in 1 10 CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 20 License Wizard Es p CLC Protein Workbench Requesting a license with id CLC LICENSE SRENMNSTED 0D43CAS Requesting and downloading a license by establishing a direct connection to the CLC bio License Web Service Your License was successfully downloaded The License is valid until 2008 08 01 If you experience any problems please contact The CLC Support Team Proxy Settings Pre Next Quit Workbench Figure 1 8 A license has been downloaded Download a license This your License Order ID CLC LICENSE SRENMNSTED 0D43CA SEDF4XXXXXD844A 4C0C 480000 AB1AEFF9 19F ur license please click the button below Download License adec a file containing the license willl b Figure 1 9 The license web page where you can download a license License Wizard zs B CLC Protein Workbench Import a license from a file Please click the button below and loca
218. dict Secondary Structure eS ES 1 Select protein sequences SEEC protein Sequence Projects Selected Elements 1 3 CLC Data As ATPSal Example Data Cloning Primers Protein analyses Protein ortholog RNA secondary Sequencing data gt xs Q nter search term gt poros ue enh a Figure 16 18 Choosing one or more protein sequences for secondary structure prediction If a sequence was selected before choosing the Toolbox action this sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements You can perform the analysis on several protein sequences at a time This will add annotations to all the sequences and open a view for each sequence Click Next if you wish to adjust how to handle the results See section 9 1 If not click Finish After running the prediction as described above the protein sequence will show predicted alpha helices and beta sheets as annotations on the original sequence see figure 16 19 Each annotation will carry a tooltip note saying that the corresponding annotation is predicted with CLC Protein Workbench Additional notes can be added through the Edit Annotation S right click mouse menu See section 10 3 2 CHAPTER 16 PROTEIN ANALYSES 253 Helix Helix Strand 20 um E ATP8al MPTMRRTVSEIRSRAEGYEKTDDVSEKTSLADQEEVRTIFINQP ba ai Helix
219. does not save the sequence e Download and save lets you choose location for saving sequence e Open at UniProt searches the sequence at UniProt s web page Double clicking a hit will download and open the sequence The hits can also be copied into the View Area or the Navigation Area from the search results by drag and drop copy paste or by using the right click menu as described below CHAPTER 11 ONLINE DATABASE SEARCH 154 Drag and drop from UniProt search results The sequences from the search results can be opened by dragging them into a position in the View Area Note A sequence is not saved until the View displaying the sequence is closed When that happens a dialog opens Save changes of sequence x Yes or No The sequence can also be saved by dragging it into the Navigation Area It is possible to select more sequences and drag all of them into the Navigation Area at the same time Download UniProt search results using right click menu You may also select one or more sequences from the list and download using the right click menu see figure 11 2 Choosing Download and Save lets you select a folder or location where the sequences are saved when they are downloaded Choosing Download and Open opens a new view for each of the selected sequences Copy paste from UniProt search results When using copy paste to bring the search results into the Navigation Area the actual files are downloaded from UniProt To copy paste
220. ds a specific position on the sequence In order to find an interval e g from position 500 to 570 enter 500 570 in the search field This will make a selection from position 500 to 570 both included Notice the two periods between the start an end number see section 10 3 2 If you enter positions including thousands separators like 123 345 the comma will just be ignored and it would be equivalent to entering 123345 e Include negative strand When searching the sequence for nucleotides or amino acids you can search on both strands e Name search Searches for sequence names This is useful for searching sequence lists mapping results and BLAST results This concludes the description of the View Preferences Next the options for selecting and editing sequences are described Text format These preferences allow you to adjust the format of all the text in the view both residue letters sequence name and translations if they are shown e Text size Five different sizes e Font Shows a list of Fonts available on your computer e Bold residues Makes the residues bold CHAPTER 10 VIEWING AND EDITING SEQUENCES 129 10 1 2 Restriction sites in the Side Panel Please see section 1 1 10 1 3 Selecting parts of the sequence You can select parts of a sequence Click Selection in Toolbar Press and hold down the mouse button on the sequence where you want the selection to start move the mouse to the end of the
221. due previous to the first residue specified which is not necessarily contained in the presented sequence and continues up to and including the ending residue lt 1 888 The region starts before the first sequenced residue and continues up to and including residue 888 1 gt 888 The region starts at the first sequenced residue and continues beyond residue 888 102 110 Indicates that the exact location is unknown but that it is one of the residues between residues 102 and 110 inclusive 1237124 Points to a site between residues 123 and 124 join 12 78 134 202 Regions 12 to 78 and 134 to 202 should be joined to form one contiguous sequence complement 34 126 Start at the residue complementary to 126 and finish at the residue complementary to residue 34 the region is on the strand complementary to the presented strand complement join 2691 4571 4918 5163 Joins regions 2691 to 4571 and 4918 to 5163 then complements the joined segments the region is on the strand complementary to the presented strand join complement 4918 5163 complement 2691 4571 Complements regions 4918 to 5163 and 2691 to 4571 then joins the complemented segments the region is on the strand complementary to the presented strand e Annotations In this field you can add more information about the annotation like comments and links Click the Add qualifier key button to enter information Select a qualifier which Note that your own
222. e Sequence project It overlaps with refseq_genomic e wgs Assemblies of Whole Genome Shotgun sequences e env_nt Sequences from environmental samples such as uncultured bacterial samples isolated from soil or marine samples The largest single source is Sagarsso Sea project This does overlap with nucleotide nr D 3 Adding more databases Besides the databases that are part of the default configuration you can add more databases located at NCBI by configuring files in the Workbench installation directory The list of databases that can be added is here http www ncbi nlm nih gov staff tao URLAPI remote blastdblist html In order to add a new database find the settings folder in the Workbench installation directory e g C Program files CLC Genomics Workbench 4 Download unzip and place the following files in this directory to replace the built in list of databases e Nucleotide databases http www clcbio com wbsettings NCBI BlastNucleotideDataba Zip e Protein databases http www clcbio com wbsettings NCBI_BlastProteinDatabases zip Open the file you have downloaded into thesettings folder e g NCBI_BlastProteinDatabases proper in a text editor and you will see the contents look like this APPENDIX D BLAST DATABASES 323 nr clcdefault Non redundant protein sequences refseq protein Reference proteins swissprot Swiss Prot protein sequences pat Patented protein sequences pdb Protein Data Bank proteins
223. e http en wikipedia org wiki Genetic_code Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents 16 10 Proteolytic cleavage detection CLC Protein Workbench offers to analyze protein sequences with respect to cleavage by a selection of proteolytic enzymes This section explains how to adjust the detection parameters and offers basic information on proteolytic cleavage in general 16 10 1 Proteolytic cleavage parameters Given a protein sequence CLC Protein Workbench detects proteolytic cleavage sites in accordance with detection parameters and shows the detected sites as annotations on the sequence and in textual format in a table below the sequence view Detection of proteolytic cleavage sites is initiated by right click a protein sequence in Navigation Area Toolbox Protein Analyses j Proteolytic Cleavage of This opens the dialog shown in figure 16 23 CHAPT
224. e will emerge if two identical or very homologous sequences are plotted against each other Dot plots can also be used to visually inspect sequences for direct or inverted repeats or regions with low sequence complexity Various smoothing algorithms can be applied to the dot plot calculation to avoid noisy background of the plot Moreover can various substitution matrices be applied in order to take the evolutionary distance of the two sequences into account To create a dot plot Toolbox General Sequence Analyses LA Create Dot Plot 4 or Select one or two sequences in the Navigation Area Toolbox in the Menu Bar General Sequence Analyses A Create Dot Plot 4 CHAPTER 14 GENERAL SEQUENCE ANALYSES 198 or Select one or two sequences in the Navigation Area right click in the Navigation Area Toolbox General Sequence Analyses 7 Create Dot Plot 2 This opens the dialog shown in figure 14 3 g Create Dot Plot ES tal 1 Select one or two _ elect one or two sequences or same type sequences of same type Projects Selected Elements 2 gt Ey CLC Data Ns 09429 Example Data fs ATP8al AX ATP8al genomic 24 ATPSal mRNA us Cloning Primers Protein analyses Protein ortholog Ss Ps P39524 Pus P57792 4 Q29449 lt a Sig QONTIZ Sw Q9SX33 RNA secondary Sequencing data y 5 5 X Cancel Figure 14 3 Selecting sequences for the dot plot If
225. e 17 6 Adding or removing enzymes from the Side Panel At the top you can choose to Use existing enzyme list Clicking this option lets you select an enzyme list which is stored in the Navigation Area See section 1 4 for more about creating and modifying enzyme lists Below there are two panels e To the left you see all the enzymes that are in the list select above If you have not chosen to use an existing enzyme list this panel shows all the enzymes available t e To the right there is a list of the enzymes that will be used Select enzymes in the left side panel and add them to the right panel by double clicking or clicking the Add button E If you e g wish to use EcoRV and BamHI select these two enzymes and add them to the right side panel The CLC Protein Workbench comes with a standard set of enzymes based on http www rebase neb com You can customize the enzyme database for your installation see section CHAPTER 17 RESTRICTION SITE ANALYSES 269 If you wish to use all the enzymes in the list Click in the panel to the left press Ctrl A 38 A on Mac Add gt The enzymes can be sorted by clicking the column headings i e Name Overhang Methylation or Popularity This is particularly useful if you wish to use enzymes which produce e g a 3 overhang In this case you can sort the list by clicking the Overhang column heading and all the enzymes producing 3 overhangs will be listed together for easy
226. e NCBI s Entrez website When conducting the search from CLC Protein Workbench the results are available and ready to work with straight away As default CLC Protein Workbench offers one text field where the search parameters can be entered Click Add search parameters to add more parameters to your search Note The search is a AND search meaning that when adding search parameters to your search you search for both or all text strings rather than any of the text strings You can append a wildcard character by clicking the checkbox at the bottom This means that you only have to enter the first part of the search text e g searching for prot will find both protein and protease The following parameters can be added to the search e All fields Text searches in all parameters in the NCBI structure database at the same time e Organism Text e Author Text e PdbAcc The accession number of the structure in the PDB database CHAPTER 11 ONLINE DATABASE SEARCH 156 The search parameters are the most recently used The All fields allows searches in all parameters in the database at the same time All fields also provide an opportunity to restrict a search to parameters which are not listed in the dialog E g writing gene Feature key AND mouse in All fields generates hits in the GenBank database which contains one or more genes and where mouse ap pears somewhere in GenBank file NB the Feature Key o
227. e Name this is the default information to be shown e Accession Sequences downloaded from databases like GenBank have an accession number e Latin name e Latin name accession e Common name e Common name accession The User Defined View Settings gives you an overview of the different Side Panel settings that are saved for each view See section 5 5 for more about how to create and save style sheets lf there are other settings beside CLC Standard Settings you can use this overview to choose which of the settings should be used per default when you open a view see an example in figure 5 4 In this example the CLC Standard Settings is chosen as default 5 2 1 Number formatting in tables In the preferences you can specify how the numbers should be formatted in tables see figure 5 5 CHAPTER 5 USER PREFERENCES AND SETTINGS 92 E EB Preferences xX _ Use r DENMEO view Seccings Available Editors amp 3D Molecule General HEE Alignment dl BLAST Graphics ES BLAST Table CLC Standard Settings Non com pact ES Motif List editor ES Multi BLAST Table No annotations No restriction sites Aer Sequence Small RNA sample ES Table ES Table Te Tree Export Import 2 Help XX Cancel Export Import Figure 5 4 Selecting the default view setting EN ALA E AICA AG MTO AAS Number of fraction digits 2 1 23 0 12
228. e able to document and reproduce previous operations This can be useful in several situations It can be used for documentation purposes where you can specify exactly how your data has been created and modified It can also be useful if you return to a project after some time and want to refresh your memory on how the data was created Also if you have performed an analysis and you want to reproduce the analysis on another element you can check the history of the analysis which will give you all parameters you set This chapter will describe how to use the History functionality of CLC Protein Workbench 8 1 Element history You can view the history of all elements in the Navigation Area except files that are opened in other programs e g Word and pdf files The history starts when the element appears for the first time in CLC Protein Workbench To view the history of an element Select the element in the Navigation Area Show in the Toolbar History LR or If the element is already open History LR at the bottom left part of the view This opens a view that looks like the one in figure 8 1 When opening an element s history is opened the newest change is submitted in the top of the view The following information is available e Title The action that the user performed e Date and time Date and time for the operation The date and time are displayed according 116 CHAPTER 8 HISTORY LOG 117 Ch Reference contig NI
229. e format of the user manual This user manual offers support to Windows Mac OS X and Linux users The software is very similar on these operating systems In areas where differences exist these will be described separately However the term right click is used throughout the manual but some Mac users may have to use Ctrl click in order to perform a right click if they have a single button mouse The most recent version of the user manuals can be downloaded from http www clcbio com usermanuals The user manual consists of four paris e The first part includes the introduction and some tutorials showing how to apply the most significant functionalities of CLC Protein Workbench e The second part describes in detail how to operate all the program s basic functionalities e The third part digs deeper into some of the bioinformatic features of the program In this part you will also find our Bioinformatics explained sections These sections elaborate on the algorithms and analyses of CLC Protein Workbench and provide more general knowledge of bioinformatic concepts e The fourth part is the Appendix and Index Each chapter includes a short table of contents CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 35 1 9 1 Text formats In order to produce a clearly laid out content in this manual different formats are applied e A feature in the program is in bold starting with capital letters Example Navigation Area e
230. e is created which also has all the annotations reversed since they now occupy the opposite strand of their previous location Note This is not the same as a reverse complement If you wish to create the reverse complement please refer to section 15 3 select a sequence in the Navigation Area Toolbox in the Menu Bar Nucleotide Analyses LA Reverse Sequence x3 This opens the dialog displayed in figure 15 4 a q Reverse Sequence Es 1 Select either protein or RBEISSSSo usa e ss nucleotide sequences Projects Selected Elements 1 CLC Data xx ATP8al mRNA gt Example Data 2 ATP8al genomic AM Sw ATP8al Cloning Primers Protein analyses Protein orthologs RNA secondary s 55 Sequencing data gt Rm y Q nter search term gt 200 aaa la RE Figure 15 4 Reversing a sequence lf a sequence was selected before choosing the Toolbox action the sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish Note This is not the same as a reverse complement If you wish to create the reverse complement please refer to section 15 3 15 5 Translation of DNA or RNA to protein In CLC Protein Workbench you can translate a nucleotide sequence into a protein sequence
231. e of the view The size of the floating Side Panel can be adjusted by dragging the hatched area in the bottom right Chapter 6 Printing Contents 6 1 Selecting which part of the view to print 2 082 ee ee eee 99 Ge PICOS eek eect weae ea ARA eS E ee E E 100 6 2 1 Header and footer 2a 4 ecu daw tba R eRe ee ED EPA RM GE we 101 6 3 Printpreview 00000 ee 101 CLC Protein Workbench offers different choices of printing the result of your work This chapter deals with printing directly from CLC Protein Workbench Another option for using the graphical output of your work is to export graphics see chapter 7 3 in a graphic format and then import it into a document or a presentation All the kinds of data that you can view in the View Area can be printed The CLC Protein Workbench uses a WYSIWYG principle What You See Is What You Get This means that you should use the options in the Side Panel to change how your data e g a sequence looks on the screen When you print it it will look exactly the same way on print as on the screen For some of the views the layout will be slightly changed in order to be printer friendly It is not possible to print elements directly from the Navigation Area They must first be opened in a view in order to be printed To print the contents of a view select relevant view Print 5 in the toolbar This will show a print dialog See figure 6 1 In
232. e output options e g a table of restriction sites and a list of restriction enzymes that can be saved for later use In this tutorial the first section describes how to use the Side Panel to show restriction sites whereas the second section describes the restriction map analysis performed from the Toolbox 2 10 1 The Side Panel way of finding restriction sites When you open a sequence there is a Restriction sites setting in the Side Panel By default 10 of the most popular restriction enzymes are shown see figure 2 29 Restriction sites Show Labels Stacked Sorting Aa LI Po 54 Non cutters Single cutters E 7 BamHI O F M Ecor1 O MN 7 ecorv o E E 7 Hindi 1 E Mirmo O MN pb O Figo O Double cutters Men o DO sma 2 ED Multiple cutters o P 7 salt 3 O Figure 2 29 Showing restriction sites of ten restriction enzymes ST TAGAGGGCCCGTTTAAACC The restriction sites are shown on the sequence with an indication of cut site and recognition sequence In the list of enzymes in the Side Panel the number of cut sites is shown in parentheses for each enzyme e g Sall cuts three times If you wish to see the recognition sequence of the enzyme place your mouse cursor on the enzyme in the list for a short moment and a tool tip will appear You can add or remove enzymes from the list by clicking the Manage enzymes button CHAPTER 2 TUTORIALS 58 2 10 2 The Toolbox way of finding
233. e printed content will be broken up vertically and split across 2 pages Note It is a good idea to consider adjusting view settings e g Wrap for sequences in the Side Panel before printing As explained in the beginning of this chapter the printed material will look like the view on the screen and therefore these settings should also be considered when adjusting Page Setup CHAPTER 6 PRINTING 101 12 34 5 6 Figure 6 6 An example where Fit to pages horizontally is set to 2 and Fit to pages vertically is set to 3 6 2 1 Header and footer Click the Header Footer tab to edit the header and footer text By clicking in the text field for either Custom header text or Custom footer text you can access the auto formats for header footer text in Insert a caret position Click either Date View name or User name to include the auto format in the header footer text Click OK when you have adjusted the Page Setup The settings are saved so that you do not have to adjust them again next time you print You can also change the Page Setup from the File menu 6 3 Print preview The preview is shown in figure 6 7 Preview CLC Main Workbench 4 0 Es E UW w tw Y Zoom 100 Figure 6 7 Print preview The Print preview window lets you see the layout of the pages that are printed Use the arrows in the toolbar to navigate between the pages Click Print lt 5 to show the print dialog which lets you choose
234. e protei o Alignment settim Hans l ig conta mueren O rs P39524 MN BBRET PPKRKPGEDO TEE NOEL a 001205 MARBNBNKON AKRESRDEDE BEEAGESwic RTEONPRECE 1 Every 10 residues P57792 MA T O s GRRRKR HE AE AA No wrap Q9sx33 Miles c TKRRRR E Consensus MAT X VERA caso BGR awe AAA gt 100 Fixed wrap Conservation 0 60 80 l l Y Numbers on sequences Q29449 2 25 se ee ee ee uu uu uu Fl Q9NT 2 descuida aer as Ce las us Relative to 1 p39524 BBTTSHSGSR SKMTNSHANC MMMPPSHMBP EETMDEDADO s Lock numbers 094296 BEREDRECSE SQMMSSSCAN STNP BRAD 6 ae PSII aa a eu do Wa eed Gaon a dra a aca aa ADE oat dl ae es 1 A ide labels Q95X33 2 222 eee eee ee eee Be ee Be eee 11 V Lock labels CONSEIA lt lt See Sa Sees SSS SSeS SS ns SS mia es Sequence label 100 N Conservation ane x 0 Domed ie Hooooblooos annal em emo Show selection boxes E y q Figure 2 27 The resulting alignment Note The new alignment is not saved automatically To save the alignment drag the tab of the alignment view into the Navigation Area Installing the Additional Alignments plugin gives you access to other alignment algorithms ClustalW Windows Mac Linux Muscle Windows Mac Linux T Coffee Mac Linux MAFFT Mac Linux and Kalign Mac Linux The Additional Alignments Module can be downloaded from http www clcbio com pl
235. e sequence than fragments in the CHAPTER 2 TUTORIALS 94 table If you conduct another proteolytic cleavage on the same sequence the output consists of possibly new annotations on the original sequence and an additional table view listing all fragments 2 8 Tutorial Align protein sequences This tutorial outlines some of the alignment functionality of the CLC Protein Workbench In addition to creating alignments of nucleotide or peptide sequences the software offers several ways to view alignments The alignments can then be used for building phylogenetic trees Sequences must be available via the Navigation Area to be included in an alignment If you have sequences open in a View that you have not saved then you just need to select the view tab and press Ctrl S or S on Mac to save them In this tutorial six protein sequences from the Example data folder will be aligned See figure 2 24 1a 094296 IP 39524 tat EYFER 11029449 Se ENE Se 95X33 Figure 2 24 Six protein sequences in Sequences from the Protein orthologs folder of the Example data To align the sequences select the sequences from the Protein folder under Sequences Toolbox Alignments and Trees Create Alignment Ez 2 8 1 The alignment dialog This opens the dialog shown in figure 2 25 r EB Create Alignment EA 1 Select sequences of same Select Seq SAO Pe Projects Selected Elements 6
236. e shown by clicking the text e Name The name of the sequence which is also shown in sequence views and in the Navigation Area e Description A description of the sequence Comments The author s comments about the sequence e Keywords Keywords describing the sequence Db source Accession numbers in other databases concerning the same sequence CHAPTER 10 VIEWING AND EDITING SEQUENCES 142 e Gb Division Abbreviation of GenBank divisions See section 3 3 in the GenBank release notes for a full list of GenBank divisions e Length The length of the sequence e Modification date Modification date from the database This means that this date does not reflect your own changes to the sequence See the history section 8 for information about the latest changes to the sequence after it was downloaded from the database e Organism Scientific name of the organism first line and taxonomic classification levels second and subsequent lines The information available depends on the origin of the sequence Sequences downloaded from database like NCBI and UniProt see section 12 have this information On the other hand some sequence formats like fasta format do not contain this information Some of the information can be edited by clicking the blue Edit text This means that you can add your own information to sequences that do not derive from databases Note that for other kinds of data the Element info will only have Name and
237. e way to manually translate coding parts of sequences CDS into protein You simply translate the new sequence into protein This is done by right click the tab of the new sequence Toolbox Nucleotide Analyses A Translate to Protein 44 CHAPTER 10 VIEWING AND EDITING SEQUENCES 130 A selection can also be copied to the clipboard and pasted into another program make a selection Ctrl C 36 C on Mac Note The annotations covering the selection will not be copied A selection of a sequence can be edited as described in the following section 10 1 4 Editing the sequence When you make a selection it can be edited by right click the selection Edit Selection W A dialog appears displaying the sequence You can add remove or change the text and click OK The original selected part of the sequence is now replaced by the sequence entered in the dialog This dialog also allows you to paste text into the sequence using Ctrl V V on Mac If you delete the text in the dialog and press OK the selected text on the sequence will also be deleted Another way to delete a part of the sequence is to right click the selection Delete Selection 1 If you wish to only correct only one residue this is possible by simply making the selection only cover one residue and then type the new residue 10 1 5 Sequence region types The various annotations on sequences cover parts of the sequence Some cover an interval some cover
238. ed by dragging it into the Navigation Area This could be views that are open elements on lists e g search hits or sequence lists and files located on your computer Finally you can add data by adding a new location see section 3 1 1 If a file or another element is dropped on a folder it is placed at the bottom of the folder If it is dropped on another element it will be placed just below that element If the element already exists in the Navigation Area you will be asked whether you wish to create CHAPTER 3 USER INTERFACE 65 a copy 3 1 2 Create new folders In order to organize your files they can be placed in folders Creating a new folder can be done in two ways right click an element in the Navigation Area New Folder H or File New Folder H0 If a folder is selected in the Navigation Area when adding a new folder the new folder is added at the bottom of this folder If an element is selected the new folder is added right above that element You can move the folder manually by selecting it and dragging it to the desired destination 3 1 3 Sorting folders You can sort the elements in a folder alphabetically right click the folder Sort Folder On Windows subfolders will be placed at the top of the folder and the rest of the elements will be listed below in alphabetical order On Mac both subfolders and other elements are listed together in alphabetical order 3 1 4 Multiselecting elements Multis
239. ed for the location To see where the folder is located on your computer place your mouse cursor on the location icon E for second This will show the path to the location Sharing data is possible of you add a location on a network drive The procedure is similar to the one described above When you add a location on a network drive or a removable drive the CHAPTER 3 USER INTERFACE 64 E q Choose folder to add as location Lookin EE Desktop 2 BE ES E Computer id Network Recent Items Desktop Documents LS Computer A Network File name C Users smoensted Desktop Add Files of type All Files Cancel RA IMavigalondrea 8 l BS Y e Ofal E o E Ej CLC Gl E E Figure 3 5 The new location has been added location will appear inactive when you are not connected Once you connect to the drive again click Update All 1 and it will become active note that there will be a few seconds delay from you connect Opening data The elements in the Navigation Area are opened by Double click the element or Click the element Show 42 in the Toolbar Select the desired way to view the element This will open a view in the View Area which is described in section 3 2 Adding data Data can be added to the Navigation Area in a number of ways Files can be imported from the file system see chapter 7 Furthermore an element can be add
240. ed on several sequences at a time the method will search for patterns in the sequences and open a new view for each of the sequences in which a pattern was discovered Each novel pattern will be represented as an annotation of the type Region More information on each found pattern is available through the tool tip including detailed information on the position of the pattern and quality scores It is also possible to get a tabular view of all found patterns in one combined table Then each found pattern will be represented with various information on obtained scores quality of the pattern and position in the sequence A table view of emission values of the actual used HMM model is presented in a table view This model can be saved and used to search for a similar pattern in new or unknown sequences 14 7 Motif Search CLC Protein Workbench offers advanced and versatile options to search for known motifs represented either by a simple Sequence or a more advanced regular expression These advanced search capabilities are available for use in both DNA and protein sequences There are two ways to access this functionality CHAPTER 14 GENERAL SEQUENCE ANALYSES 218 e When viewing sequences it is possible to have motifs calculated and shown on the sequence in a similar way as restriction sites see section 1 1 This approach is called Dynamic motifs and is an easy way to spot known sequence motifs when working with sequences for cloning etc
241. editing the image after export CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 112 Vector graphics Vector graphic is a collection of shapes Thus what is stored is e g information about where a line starts and ends and the color of the line and its width This enables a given viewer to decide how to draw the line no matter what the zoom factor is thereby always giving a correct image This format is good for e g graphs and reports but less usable for e g dot plots If the image is to be resized or edited vector graphics are by far the best format to store graphics If you open a vector graphics file in an application like e g Adobe Illustrator you will be able to manipulate the image in great detail Graphics files can also be imported into the Navigation Area However no kinds of graphics files can be displayed in CLC Protein Workbench See section 7 2 for more about importing external files into CLC Protein Workbench 1 3 3 Graphics export parameters When you have specified the name and location to save the graphics file you can either click Next or Finish Clicking Next allows you to set further parameters for the graphics export whereas clicking Finish will export using the parameters that you have set last time you made a graphics export in that file format if it is the first time it will use default parameters Parameters for bitmap formats For bitmap files clicking Next will display the dialog shown in figure 7 12
242. ee 27 APR 1993 PERH3BA M15291 P maniculatus dee 27 APR 1993 AAA 2 A A AAA Figure 10 16 A sequence list containing multiple sequences can be viewed in either a table or in a graphical sequence list The graphical view is useful for viewing annotations and the sequence itself while the table view provides other information like sequence lengths and the number of sequences in the list number of Rows reported e Name Accession Description Modification date Length The number of sequences in the list is reported as the number of Rows at the top of the table view Learn more about tables in section C Adding and removing sequences from the list is easy adding is done by dragging the sequence from another list or from the Navigation Area and drop it in the table To delete sequences simply select them and press Delete 4 You can also create a subset of the sequence list select the relevant sequences right click Create New Sequence List This will create a new sequence list which only includes the selected sequences 10 7 3 Extract sequences It is possible to extract individual sequences from a sequence list in two ways If the sequence list is opened in the tabular view it is possible to drag with the mouse one or more sequences CHAPTER 10 VIEWING AND EDITING SEQUENCES 146 into the Navigation Area This allows you to extract specific sequences from the entire list Another option is to extract
243. een specified in the Hierarchical view below see section 13 3 2 e Atom type Colors the atoms individually Carbon Light grey Oxygen Red Hydrogen White Nitrogen Light blue Sulphur Yellow Chlorine Boron Green Phosphorus Iron Barium Orange Sodium Blue Magnesium Forest green Zn Cu Ni Br Brown Ca Mn Al Ti Cr Ag Dark grey CHAPTER 13 3D MOLECULE VIEWING 189 F Si Au Goldenrod lodine Purple Lithium firebrick Helium Pink Other Deep pink e Entities This will color protein subunits and additional structures individually Using the view table the user may select which colors are used to color subunits e Rainbow This color mode will color the structure with rainbow colors along the sequence e Residue hydrophobicity Colors the residues according to hydrophobicity In the Settings group you can specify the background color to use Default is black 13 3 2 Hierarchical view changing how selections of the structure are displayed In the bottom of the Side Panel you see the hierarchical view of the 3D structure see an example in figure 13 3 IL PTO ES fa Fr e Default colors k Settings Hierarchical view Settings Clear Settings El a Molecule a A Subunit protein 4 E Subunit protein 4 C Subunit protein 4 D Subunit protein 4 E Subunit protein At F Subunit protein 4 G Subunit protein At H Subunit protein feds HOH Subunit wate
244. efault settings for the view 2 4 Tutorial GenBank search and download The CLC Protein Workbench allows you to search the NCBI GenBank database directly from the program giving you the opportunity to both open view analyze and save the search results without using any other applications To conduct a search in NCBI GenBank from CLC Protein Workbench you must be connected to the Internet This tutorial shows how to find a complete human hemoglobin DNA sequence in a situation where you do not know the accession number of the sequence To start the search Search Search for Sequences at NCBI i This opens the search view We are searching for a DNA sequence hence Nucleotide Now we are going to adjust parameters for the search By clicking Add search parameters you activate an additional set of fields where you can enter search criteria Each search criterion consists of a drop down menu and a text field In the drop down menu you choose which part of the NCBI database to search and in the text field you enter what to search for CHAPTER 2 TUTORIALS 44 Click Add search parameters until three search criteria are available choose Organism in the first drop down menu write human in the adjoining text field choose All Fields in the second drop down menu write hemoglobin in the adjoining text field choose All Fields in the third drop down menu write complete in the adjoining text field NCBI search O
245. egends e Tick type Determine whether tick lines should be shown outside or inside the frame Outside Inside e Tick lines at Choosing Major ticks will show a grid behind the graph None Major ticks e Horizontal axis range Sets the range of the horizontal axis x axis Enter a value in Min and Max and press Enter This will update the view If you wait a few seconds without pressing Enter the view will also be updated e Vertical axis range Sets the range of the vertical axis y axis Enter a value in Min and Max and press Enter This will update the view If you wait a few seconds without pressing Enter the view will also be updated e X axis at zero This will draw the x axis at y O Note that the axis range will not be changed e Y axis at zero This will draw the y axis at x O Note that the axis range will not be changed e Show as histogram For some data series it is possible to see the graph as a histogram rather than a line plot 316 APPENDIX B GRAPH PREFERENCES 317 The Lines and plots below contains the following settings e Dot type None Cross Plus Square Diamond Circle Triangle Reverse triangle Dot Dot color Allows you to choose between many different colors Click the color box to select a color Line width Thin Medium Wide e Line type None Line Long dash Short dash e Line color Allows you to choose betwe
246. eins Eisenberg scale The Eisenberg scale is a normalized consensus hydrophobicity scale which CHAPTER 16 PROTEIN ANALYSES 248 shares many features with the other hydrophobicity scales Eisenberg et al 1984 Hopp Woods scale Hopp and Woods developed their hydrophobicity scale for identification of potentially antigenic sites in proteins This scale is basically a hydrophilic index where apolar residues have been assigned negative values Antigenic sites are likely to be predicted when using a window size of 7 Hopp and Woods 1983 Cornette scale Cornette et al computed an optimal hydrophobicity scale based on 28 published scales Cornette et al 1987 This optimized scale is also suitable for prediction of alpha helices in proteins Rose scale The hydrophobicity scale by Rose et al is correlated to the average area of buried amino acids in globular proteins Rose et al 1985 This results in a scale which is not showing the helices of a protein but rather the surface accessibility Janin scale This scale also provides information about the accessible and buried amino acid residues of globular proteins Janin 1979 Welling scale Welling et al used information on the relative occurrence of amino acids in antigenic regions to make a scale which is useful for prediction of antigenic regions This method is better than the Hopp Woods scale of hydrophobicity which is also used to identify antigenic regions Kolaskar Tongao
247. electing elements means that you select more than one element at the same time This can be done in the following ways e Holding down the lt Ctrl gt key 38 on Mac while clicking on multiple elements selects the elements that have been clicked e Selecting one element and selecting another element while holding down the lt Shift gt key selects all the elements listed between the two locations the two end locations included e Selecting one element and moving the curser with the arrow keys while holding down the lt Shift gt key enables you to increase the number of elements selected 3 1 5 Moving and copying elements Elements can be moved and copied in several ways Using Copy 1 Cut and Paste S from the Edit menu Using Ctrl C 38 C on Mac Ctrl X 48 X on Mac and Ctrl V 48 V on Mac Using Copy 5 Cut 2 and Paste in the Toolbar Using drag and drop to move elements CHAPTER 3 USER INTERFACE 66 e Using drag and drop while pressing Ctrl Command to copy elements In the following all of these possibilities for moving and copying elements are described in further detail Copy cut and paste functions Copies of elements and folders can be made with the copy paste function which can be applied in a number of ways select the files to copy right click one of the selected files Copy 55 right click the location to insert files into Paste or select the files
248. en many different colors Click the color box to select a color For graphs with multiple data series you can select which curve the dot and line preferences Should apply to This setting is at the top of the Side Panel group Note that the graph title and the axes titles can be edited simply by clicking with the mouse These changes will be saved when you Save the graph whereas the changes in the Side Panel need to be saved explicitly see section 5 5 For more information about the graph view please see section B Appendix C Working with tables Tables are used in a lot of places in the CLC Protein Workbench The contents of the tables are of course different depending on the context but there are some general features for all tables that will be explained in the following Figure C 1 shows an example of a typical table This is the table result of Find Open Reading Frames xx We will use this table as an example in the following to illustrate the concepts that are relevant for all kinds of tables Find reading Rows 169 Find reading Frame output Filter Do a n ia Settings mid Column width Found at strand Start codon positive ACT negative MEIN Show column negative TT positive Tac aah positive ACC End negative TAT Length negative AT E E CAC Found at strand positive AGG Start codon positive Baia eo postive TTG negative AG Deselect All negative ETE positive AG negative GT Fi
249. en started in safe mode some of the functionalities are missing and you will have to restart the CLC Protein Workbench again without pressing Shift 1 5 3 CLC Sequence Viewer vs Workbenches The advanced analyses of the commercial workbenches CLC Protein Workbench CLC RNA Workbench and CLC DNA Workbench are not present in CLC Sequence Viewer Likewise some advanced analyses are available in CLC DNA Workbench but not in CLC RNA Workbench or CLC Protein Workbench and vice versa All types of basic and advanced analyses are available in CLC Main Workbench However the output of the commercial workbenches can be viewed in all other workbenches This allows you to share the result of your advanced analyses from e g CLC Main Workbench with people working with e g CLC Sequence Viewer They will be able to view the results of your analyses but not redo the analyses The CLC Workbenches and the CLC Sequence Viewer are developed for Windows Mac and Linux platforms Data can be exported imported between the different platforms in the same easy way as when exporting importing between two computers with e g Windows 1 6 When the program is installed Getting started CLC Protein Workbench includes an extensive Help function which can be found in the Help menu of the program s Menu bar The Help can also be shown by pressing F1 The help topics are sorted in a table of contents and the topics can be searched We also recommend our Online pre
250. ening in order to have a steady production of proteins needed for the survival of the cell In bioinformatics analysis of proteins it is sometimes useful to know the ancestral DNA sequence in order to find the genomic localization of the gene Thus the translation of proteins back to DNA RNA is of particular interest and is called reverse translation or back translation The Genetic Code In 1968 the Nobel Prize in Medicine was awarded to Robert W Holley Har Gobind Khorana and Marshall W Nirenberg for their interpretation of the Genetic Code http nobelprize org medicine laureates 1968 The Genetic Code represents translations of all 64 different codons into 20 different amino acids Therefore it is no problem to translate a DNA RNA sequence into a specific protein But due to the degeneracy of the genetic code several codons may code for only one specific amino acid This can be seen in the table below After the discovery of the genetic code it has been concluded that different organism and organelles have genetic codes which are different from the standard genetic code Moreover the amino acid alphabet is no longer limited to 20 amino acids The 21 st amino acid selenocysteine is encoded by an UGA codon which is normally a stop codon The discrimination of a selenocysteine over a stop codon is carried out by the translation machinery Selenocysteines are very rare amino acids The table below shows the Standard Genetic Code w
251. ents window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements You can perform the analysis on several protein sequences at a time This will add annotations to all the sequences and open a view for each sequence Click Next to adjust parameters see figure 16 16 16 6 1 Pfam search parameters e Choose database and search type When searching for Pfam domains it is possible to choose different databases and specify the search for full domains or fragments of domains Only the 100 most frequent domains are included as default in CLC Protein Workbench Additional databases can be downloaded directly from CLC bio s web site at http www clcbio com resources Search full domains and fragments This option allows you to search both for full domain but also for partial domains This could be the case if a domain extends beyond the ends of a sequence Search full domains only Selecting this option only allows searches for full domains CHAPTER 16 PROTEIN ANALYSES 251 Search fragments only Only partial domains will be found Database Only the 100 most frequent domains are included as default in CLC Protein Workbench but additional databases can be downloaded and installed as described in section 16 6 2 e Set significance cutoff The E value expectation value is the number of hits that would be expected to have a score equal to or better than this value by chance alone
252. epresented directly in a graphical view The top sequence is the query sequence and is shown with a selection of annotations 12 5 8 What you cannot get out of BLAST Don t expect BLAST to produce the best available alignment BLAST is a heuristic method which does not guarantee the best results and therefore you cannot rely on BLAST if you wish to find all the hits in the database Instead use the Smith Waterman algorithm for obtaining the best possible local alignments Smith and Waterman 1981 BLAST only makes local alignments This means that a great but short hit in another sequence may not at all be related to the query sequence even though the sequences align well in a small region It may be a domain or similar It is always a good idea to be cautious of the material in the database For instance the sequences may be wrongly annotated hypothetical proteins are often simple translations of a found ORF on a sequenced nucleotide sequence and may not represent a true protein Don t expect to see the best result using the default settings As described above the settings should be adjusted according to the what kind of query sequence is used and what kind of results you want It is a good idea to perform the same BLAST search with different settings to get an idea of how they work There is not a final answer on how to adjust the settings for your particular sequence 12 5 9 Other useful resources The BLAST web page hosted at
253. equence statistics is less extensive than that of the protein sequence statistics Note The headings of the tables change depending on whether you calculate individual or comparative sequence statistics The output of comparative protein sequence statistics include e Sequence information Sequence type Length Organism CHAPTER 14 GENERAL SEQUENCE ANALYSES 210 Name Description Modification Date Weight This is calculated like this swimunitsinsequence wetght unit links x weight H20 where links is the sequence length minus one and units are amino acids The atomic composition is defined the same way Isoelectric point Aliphatic index e Half life e Extinction coefficient e Counts of Atoms e Frequency of Atoms e Count of hydrophobic and hydrophilic residues e Frequencies of hydrophobic and hydrophilic residues e Count of charged residues e Frequencies of charged residues e Amino acid distribution e Histogram of amino acid distribution e Annotation table e Counts of di peptides e Frequency of di peptides The output of nucleotide sequence statistics include e General statistics Sequence type Length Organism Name Description Modification Date Weight This is calculated like this swimunitsinsequence wetght unit links x weight H20 where links is the sequence length minus one for linear sequences and sequence length fo
254. exceptions See section 7 1 1 To export a file select the element to export Export E choose where to export to select File of type enter name of file Save When exporting to CSV and tab delimited files decimal numbers are formatted according to the Locale setting of the Workbench see section 5 1 If you open the CSV or tab delimited file with spreadsheet software like Excel you should make sure that both the Workbench and the spreadsheet software are using the same Locale Note The Export dialog decides which types of files you are allowed to export into depending on what type of data you want to export E g protein sequences can be exported into GenBank Fasta Swiss Prot and CLC formats Export of folders and multiple elements The zip file type can be used to export all kinds of files and is therefore especially useful in these situations e Export of one or more folders including all underlying elements and folders e f you want to export two or more elements into one file Export of folders is similar to export of single files Exporting multiple files of different formats is done in zip format This is how you export a folder select the folder to export Export E choose where to export to enter name Save CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 108 You can export multiple files of the same type into formats other than ZIP zip E g two DNA sequences can be exported in GenBank format
255. ext if you wish to adjust how to handle the results see section 9 1 If not click Finish 12 1 2 BLAST a partial sequence against NCBI You can search a database using only a part of a sequence directly from the sequence view select the sequence region to send to BLAST right click the selection BLAST Selection Against NCBI This will go directly to the dialog shown in figure 12 3 and the rest of the options are the same as when performing a BLAST search with a full sequence 12 1 3 BLAST against local data Running BLAST searches on your local machine can have several advantages over running the searches remotely at the NCBI e It can be faster e It does not rely on having a stable internet connection e It does not depend on the availability of the NCBI BLAST blast servers e You can use longer query sequences e You use your own data sets to search against On a technical level the CLC Protein Workbench uses the NCBI s blast software see ftp ftp ncbi nlm nih gov blast executables blast LATEST Thus the results of using a particular data set to search the same database with the same search parameters would give the same results whether run locally or at the NCBI There are a number of options for what you can search against e You create a database based on data already imported into your Workbench see sec tion 12 3 3 e You can add pre formatted databases see section 12 3 1 e You can use sequence data fro
256. f quickly editing annotations which is particularly useful when you wish to edit several annotations To edit the information simply double click and you will be able to edit e g the name or the annotation type If you wish to edit the qualifiers and double click in this column you will see the dialog for editing annotations Advanced editing of annotations Sometimes you end up with annotations which do not have a meaningful name In that case there is an advanced batch rename functionality Open the Annotation Table select the annotations that you want to rename right click the selection Advanced Rename This will bring up the dialog shown in figure 10 11 El Rename o Use this qualifier if exists organism Lise annotation type as name Xena Figure 10 11 The Advanced Rename dialog In this dialog you have two options CHAPTER 10 VIEWING AND EDITING SEQUENCES 140 e Use this qualifier Use one of the qualifiers as name A list of all qualifiers of all the selected annotations is shown Note that if one of the annotations do not have the qualifier you have chosen it will not be renamed If an annotation has multiple qualifiers of the same type the first is used for naming e Use annotation type as name The annotation s type will be used as name e g if you have an annotation of type Promoter it will get Promoter as its name by using this option A similar functionality is available for batch re typ
257. f the element is dragged from the Navigation Area and dropped next to the tab s in that View Area e Drag from the View Area to the Navigation Area The element e g a sequence alignment search report etc is saved where it is dropped If the element already exists you are asked whether you want to save a copy You drag from the View Area by dragging the tab of the desired element CHAPTER 3 USER INTERFACE 67 Use of drag and drop is supported throughout the program also to open and re arrange views see section 3 2 6 Note that if you move data between locations the original data is kept This means that you are essentially doing a copy instead of a move operation Copy using drag and drop To copy instead of move using drag and drop hold the Ctrl on Mac key while dragging click the element click on the element again and hold left mouse button drag the element to the desired location press Ctrl 38 on Mac while you let go of mouse button release the Ctrl 3 button 3 1 6 Change element names This section describes two ways of changing the names of sequences in the Navigation Area In the first part the sequences themselves are not changed it s their representation that changes The second part describes how to change the name of the element Change how sequences are displayed Sequence elements can be displayed in the Navigation Area with different types of information e Name this is the default information
258. f window on sequence In Step 7 you can adjust parameters for BLAST search e Program Lets you choose between different BLAST programs e Database Lets you limit your search to a particular database 16 8 1 Protein report output An example of Protein report can be seen in figure 16 20 By double clicking a graph in the output this graph is shown in a different view CLC Protein Workbench generates another tab The report output and the new graph views can be saved by dragging the tab into the Navigation Area The content of the tables in the report can be copy pasted out of the program and e g into Microsoft Excel To do so Select content of table Right click the selection Copy You can also Export ES the report in Excel format CHAPTER 16 PROTEIN ANALYSES 255 m C 4024102 report E 1 Protein statistics ME 1 1 Sequence information Mus muscu house mouse CARZH102 Modification Date 18 4P R 2005 1 2 Half life M tenninal aa Hal life mammals Halife yeast Half lif E Coli 1 3 Extinction coefficient Conditions Extinction coefficient at Absorption at 280nm 0 1 II gt Figure 16 20 A protein report There is a Table of Contents in the Side Panel that makes it easy to browse the report 16 9 Reverse translation from protein into DNA A protein sequence can be back translated into DNA using CLC Protein Workbench Due to degeneracy of the genetic code every amino acid could translate
259. fast option is particularly useful for data sets with very long sequences e Slow very accurate This is the recommended choice unless you find the processing time too long CHAPTER 18 SEQUENCE ALIGNMENT 285 40 20 P49342 MNP TETRA BP US QQMES PH PNEKKHKEKOA UK TE EKKSQ STKES UMHEK P20810 E PNKKKHKEKOA WETEPERESO STRESMMHER P27321 MSTTCABA MEMBESEBK so ssErrPmEHER PO8855 1MNPABABAMP MsKEMBECPH P HSKKRHAROM AKTEPER SO sTKPPMEHER P12675 MNPTETKA BM MsKOBECPHS PNEKEHKEKOA METEPENKSO STEPSENHER P20811 loro 002200000 METEPEKKPO ssKPSEMNHER Q95208 MNPTBAKA MP CSMOBBCPHS PNEKRHEKOA WMETEPERESsO sTEPSENHER 20 40 P49342 MNP TETRA BP MS QQMBES P HI PNEKKHKKOA WRTEPERRSQ STRES UMHEK P20810 MNPTETKA MP MsooMEcPHE PNEREHEROL P27321 1MBMBcCABAB PO8855 1MNPABABAMP MsKBvBcrHr HSEREHEROS P12675 MNPTETKA Mr MSKOBECPHS PNEERHEROL P20811 1 MPAA Q95208 MNPTBAKAMr csKQMEcPHS PNEKRHKEKOA MATH ENKSO sTEPSENHER Figure 18 3 The first 50 positions of two different alignments of seven calpastatin sequences The top alignment is made with cheap end gaps while the bottom alignment is made with end gaps having the same price as any other gaps In this case it seems that the latter scoring scheme gives the best result STKES WHER SS HNHER STH P MBHER NM_173881_CD5 1 NM_000559 Fs NM_1 3881_CD5 1 NM 000559 1 Figure 18 4 The alignment of
260. figure 11 4 amp NCBI structur Database Structure 155 al Fields insulin E Fields v human Add search parameters Append wildcard to search words 8 Start search Rows 50 Search results Filter Accession Description Resolution C Terminal Domain O Enhancing The Activi 1 Method Nmr 20 Structures X Ray Diffraction Protein Chains Release Date 2004 11 9 2005 2 15 Nmr Structure OF Hu Mmr 15 Structures 2004 6 22 Solution Structure O Nmr 30 Structures 2005 8 30 Nmr Structure OF Hu Nmr 15 Structures 2004 8 10 Nmr Structure OF Hu Nmr 15 Structures 2004 8 10 Nmr Structure OF Hu Nmr 15 Structures 2004 8 10 2004 12 28 2005 3 3 2005 2 3 2005 6 7 2005 3 1 X Ray Diffraction X Ray Diffraction X Ray Diffraction X Ray Diffraction X Ray Diffraction X Ray Diffraction Crystal Structure OF 1 Crystallographic And lt Structural Properties 2 Crystal Structure OF Lo 2 Structural Basis For 1 Diabetes Associated 2 mm I H Download and Open Y Download and Save Efe TRIN MTR TM MMM Nh mim a Total number of hits 166 Figure 11 4 The structure search view 11 3 1 Structure search options Conducting a search in the NCBI Database from CLC Protein Workbench corresponds to conducting search for structures on th
261. g the BLAST algorithm is described in more detail Seeding When finding a match between a query sequence and a hit sequence the starting point is the words that the two sequences have in common A word is simply defined as a number of letters CHAPTER 12 BLAST SEARCH 1 8 For blastp the default word size is 3 W 3 If a query sequence has a QWRTG the searched words are QWR WRT RIG See figure 12 15 for an illustration of words in a protein sequence Query word W 3 GSVEDTTGSQSLAALLNKCKTPOGORLVNOWIKQPLMDKNRIEERLNLVEAFVEDAELROTILQEDL Figure 12 15 Generation of exact BLAST words with a word size of W 3 During the initial BLAST seeding the algorithm finds all common words between the query sequence and the hit sequence s Only regions with a word hit will be used to build on an alignment BLAST will start out by making words for the entire query sequence see figure 12 15 For each word in the query sequence a compilation of neighborhood words which exceed the threshold of T is also generated A neighborhood word is a word obtaining a score of at least T when comparing using a selected scoring matrix see figure 12 16 The default scoring matrix for blastp is BLOSUM62 for explanation of scoring matrices see www clcbio com be The compilation of exact words and neighborhood words is then used to match against the database sequences Query word W 3 GSVEDTTGSOSLAALINKCKTPOGORLVNOWIKOPLMDKNRIEERLNLVEAFVEDAEL
262. g at the second residue Every 3 residues frame 3 There is a space every 3 residues corresponding to the reading frame starting at the third residue e Wrap sequences Shows the sequence on more than one line No wrap The sequence is displayed on one line Auto wrap Wraps the sequence to fit the width of the view not matter if it is Zoomed in our out displays minimum 10 nucleotides on each line Fixed wrap Makes it possible to specify when the sequence should be wrapped In the text field below you can choose the number of residues to display on each line e Double stranded Shows both strands of a sequence only applies to DNA sequences e Numbers on sequences Shows residue positions along the sequence The starting point can be changed by setting the number in the field below If you set it to e g 101 the first residue will have the position of 100 This can also be done by right clicking an annotation and choosing Set Numbers Relative to This Annotation e Numbers on plus strand Whether to set the numbers relative to the positive or the negative strand in a nucleotide sequence only applies to DNA sequences e Follow selection When viewing the same sequence in two separate views Follow selection will automatically scroll the view in order to follow a selection made in the other view e Lock numbers When you scroll vertically the position numbers remain visible Only possible when the sequence i
263. g the sequence with the enzymes Click the Fragments button at the bottom of the view In a similar way the fragments can be shown on a virtual gel Click the Gel button E at the bottom of the view Part Il Core Functionalities 60 Chapter 3 User interface Contents Sb Wavigauon Aled scales aa 62 Suit DGM sumada EE ee ee A e a 63 3 1 2 Create new folders 65 3 1 3 Sorting folders iw ss e E Boke we ew S amp S 65 3 1 4 Multiselecting elements lt lt ee ee 65 3 1 5 Moving and copying elementS ee eee 65 3 1 6 Change elementnames 67 3 1 7 Delete elements ee ee 68 3 1 8 Show folder elements in a table ee eee 68 3 2 View rea a o 69 SL DNC cierras EE A A GX 70 3 2 2 Show element in another view 0 11 3 2 3 CloseviewS 2 ee ee ee 4 11 3 2 4 Save changes in a view eoa ee 2 0 12 3 2 0 MO MEDO EE 12 3 2 6 Arrange views in View rea 13 3 A ME Panel aaa eh ee ee ORE Ea eh ae ed E 15 3 3 Zoom and selection in View Area lt 76 al ON lt lt redada asar sa 16 Soe POW cb bene ede Ge OSS Sw AA DGS a Soe E O 16 Su PM sides een he Owe Bho ek E 17 Pe LOMO E aia aaa 17 Sea Mis aus dias
264. gainst database sequences Mask lower case If you have a sequence with regions denoted in lower case and other regions in upper case then choosing this option would keep any of the regions in lower case from being considered in your BLAST search e Expect The threshold for reporting matches against database sequences the default value is 10 meaning that under the circumstances of this search 10 matches are expected to be found merely by chance according to the stochastic model of Karlin and Altschul 1990 Details of how E values are calculated can be found at the NCBI http www ncbi nlm nih gov BLAST tutorial Altschul 1 html If the E value ascribed to a match is greater than the EXPECT threshold the match will not be reported Lower EXPECT thresholds are more stringent leading to fewer chance matches being reported Increasing the threshold results in more matches being reported but many may just matching by chance not due to any biological similarity Values of E less than one can be entered as decimals or in scientific notiation For example 0 001 1e 3 and 10e 4 would be equivalent and acceptable values e Word Size BLAST is a heuristic that works by finding word matches between the query and database sequences You may think of this process as finding hot spots that BLAST can then use to initiate extensions that might lead to full blown alignments For nucleotide nucleotide searches i e BLASTn an exact match of the en
265. ge 180 12 5 6 Explanation of the BLAST output 2 44 2 0 lt 4 oe ed eee weed eo ADE 181 12 5 7 I want to BLAST against my own sequence database is this possible 183 12 5 8 What you cannot get out of BLAST 24 184 12 5 9 Other useful resources a eee 184 CLC Protein Workbench offers to conduct BLAST searches on protein and DNA sequences In short a BLAST search identifies homologous sequences between your input query query sequence and a database of sequences McGinnis and Madden 2004 BLAST Basic Local 160 CHAPTER 12 BLAST SEARCH 161 Alignment Search Tool identifies homologous sequences using a heuristic method which finds short matches between two sequences After initial match BLAST attempts to start local alignments from these initial matches If you are interested in the bioinformatics behind BLAST there is an easy to read explanation of this in section 12 5 With CLC Protein Workbench there are two ways of performing BLAST searches You can either have the BLAST process run on NCBI s BLAST servers http www ncbi nlm nih gov or perform the BLAST search on your own computer The advantage of running the BLAST search on NCBI servers is that you have readily access to the most popular BLAST databases without having to download them to your own computer The advantage of running BLAST on your own computer is that you can use your own Sequence data and that this can s
266. gure C 1 A table showing open reading frames First of all the columns of the table are listed in the Side Panel to the right of the table By clicking the checkboxes you can hide show the columns in the table Furthermore you can sort the table by clicking on the column headers Pressing Ctrl on Mac while you click will refine the existing sorting 318 APPENDIX C WORKING WITH TABLES 319 C 1 Filtering tables The final concept to introduce is Filtering The table filter as an advanced and a simple mode The simple mode is the default and is applied simply by typing text or numbers see an example in figure C 2 ES Find reading Rows 91 169 Find reading Frame output Filter Length Found at strand Start codon 14 306 57a negative PIIN AM 405 B00 396 negative TT 1378 1 52 375 negative TAT E 1995 2403 2309 alz negative AAT la dae A 2 mo Figure C 2 Typing neg in the filter in simple mode Typing neg in the filter will only show the rows where neg is part of the text in any of the columns also the ones that are not shown The text does not have to be in the beginning thus ega would give the same result This simple filter works fine for fast textual and non complicated filtering and searching However if you wish to make use of numerical information or make more complex filters you can switch to the advanced mode by clicking the Advanced filter j button The advanced filter is
267. h option you chose above you will now see the dialog shown in figure 1 18 License Wizard ES BD CLC Protein Workbench License Agreement Please read and accept the license agreement below to begin using you license END USER LICENSE AGREEMENT FOR CLC BIO SOFTWARE a CLC Genomics Workbench 1 0 E 1 Recitals 1 1 This End User License Agreement EULA is a legal agreement between you either an individual person or a single legal entity who will be referred to in this EULA as You and CLC bio A S CVR no 28 30 50 87 for the software products that accompanies this EULA including any associated media printed materials and electronic documentation the Software Product I accept these terms If you experience any problems please contact The CLC Support Team Figure 1 18 Read the license agreement carefully Please read the License agreement carefully before clicking accept these terms and Finish 1 4 5 Configure license server connection If your organization has installed a license server you can use a floating license The license server has a set of licenses that can be used on all computers on the network If the server has e g 10 licenses it means that maximum 10 computers can use a license simultaneously When you have selected this option and click Next you will see the dialog shown in figure 1 19 This dialog lets you specify how to connect to the license server e Connect to a license
268. he Annotation Layout and the Annotation Types in the Side Panel Check the Region annotation type and you will see the regions as red annotations on the CHAPTER 2 TUTORIALS 42 sequences Next we will change the way the residues are colored Click the Alignment Info group and under Conservation check Background color This will use a gradient as background color for the residues You can adjust the coloring by dragging the small arrows above the color box 2 3 1 Saving the settings in the Side Panel Now the alignment should look similar to figure 2 7 Ez ATPase protei O 540 560 E ie Alignment info Transmembrane region Topological domain w Consensus 4 Show Limit Majority 7 No gaps Ambiguous symbol 094296 M KGLOBFwifl ysNLVBMBEF MTFELVRYIM AaL_ ss oi ar Conservation Foreground color V Background color Transmembrane region Topological domain 100 Height low v Bar plot X P57792 APMAA IYHFERALME nsyF ii ENSEy Msi Bi VKvLA siriiNoBiHi gt Gap Fraction Color different residues gt aya Figure 2 1 The alignment when all the above settings have been changed At this point if you just close the view the changes made to the Side Panel will not be saved This means that you would have to perform the changes again next time you open the alignment To save the changes to the Side Panel click the Save Restore Settings bu
269. he Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements Click Next to determine how the shuffling should be performed In this step shown in figure 14 2 For nucleotides the following parameters can be set r q Shuffle Sequence la 1 Select one or more sequences of same type 2 Set parameters Resampling methods Mononucleotide shuffling Mononucleotide sampling from zero order Markov chain Dinucleotide shuffling Dinucleotide sampling From first order Markov chain Number of sequences 10 CJS Cees are le Xena Figure 14 2 Parameters for shuffling e Mononucleotide shuffling Shuffle method generating a sequence of the exact same mononucleotide frequency e Dinucleotide shuffling Shuffle method generating a sequence of the exact same dinu cleotide frequency e Mononucleotide sampling from zero order Markov chain Resampling method generating a sequence of the same expected mononucleotide frequency CHAPTER 14 GENERAL SEQUENCE ANALYSES 197 e Dinucleotide sampling from first order Markov chain Resampling method generating a sequence of the same expected dinucleotide frequency For proteins the following parameters can be set e Single amino acid shuffling Shuffle method generating a sequence of the exact same amino acid frequency e Single amino acid sampling from zero order Markov chain Resampling method
270. he color red is used to indicate high scores of hydrophobicity A color slider CHAPTER 16 PROTEIN ANALYSES 247 allows you to amplify the scores thereby emphasizing areas with high or low blue levels of hydrophobicity The color settings mentioned are default settings By clicking the color bar just below the color slider you get the option of changing color settings Graphs along sequences When selecting graphs you choose to display the hydrophobicity scores underneath the sequence This can be done either by a line plot or bar plot or by coloring The latter option offers you the same possibilities of amplifying the scores as applies for coloring of letters The different ways to display the scores when choosing graphs are displayed in figure 16 14 Notice that you can choose the height of the graphs underneath the sequence 16 5 3 Bioinformatics explained Protein hydrophobicity Calculation of hydrophobicity is important to the identification of various protein features This can be membrane spanning regions antigenic sites exposed loops or buried residues Usually these calculations are shown as a plot along the protein sequence making it easy to identify the location of potential protein features 20 40 Q6H1U7 mvh I BBBSRA aitsiwgkva ie Bogealg Fl livypWts pffdhfc Figure 16 15 Plot of hydrophobicity along the amino acid sequence Hydrophobic regions on the sequence have higher numbers according to
271. he full sequence from NCBI and save it When you click the button there will be a save dialog letting you specify a folder to save the sequences If multiple sequences are selected they will all open if the same sequence is listed several times only one copy of the sequence is downloaded and opened e Open at NCBI Opens the corresponding sequence s at GenBank at NCBI Here is stored additional information regarding the selected sequence s The default Internet browser is used for this purpose e Open structure If the hit sequence contain structure information the sequence is opened in a text view or a 3D view 3D view in CLC Protein Workbench and CLC Main Workbench You can do a text based search in the information in the BLAST table by using the filter at the upper right part of the view In this way you can search for e g species or other information which is typically included in the Description field The table is integrated with the graphical view described in section 12 2 3 so that selecting a hit in the table will make a selection on the corresponding sequence in the graphical view 12 3 Local BLAST databases BLAST databases on your local system can be made available for searches via your CLC Protein Workbench section 12 3 1 To make adding databases even easier you can download pre formatted BLAST databases from the NCBI from within your CLC Protein Workbench section 12 3 2 You can also easily create your own local
272. he license agreement carefully 1 4 3 Import a license from a file lf you are provided a license file instead of a license ID you will be able to import the file using this option When you have clicked Next you will see the dialog shown in 1 12 License Wizard FEZ p CLC Protein Workbench Import a license from a file Please click the button below and locate the file containing your license No file selected Choose License File If you experience any problems please contact The CLC Support Team Proxy Settings Previous Next Quit Workbench Figure 1 12 Selecting a license file Click the Choose License File button and browse to find the license file provided by CLC bio When you have selected the file click Next Accepting the license agreement Regardless of which option you chose above you will now see the dialog shown in figure 1 13 Please read the License agreement carefully before clicking I accept these terms and Finish 1 4 4 Upgrade license If you already have used a previous version of CLC Protein Workbench and you are entitled to upgrading to the new CLC Protein Workbench 5 8 select this option to get a license upgrade When you click Next the workbench will search for a previous installation of CLC Protein Workbench It will then locate the old license CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 22 License Wizard EA p CLC Protein Workben
273. hes Note Dragging in a tree will change it You are therefore asked if you want to save this tree when the Tree View is closed You may select part of a Tree by clicking on the nodes that you want to select Right click a selected node opens a menu with the following options Set root above node defines the root of the tree to be just above the selected node Set root at this node defines the root of the tree to be at the selected node Toggle collapse collapses or expands the branches below the node Change label allows you to label or to change the existing label of a node Change branch label allows you to change the existing label of a branch You can also relocate leaves and branches in a tree or change the length It is possible to modify the text on the unit measurement at the bottom of the tree view by right clicking the text In this way you can specify a unit e g years Branch lengths are given in terms of expected numbers of substitutions per site Note To drag branches of a tree you must first click the node one time and then click the node again and this time hold the mouse button In order to change the representation CHAPTER 19 PHYLOGENETIC TREES 306 e Rearrange leaves and branches by Select a leaf or branch Move it up and down Hint The mouse turns into an arrow pointing up and down e Change the length of a branch by Select a leaf or branch Press Ctrl Move left and right Hint The m
274. hich is the default translation table CHAPTER 16 PROTEIN ANALYSES 258 TTT F Phe TCT S Ser TAT Y Tyr TGT C Cys TTC F Phe TCC S Ser TAC Y Tyr TGC C Cys TTA L Leu TCA S Ser TAA Ter TGA Ter TTG L Leu i TCG S Ser TAG Ter TGG W Trp CTT L Leu CCT P Pro CAT H His CGT R Arg CTC L Leu CCC P Pro CAC H His CGC R Arg CTA L Leu CCA P Pro CAA Q Gin CGA R Arg CTG L Leu i CCG P Pro CAG Q Gin CGG R Arg ATT I lle ACT T Thr AAT N Asn AGT S Ser ATC I lle ACC T Thr AAC N Asn AGC S Ser ATA I Ile ACA T Thr AAA K Lys AGA R Arg ATG M Met i ACG T Thr AAG K Lys AGG R Arg GTT V Val GCT A Ala GAT D Asp GGT G Gly GTC V Val GCC A Ala GAC D Asp GGC G Gly GTA V Val GCA A Ala GAA E Glu GGA G Gly GTG V Val GCG A Ala GAG E Glu GGG G Gly Challenge of reverse translation A particular protein follows from the translation of a DNA sequence whereas the reverse translation need not have a specific solution according to the Genetic Code The Genetic Code is degenerate which means that a particular amino acid can be translated into more than one codon Hence there are ambiguities of the reverse translation Solving the ambiguities of reverse translation In order to solve these ambiguities of reverse translation you can define how to prioritize the codon selection e g e Choose a codon randomly e Select the most frequent codon in a given organism e Randomize a codon but with respect to its frequency in the organism As an example we want to translate an alanine to
275. homologs DNA or protein sequences However despite their frequent use the development of multiple alignment algorithms remains one of the algorithmically most challenging areas in bioinformatical research Constructing a multiple alignment corresponds to developing a hypothesis of how a number of sequences have evolved through the processes of character substitution insertion and deletion The input to multiple alignment algorithms is a number of homologous sequences i e sequences that share a common ancestor and most often also share molecular function The generated alignment is a table see figure 18 16 where each row corresponds to an input sequence and each column corresponds to a position in the alignment An individual column in this table represents residues that have all diverged from a common ancestral residue Gaps in the table commonly represented by a represent positions where residues have been inserted or deleted and thus do not have ancestral counterparts in all sequences 18 6 1 Use of multiple alignments Once a multiple alignment is constructed it can form the basis for a number of analyses e The phylogenetic relationship of the sequences can be investigated by tree building methods based on the alignment e Annotation of functional domains which may only be known for a subset of the sequences can be transferred to aligned positions in other un annotated sequences e Conserved regions in the alignment can be found
276. how to use the program to align sequences The chapter also describes alignment algorithms in more general terms 282 CHAPTER 18 SEQUENCE ALIGNMENT 283 18 1 Create an alignment Alignments can be created from sequences sequence lists see section 10 7 existing align ments and from any combination of the three To create an alignment in CLC Protein Workbench select sequences to align Toolbox in the Menu Bar Alignments and Trees Create Alignment or select sequences to align right click any selected sequence Toolbox Alignments and Trees Create Alignment Ez This opens the dialog shown in figure 18 1 a E Create Alignment 88 1 Select sequences of same Select sequences of same type SS s C Cs SSSCSCSCSSS type Projects Selected Elements 6 CLC Data Ae 094296 Example Data Ae P39524 XX ATP8al genomi As P57792 XX ATPSal mRNA Ms Q29449 fht ATP8al As QONTIZ Cloning fie Q95X33 5 Primers Protein analyse Protein ortholog EZ ATPBal orth gt Le 222222 RNA secondary Sequencing dat 4 HI b Qy lt enter search term gt 4 Figure 18 1 Creating an alignment If you have selected some elements before choosing the Toolbox action they are now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences sequence lists or alignments from the selected element
277. ht click a sequence in Navigation Area Toolbox Nucleotide Analyses 4 Reverse Complement x This opens the dialog displayed in figure 15 3 a q Reverse Complement Sequence Es al 1 Select nucleotide Ms ias sequences Projects Selected Elements 1 3 CLC Data XX ATPSal mRNA Example Data Xc ATP8al genomic sec Cloning Cloning vector liti Enzyme lists Xc pcDNA3 atp8al xXx pcDNA4_TO Processed data i Cloning expe Primers Protein analyses Protein orthologs RNA secondary strui Sequencing data j 4 HE p Qy lt enter search term gt A gt Next X Cancel Figure 15 3 Creating a reverse complement sequence p am m If a sequence was selected before choosing the Toolbox action the sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish This will open a new view in the View Area displaying the reverse complement of the selected sequence The new sequence is not saved automatically To save the sequence drag it into the Navigation Area or press Ctrl S S on Mac to activate a save dialog CHAPTER 15 NUCLEOTIDE ANALYSES 228 15 4 Reverse sequence CLC Protein Workbench is able to create the reverse of a nucleotide sequence By doing that a new sequenc
278. ic trees External files are files or links which are stored in CLC Protein Workbench but are opened by other applications e g pdf files Microsoft Word files Open Office spreadsheet files or links to programs and web pages etc This chapter first deals with importing and exporting data in bioinformatic data formats and as external files Next comes an explanation of how to export graph data points to a file and how export graphics 7 1 Bioinformatic data formats The different bioinformatic data formats are imported in the same way therefore the following description of data import is an example which illustrates the general steps to be followed regardless of which format you are handling 102 CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 103 1 1 1 Import of bioinformatic data CLC Protein Workbench has support for a wide range of bioinformatic data such as sequences alignments etc See a full list of the data formats in section F 1 The CLC Protein Workbench offers a lot of possibilities to handle bioinformatic data Read the next sections to get information on how to import different file formats or to import data from a Vector NTI database Import using the import dialog To start the import using the import dialog click Import amp in the Toolbar This will show a dialog similar to figure 7 1 depending on which platform you use You can change which kind of file tyoes that should be shown by selecting a file format i
279. ides available are these e Wildcard search Appending an asterisk to the search term will find matches starting with the term E g searching for brca will find both brca1 and brca2 e Search related words If you don t know the exact spelling of a word you can append a question mark to the search term E g brac1 will find sequences with a brcal gene CHAPTER 4 SEARCHING YOUR DATA 86 e Include both terms AND If you write two search terms you can define if your results have to match both search terms by combining them with AND E g search for brca1 AND human will find sequences where both terms are present e Include either term OR If you write two search terms you can define that your results have to match either of the search terms by combining them with OR E g search for brcat OR brca2 will find sequences where either of the terms is present e Name search name Search only the name of element e Organism search organism For sequences you can specify the organism to search for This will look in the Latin name field which is seen in the Sequence Info view see section 10 4 e Length search length START TO END Search for sequences of a specific length E g search for sequences between 1000 and 2000 residues length 1000 TO 2000 If you do not use this special syntax you will automatically search for both name description organism etc and search terms will be combined as if you ha
280. ient color box can be dragged to highlight relevant levels of G C content The colors can be changed by clicking the box This will show a list of gradients to choose from Background color Sets a background color of the residues using a gradient in the same way as described above Graph The G C content level is displayed on a graph Learn how to export the data behind the graph in section 4 x Height Specifies the height of the graph x Type The graph can be displayed as Line plot Bar plot or as a Color bar Color box For Line and Bar plots the color of the plot can be set by clicking the color box For Colors the color box is replaced by a gradient color box as described under Foreground color Protein info These preferences only apply to proteins The first nine items are different hydrophobicity scales and are described in section 16 5 2 e Kyte Doolittle The Kyte Doolittle scale is widely used for detecting hydrophobic regions in proteins Regions with a positive value are hydrophobic This scale can be used for identifying both surface exposed regions as well as transmembrane regions depending on the window size used Short window sizes of 5 generally work well for predicting CHAPTER 10 VIEWING AND EDITING SEQUENCES 127 putative surface exposed regions Large window sizes of 19 21 are well suited for finding transmembrane domains if the values calculated are above 1 6 Kyte and Doolittle 1982 These values
281. iewer Viewer Viewer y Viewer y Protein E Protein Protein y Protein E y Protein y DNA RNA Main DNA RNA Main DNA RNA Main DNA RNA Main DNA RNA Main 314 Genomics E Genomics Ly Genomics E Genomics E Genomics E APPENDIX A COMPARISON OF WORKBENCHES Primer design Viewer Protein Advanced primer design tools Detailed primer and probe parameters Graphical display of primers Generation of primer design output Support for Standard PCR Support for Nested PCR Support for TaqMan PCR Support for Sequencing primers Alignment based primer design Alignment based TaqMan probedesign Match primer with sequence Ordering of primers Advanced analysis of primer properties Molecular cloning Viewer Protein Advanced molecular cloning Graphical display of in silico cloning Advanced sequence manipulation Virtual gel view Viewer Protein Fully integrated virtual 1D DNA gel simulator For a more detailed comparison we refer to http www clcbio com compare DNA RNA Main DNA RNA Main DNA RNA Main 315 Genomics E Genomics Genomics E Appendix B Graph preferences This section explains the view settings of graphs The Graph preferences at the top of the Side Panel includes the following settings e Lock axes This will always show the axes even though the plot is zoomed to a detailed level e Frame Shows a frame around the graph e Show legends Shows the data l
282. ilable for download a a Clicking a plug in will display additional information at the right side of the dialog This will also display a button Download and Install Click the plug in and press Download and Install A dialog displaying progress is now shown and the plug in is downloaded and installed If the plug in is not shown on the server and you have it on your computer e g if you have downloaded it from our web site you can install it by clicking the Install from File button at the bottom of the dialog This will open a dialog where you can browse for the plug in The plug in file should be a file of the type cpa When you close the dialog you will be asked whether you wish to restart the CLC Protein Workbench The plug in will not be ready for use before you have restarted 1 7 2 Uninstalling plug ins Plug ins are uninstalled using the plug in manager Help in the Menu Bar Plug ins and Resources or Plug ins 5 in the Toolbar This will open the dialog shown in figure 1 25 The installed plug ins are shown in this dialog To uninstall CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 32 Manage Plug ins and Resources Manage Plug ins Download Plug ins Manage Resources Download Resources Additional Alignments O CLC bio support clcbio com Version 1 02 Perform alignments with many different programs From within the workbench ClustalW Windows Mac Linux Muscle Windows Mac L
283. ill automatically display the corresponding translation Read more about selecting in section 10 1 3 1 to 1 Select one of the six reading frames All forward All reverse Shows either all forward or all reverse reading frames All Select all reading frames at once The translations will be displayed on top of each other x Table The translation table to use in the translation For more about translation tables see section 15 5 Only AUG start codons For most genetic codes a number of codons can be start codons Selecting this option only colors the AUG codons green Single letter codes Choose to represent the amino acids with a single letter instead of three letters e G C content Calculates the G C content of a part of the sequence and shows it as a gradient of colors or as a graph below the sequence Window length Determines the length of the part of the sequence to calculate A window length of 9 will calculate the G C content for the nucleotide in question plus the 4 nucleotides to the left and the 4 nucleotides to the right A narrow window will focus on small fluctuations in the G C content level whereas a wider window will show fluctuations between larger parts of the sequence Foreground color Colors the letter using a gradient where the left side color is used for low levels of G C content and the right side color is used for high levels of G C content The sliders just above the grad
284. ilter Expect value Standard BLAST 1000 These settings are shown in figure 2 19 fF EB Local BLAST 53 pm 1 Select sequences of same RSS UE type 2 Set program parameters 3 Set input parameters Choose parameters Low Complexity Choose filter Mask lower case Expect 1000 Word size 7 No of processors 2 Match Mismatch Match 1 Mismatch 3 7 Gap cost Open 5 Extension 2 Command line options IS Previous gt Next Enh MX Cancel Figure 2 19 Settings for searching for primer binding sites 2 6 3 Finding remote protein homologues If you look for short identical peptide sequences in a database the standard BLAST param eters will have to be reconfigured Using the parameters described below you are likely to be able to identify whether antigenic determinants will cross react to other proteins Low complexity filter Expect value Standard BLAST blastp BLSUM62 Remote homologues blastp 20000 PAM30 These settings are shown in figure 2 20 2 6 4 Further reading A valuable source of information about BLAST can be found athttp blast ncbi nlm nih gov Blast cgi CMD Web amp PAGE_TYPE BlastDocs amp DOC_TYPE ProgSelectionGuide Remember that BLAST is a heuristic method This means that certain assumptions are made to allow searches to be done in a reasonable amount of time Thus you cannot trust BLAST search results to be accurate Fo
285. in figure 2 18 sas RD ID 0_reverse E MC_000011 sel E gt 000011 selection AA oi RD IDJD reverse UI RD IDJO reverse IRD ID O reverse RD ID 0_reverse rm LPRA a lt 000011 sel amp 000011 selection GGCAGACTTCTCCTCAGGAGTCAGATGCACCATGGTGTC ROD_ID JO_reverse Ala Ser Lys Glu Glu Pro Thr Leu His Val Met RD OJO reverse RD_ID O_ reverse RO OJO reverse RD IDO reverse Figure 2 18 Verification of the result at the top a view of the whole BLAST result At the bottom the same view is zoomed in on exon 3 to show the amino acids In theory you could use the chromosome sequence as query but the performance would not be optimal it would take a long time and the computer might run out of memory In this example you have used well annotated sequences where you could have searched for the name of the gene instead of using BLAST However there are other situations where you either do not know the name of the gene or the genomic sequence is poorly annotated In these cases the approach described in this tutorial can be very productive 2 6 2 BLAST for primer binding sites You can adjust the BLAST parameters so it becomes possible to match short primer sequences against a larger sequence Then it is easy to examine whether already existing lab primers can CHAPTER 2 TUTORIALS 91 be reused for other purposes or if the primers you designed are specific Low complexity f
286. ined can either be shown as annotations on the sequence in a table or as the detailed and text output from the TMHMM method e Add annotations to sequence e Create table e Text Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish You can perform the analysis on several protein sequences at a time This will add annotations to all the sequences and open a view for each sequence if a transmembrane helix is found If a transmembrane helix is not found a dialog box will be presented After running the prediction as described above the protein sequence will show predicted transmembrane helices as annotations on the original sequence see figure 16 8 Moreover annotations showing the topology will be shown That is which part the proteins is located on the inside or on the outside Each annotation will carry a tooltip note saying that the corresponding annotation is predicted with TMHMM version 2 0 Additional notes can be added through the Edit annotation 3 right click mouse menu See section 10 3 2 Undesired annotations can be removed through the Delete Annotation right click mouse menu See section 10 3 4 16 4 Antigenicity CLC Protein Workbench can help to identify antigenic regions in protein sequences in different ways using different algorithms The algorithms provided in the Workbench merely plot an index of antigenicity over the sequence Two different methods are availab
287. ing annotations is available in the right click menu as well in case your annotations are not typed correctly Open the Annotation Table 5 select the annotations that you want to retype right click the selection Advanced Retype This will bring up the dialog shown in figure 10 12 Y Retype Options 0 Use this qualifier if exists db_xref o New type O Use annotation name as type Figure 10 12 The Advanced Retype dialog In this dialog you have two options e Use this qualifier Use one of the qualifiers as type A list of all qualifiers of all the selected annotations is shown Note that if one of the annotations do not have the qualifier you have chosen it will not be retyped If an annotation has multiple qualifiers of the same type the first is used for the new type e New type You can select from a list of all the pre defined types as well as enter your own annotation type All the selected annotations will then get this type e Use annotation name as type The annotation s name will be used as type e g if you have an annotation named Promoter it will get Promoter as its type by using this option 10 3 4 Removing annotations Annotations can be hidden using the Annotation Types preferences in the Side Panel to the right of the view see section 10 3 1 In order to completely remove the annotation right click the annotation Delete Delete Annotation If you want to remove all annota
288. ing fixpoints sequence A will align to the first copy of the domain in sequence C while sequence B would align to the second copy of the domain in sequence C You can name fixpoints by right click the Fixpoint annotation Edit Annotation S type the name in the Name field 18 2 View alignments Since an alignment is a display of several sequences arranged in rows the basic options for viewing alignments are the same as for viewing sequences Therefore we refer to section 10 1 for an explanation of these basic options However there are a number of alignment specific view options in the Alignment info and the Nucleotide info in the Side Panel to the right of the view Below is more information on these view options Under Translation in the Nucleotide info there is an extra checkbox Relative to top sequence Checking this box will make the reading frames for the translation align with the top sequence so that you can compare the effect of nucleotide differences on the protein level The options in the Alignment info relate to each column in the alignment e Consensus Shows a consensus sequence at the bottom of the alignment The consensus sequence is based on every single position in the alignment and reflects an artificial sequence which resembles the sequence information of the alignment but only as one single sequence If all sequences of the alignment is 100 identical the consensus sequence will be identical to all seque
289. into one Note that wnen sequences are joined all their annotations are carried over to the new spliced sequence Two or more sequences can be joined by select sequences to join Toolbox in the Menu Bar General Sequence Analyses Join sequences 258 or select sequences to join right click any selected sequence Toolbox General Sequence Analyses Join sequences 3 58 This opens the dialog shown in figure 14 17 If you have selected some sequences before choosing the Toolbox action they are now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences from the selected elements Click Next opens the dialog shown in figure 14 18 In step 2 you can change the order in which the sequences will be joined Select a sequence and use the arrows to move the selected sequence up or down CHAPTER 14 GENERAL SEQUENCE ANALYSES 215 f g Join Sequences E 1 Select sequences of same e oF same ype type Projects Selected Elements 2 p CLC Data Ms 094296 EE Example Data ss P39524 XxX ATP8al genomit 24 ATPSal mRNA Sw ATPSal H E Cloning H Primers H E Protein analyse EE Protein ortholog Se on 84 P57792 oN Q29449 olf QONTIZ ofthe Q9SX33 RNA secondary H Sequencing dat 4 mW Qy zenter search term gt 4 amp Previous gt Next Finish Figure 14 17 Selecting two sequences to be joined f E Join Sequences Eg 1
290. into several different codons only 20 amino acids but 64 different codons Thus the program offers a number of choices for determining which codons should be used These choices are explained in this section In order to make a reverse translation Select a protein sequence Toolbox in the Menu Bar Protein Analyses la Reverse Translate or right click a protein sequence Toolbox Protein Analyses j Reverse translate 3 This opens the dialog displayed in figure 16 21 E EB Reverse Translate 88 z m 1 Select protein sequences P detect proce A Projects Selected Elements 1 J CLC Data e ATP8al Example Data su Cloning Primers Protein analyses ES Protein ortholog HS RNA secondary H Sequencing data 4 m Qy lt enter search term gt Eis e E Figure 16 21 Choosing a protein sequence for reverse translation CHAPTER 16 PROTEIN ANALYSES 256 If a sequence was selected before choosing the Toolbox action the sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements You can translate several protein sequences at a time Click Next to adjust the parameters for the translation 16 9 1 Reverse translation parameters Figure 16 22 shows the choices for making the translation fr G Reverse Translate ES pi 1 Select pro
291. inux T Coffee Mac Linux MAFFT Mac Linux Kalign Mac Linux Annotate with GFF file CLC bio support clcbio com Version 1 03 Using this plug in it is possible to annotate a sequence from list of annotations found in a GFF file Located in the Toolbox Extract Annotations O CLC bio supportiocicbio com version 1 02 Extracts annotations from one or more sequences The result is a sequence list containing sequences covered by the specified annotations Uninstall v Figure 1 25 The plug in manager with plug ins installed Click the plug in Uninstall If you do not wish to completely uninstall the plug in but you don t want it to be used next time you start the Workbench click the Disable button When you close the dialog you will be asked whether you wish to restart the workbench The plug in will not be uninstalled before the workbench is restarted 1 7 3 Updating plug ins If a new version of a plug in is available you will get a notification during start up as shown in figure 1 26 In this list select which plug ins you wish to update and click Install Updates If you press Cancel you will be able to install the plug ins later by clicking Check for Updates in the Plug in manager see figure 1 25 1 7 4 Resources Resources are downloaded installed un installed and updated the same way as plug ins Click the Download Resources tab at the top of the plug in manager and you will see
292. ion on the selected structure at NCBI s web page Double clicking a hit will download and open the structure The hits can also be copied into the View Area or the Navigation Area from the search results by drag and drop copy paste or by using the right click menu as described below CHAPTER 11 ONLINE DATABASE SEARCH 157 Drag and drop from structure search results The structures from the search results can be opened by dragging them into a position in the View Area Note A structure is not saved until the View displaying the structure is closed When that happens a dialog opens Save changes of structure x Yes or No The structure can also be saved by dragging it into the Navigation Area It is possible to select more structures and drag all of them into the Navigation Area at the same time Download structure search results using right click menu You may also select one or more structures from the list and download using the right click menu see figure 11 5 Choosing Download and Save lets you select a folder or location where the structures are saved when they are downloaded Choosing Download and Open opens a new view for each of the selected structures Definition p File Edit View Toolbox Show T F F F F 4 Download and Open o ral Download and Save Open at NCBI I Figure 11 5 By right clicking a search result it is possible to choose how to handle the relevant structure The selected stru
293. ions where you wish to use another license or see information about the license you currently use In this case open the license manager Help License Manager E The license manager is shown in figure 1 22 Besides letting you borrow licenses see section 1 4 5 this dialog can be used to e See information about the license e g what kind of license when it expires CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 21 License Manager p CLC Protein Workbench License overview Feature na License type Expires in Status Borrow limit cleproteinwb Network 192 168 1 200 50 days Valid 3 days License borrowing IF you use a license server and need to work outside of your organization network for an extended period of time your can borrow a copy of your licenses From the license server The borrowed license will allow you to use the application for the specified number of hours Borrow the selected licenses for a period of 11 hour o Configure Network License Upgrade License Figure 1 22 The license manager e Configure how to connect to a license server Configure License Server the button at the lower left corner Clicking this button will display a dialog similar to figure 1 19 e Upgrade from an evaluation license by clicking the Upgrade license button This will display the dialog shown in figure 1 1 If you wish to switch away from using a floating license click Configure License Server a
294. irect plas mamembrane translocation in prokaryotes or is routed through the Endoplasmatic Reticulum in eukaryotic cells The signal peptide is removed from the resulting mature protein during translo cation across the membrane For prediction of signal peptides we query SignalP Nielsen et al 1997 Bendtsen et al 2004b located at http www cbs dtu dk services SignalP Thus an active internet connection is required to run the signal peptide prediction Additional information on SignalP and Center for Biological Sequence analysis CBS can be found at http www cbs dtu dk and in the original research papers Nielsen et al 1997 Bendtsen et al 2004b In order to predict potential signal peptides of proteins the D score from the SignalP output is used for discrimination of signal peptide versus non signal peptide see section 16 1 3 This score has been shown to be the most accurate Klee and Ellis 2005 in an evaluation study of signal peptide predictors In order to use SignalP you need to download the SignalP plug in using the plug in manager see section 1 7 1 When the plug in is downloaded and installed you can use it to predict signal peptides Select a protein sequence Toolbox in the Menu Bar Protein Analyses j Signal Peptide Prediction or right click a protein sequence Toolbox Protein Analyses 4 Signal Peptide Prediction 4s If a sequence was selected before choosing the Toolbox action this seq
295. is is done to take care of situations where the two closest nodes are not neighbors in the real tree The neighbor join algorithm is generally considered to be fairly good and is widely used Algorithms that improves its cubic time performance exist The improvement is only significant for quite large datasets Character based methods Whereas the distance based methods compress all sequence information into a single number the character based methods attempt to infer the phylogeny based on all the individual characters nucleotides or amino acids Parsimony In parsimony based methods a number of sites are defined which are informative about the topology of the tree Based on these the best topology is found by minimizing the number of substitutions needed to explain the informative sites Parsimony methods are not based on explicit evolutionary models Maximum Likelihood Maximum likelinood and Bayesian methods see below are probabilistic methods of inference Both have the pleasing properties of using explicit models of molecular evolution and allowing for rigorous statistical inference However both approaches are very computer intensive A stochastic model of molecular evolution is used to assign a probability likelinood to each phylogeny given the sequence data of the OTUs Maximum likelihood inference Felsenstein CHAPTER 19 PHYLOGENETIC TREES 309 1981 then consists of finding the tree which assign the highest probability to
296. ish Chapter 11 Online database search Contents 11 1 GenBank SET tac ve eae eke eee ewe hee ER 148 11 1 1 GenBank search options 0 00 ee eee a 149 11 1 2 Handling of GenBank search results 2 550208 150 11 1 3 Save GenBank search parameters 151 11 2 UniProt Swiss Prot TrEMBL search 00 08 2 eee een eee 152 11 2 1 UniProt search options a 2 eee ee ee ee 152 11 2 2 Handling of UniProt search results lt lt 153 11 2 3 Save UniProt search parameters 154 11 3 Search for structures at NCBI lt lt lt 154 11 3 1 Structure search options a a ee a 155 11 3 2 Handling of NCBI structure search results 156 11 3 3 Save structure search parameters 2 a a a a a 157 11 4 Sequence web info lt lt lt ee te 4 158 11 4 1 Google sequence 2 uo 158 ERE WED co aserrada tenet eee ea eee a ee E 158 11 4 3 PubMed References eee ee ee ee 159 ELA UMOL setae ene we ss ass 159 11 4 5 Additional annotation information eae 159 CLC Protein Workbench offers different ways of searching data on the Internet You must be online when initiating and performing the following searches 11 1 GenBank search This section describes searches for
297. it from this table in several ways e It provides an intelligible overview of all the annotations on the sequence e You can use the filter at the top to search the annotations Type e g UCP into the filter and you will find all annotations which have UCP in either the name the type the region or the qualifiers Combined with showing or hiding the annotation types in the Side Panel this makes it easy to find annotations or a subset of annotations e You can copy and paste annotations e g from one sequence to another e f you wish to edit many annotations consecutively the double click editing makes this very fast see section 10 3 2 10 3 2 Adding annotations Adding annotations to a sequence can be done in two ways open the sequence in a sequence view double click in the Navigation Area make a selection covering the part of the sequence you want to annotate right click the selection Add Annotation or select the sequence in the Navigation Area Show 2 Annotations E Add Annotation This will display a dialog like the one in figure 10 10 E gu Add annotation Annotation types Properties a Protein Features Name Test Protein Functional Features Protein Sequence Features DNA RNA Sequence Features Region 10 16 E gt Misc EMO sc ay Alignment fixpoint Type Misc Feature Annotation notes Add qualifier key da MX Can
298. kely generate more hits Below are some rules of thumb which can be used as a guide but should be considered with common sense e E value lt 10e 100 Identical sequences You will get long alignments across the entire query and hit sequence e 10e 100 lt E value lt 10e 50 Almost identical sequences A long stretch of the query protein is matched to the database e 10e 50 lt E value lt 10e 10 Closely related sequences could be a domain match or similar e 10e 10 lt E value lt 1 Could be a true homologue but it is a gray area e E value gt 1 Proteins are most likely not related e E value gt 10 Hits are most likely junk unless the query sequence is very short Gap costs For blastp it is possible to specify gap cost for the chosen substitution matrix There is only a limited number of options for these parameters The open gap cost is the price of introducing gaps in the alignment and extension gap cost is the price of every extension past the initial opening gap Increasing the gap costs will result in alignments with fewer gaps CHAPTER 12 BLAST SEARCH 181 Filters It is possible to set different filter options before running the BLAST search Low complexity regions have a very simple composition compared to the rest of the sequence and may result in problems during the BLAST search Wootton and Federhen 1993 A low complexity region of a protein can for example look like this fftfflllsss which in this case is a
299. king regions are shown In this example a consensus sequence would only display ATG as the start codon in position 1 but when looking at the sequence logo it is seen that a GTG is also allowed as a Start codon CHAPTER 18 SEQUENCE ALIGNMENT 291 20 1 20 l tlA CTTTTCAAGG AGTATTTCCT ATGAACGAGT TAGACGGCAT evgA CATTGCAAAG GGAATAATCT ATGAACGCAA TAATTATTGA ypdl CATTTTCAGG ATAACTTTCT ATGAAAGTAA ACTTAATACT nrB GAAAAGAAAT CGAGGCAAAA ATGAGCAAAG TCAGACTCGC hmpA TGCAAAAAAA GGAAGACCAT ATGCTTGACG CTCAAACCAT narQ TTTTTGTGGA GAAGACGCGT GTGATTGTTA AACGACCCGT gtf GTTATTAAGG ATATGTTCAT ATGTTTTTCA AAAAGAACCT intS TACCCACCGG ATTTTTACCC ATGCTCACCG TTAAGCAGAT yidF AATCAAAATG GAATAAAATC ATGCTACCAT CTATTTCAAT dsdX ATCACAGGGG AAGGTGAGAT ATGCACTCTC AAATCTGGGT sunB ACATCCAGTG AGAGAGACCG ATGCATCCGA TGCTGAACAT Consensus AATTTAAAGG AGAATTACCT ATGAACGCAA TAATAAACAT Sequence Logo 5 RABG faha xea ASt ea 8 58 x efl Conservation i i o oa eta coca Figure 18 8 Ungapped sequence alignment of eleven E coli sequences defining a start codon The start codons start at position 1 Below the alignment is shown the corresponding sequence logo As seen a GTG start codon and the usual ATG start codons are present in the alignment This can also be visualized in the logo at position 1 Calculation of sequence logos A comprehensive walk through of the calculation of the information content in sequence logos is beyond the scope of this document but can
300. l 2 5 1 Performing the BLAST search Start out by select protein ATP8a1 Toolbox BLAST Search NCBI BLAST 2 In Step 1 you can choose which sequence to use as query sequence Since you have already chosen the sequence it is displayed in the Selected Elements list Click Next In Step 2 figure 2 12 choose the default BLAST program blastp Protein sequence and database and select the Swiss Prot database in the Database drop down menu Ml nce BLAST 53 m 1 Select sequences of same set program parameters 2 Set program parameters Choose program and database Program blastp Protein sequence and database X Database Swiss Prot protein sequences swissprot v Genetic code 1 Standard Database genetic code 1 Standard EJA tros Sue Jos Xena Figure 2 12 Choosing BLAST program and database Click Next In the Limit by Entrez query in Step 3 choose Homo sapiens ORGN from the drop down menu to arrive at the search configuration seen in figure 2 13 Including this term limits the query to proteins of human origin Choose to Open your results Click Finish to accept the parameter settings and begin the BLAST search The computer now contacts NCBI and places your query in the BLAST search queue After a short while the result should be received and opened in a new view CHAPTER 2 TUTORIALS 46 E Ml nece BLAST 5 1 Select sequences of same Se input pa amete type
301. l peptides as vehicles for chimeric proteins for biomedical and pharmaceutical industry Many papers describe statistical or machine learning methods for prediction of signal peptides and prediction of subcellular localization in general After the first published method for signal peptide prediction von Heijne 1986 more and more methods have surfaced although not all methods have been made available publicly Different types of signal peptides Soon after Gunter Blobel s initial discovery of signal peptides more targeting signals were found Most cell types and organisms employ several ways of targeting proteins to the extracellular environment or subcellular locations Most of the proteins targeted for the extracellular space or subcellular locations carry specific sequence motifs signal peptides characterizing the type of secretion targeting it undergoes Several new different signal peptides or targeting signals have been found during the later years and papers often describe a small amino acid motif required for secretion of that particular protein In most of the latter cases the identified sequence motif is only found in this particular protein and as such cannot be described as a new group of signal peptides Describing the various types of signal peptides is beyond the scope of this text but several review papers on this topic can be found on PubMed Targeting motifs can either be removed from or retained in the mature protein af
302. lay bonds Coloring options Default color Color C Opacity 20 40 60 Figure 13 6 Customize appearance for bonds At the top you can choose to show bonds and below you can specify their appearance e Color Clicking the color box allows you to select a color e Opacity Determines the level of opacity Representations The Representations tab is shown in figure 13 7 At the top you can choose to between four display models e Secondary structure CHAPTER 13 3D MOLECULE VIEWING 192 Customize appearance Atoms Bonds Representations Models Display model Stick model Both None Coloring options Default color Color LJ Opacity Figure 13 7 Customize appearance for representations Stick model Both Displaying both secondary structure and stick model None Will not display representations Color Clicking the color box allows you to select a color Opacity Determines the level of opacity Models The Models tab is shown in figure 13 8 At the top you can choose to between three display Customize appearance Atoms Bonds Representations Models Display models All models None Coloring options Default color Color LJ Opacity Figure 13 8 Customize appearance for models modes This functionality is only applicable to NMR structures which have multiple resolved structures for X ray structures only one structure is av
303. layout Annotation layout Show annotations Position Next to sequence 7 Offset Little offset Label Stacked 7 Show arrows Use gradients Annotation types EM 7 cos Exon CD E 4 Gene DD Source osso Select All k Restriction sites Residue coloring k Nucleotide info k Find P Text Format Figure 10 7 Changing the layout of annotations in the Side Panel The two groups are shown in figure 10 7 In the Annotation layout group you can specify how the annotations should be displayed notice that there are some minor differences between the different sequence views e Show annotations Determines whether the annotations are shown e Position On sequence The annotations are placed on the sequence The residues are visible through the annotations if you have zoomed in to 100 CHAPTER 10 VIEWING AND EDITING SEQUENCES 135 Next to sequence The annotations are placed above the sequence Separate layer The annotations are placed above the sequence and above restriction sites only applicable for nucleotide sequences e Offset If several annotations cover the same part of a sequence they can be spread out Piled The annotations are piled on top of each other Only the one at front is visible Little offset The annotations are piled on top of each other but they have been offset a little More offset Same as above but with more spreading Most offset
304. le Welling et al 1985 Welling et al used information on the relative occurrence of amino acids in antigenic regions to make a scale which is useful for prediction of antigenic regions This method CHAPTER 16 PROTEIN ANALYSES 243 outside membrane membrane inside inside ATP Ba en arraal _ _ __ _ __ _ __ _ _ Inside outside outside membrane outside membrane membrana membrane membrane ATF Bal Figure 16 8 Transmembrane segments shown as annotation on the sequence and the topology is better than the Hopp Woods scale of hydrophobicity which is also used to identify antigenic regions semi empirical method for prediction of antigenic regions has been developed Kolaskar and Tongaonkar 1990 This method also includes information of surface accessibility and flexibility and at the time of publication the method was able to predict antigenic determinants with an accuracy of 5 Note Similar results from the two method can not always be expected as the two methods are based on different training sets 16 4 1 Plot of antigenicity Displaying the antigenicity for a protein sequence in a plot is done in the following way select a protein sequence in Navigation Area Toolbox in the Menu Bar Protein Analyses a Create Antigenicity Plot l This opens a dialog The first step allows you to add or remove sequences Clicking Next takes
305. lect enzymes Include Name Cyanogen bromide CNBr Asp N endopeptidase Chymotrypsin high spec Chymotrypsin low spec o Iodosobenzoate Thermolysin Post Pro Glu C Asp N Proteinase K Thrombin Factor Xa F Granzyme B A emite 1 Er A J 3 Select Al De selectAll ID e ae ve Figure 2 21 Selecting trypsin as the cleaving enzyme Click Next to go to Step 3 of the dialog CHAPTER 2 TUTORIALS 99 In Step 3 you can adjust the parameters for which fragments of the cleavage you want to include in the table output of the analysis Type 10 in the Min fragment length Check the box Max fragment length enter 15 in the corresponding text field These parameter adjustments are shown in figure 2 22 fr q Proteolytic Cleavage ES 1 Select protein sequences BE amet 2 Select enzymes 3 Set parameters Enzyme criteria Min number of cleavage sites 15 Max number of cleavage sites 1 Criteria For the table list of Fragments Min fragment length 10 Y Max fragment length 155 Min fragment mass Da oF Max Fragment mass Da 100 25 Ds DEE DES Figure 2 22 Adjusting the output from the cleavage to include fragments which are between 10 and 15 amino acids long Click Finish to make the analysis The re
306. lgorithm The lines in the BLAST view are the actual sequences which are downloaded This means that you Can zoom in and see the actual alignment Zoom in in the Tool Bar 550 Click in the BLAST view a number of times until you see the residues CHAPTER 2 TUTORIALS 41 Now we will focus our attention on sequence 09Y200 the BLAST hit that is at the top of the list To download the full sequence right click the line representing sequence Q9Y2Q0 Download Full Hit Sequence from NCBI This opens the sequence However the sequence is not saved yet Drag and drop the sequence into the Navigation Area to save it This homologous sequence is now stored in the CLC Protein Workbench and you can use it to gain information about the query sequence by using the various tools of the workbench e g by studying its textual information by studying its annotation or by aligning it to the query sequence 2 5 3 Using the BLAST table view As an alternative to the graphic BLAST view you can click the Table View H This will display a tabular view of the BLASt hits as shown in figure 2 15 at the bottom EE ATPBal BLAST Rows 54 Summary of hits from query ATPBal Filter All nal Description E value Probable phospholipid transporting ATPase IB ATPase class I type 8A me 0 00 4 058 00 Probable phospholipid transporting ATPase ID ATPase class I type 8B me 0 00 2 120 00 Probable phospholipid transporting ATPa
307. ll Figure 3 6 Viewing the elements in a folder Sorting the elements in a view does not affect the ordering of the elements in the Navigation Area Note The view only displays one layer at a time the content of subfolders is not visible in this view Also note that only sequences have the full span of information like organism etc Batch edit folder elements You can select a number of elements in the table right click and choose Edit to batch edit the elements In this way you can change the e g the description or common name of several elements in one go In figure 3 you can see an example where the common name of five sequence are renamed in one go In this example a dialog with a text field will be shown letting you enter a new common name for these five sequences Note This information is directly saved and you cannot undo 3 2 View Area The View Area is the right hand part of the screen displaying your current work The View Area may consist of one or more Views represented by tabs at the top of the View Area This is illustrated in figure 3 8 The tab concept is central to working with CLC Protein Workbench because several operations can be performed by dragging the tab of a view and extended right click menus can be activated from the tabs CHAPTER 3 USER INTERFACE Type Mame Modified Modifi Descri A MM13mp8 Tue Jun smoensted Mimp A M13mp9 Tue Jun smoensted Mi3mpa A Aa
308. llowing problems were encountered while trying to locate a valid license Click on each error for a more detailed description License Server 192 168 1 200 port 6200 a No license available at the moment All licenses obtainable from the server are currently in use If the problem persists please contact your local license server administrator Additional licenses can be purchased by contacting the CLC bio sales team on sales clcbio com To import a new license or change your license server settings please click the License Assistant button If you experience any problems please contact The CLC Support Team Figure 1 20 No more licenses available on the server In this case please contact your organization s license server administrator To purchase additional licenses contact sales clcbio com You can also click the Limited Mode button see section 1 4 6 lf your connection to the license server is lost you will see a dialog as shown in figure 1 21 License Server Error CLC Network Licensing Unable to locate a license server A license server could not be located on your network Ifthe problem persists please contact your local license server administrator Configure License Server Figure 1 21 Unable to contact license server In this case you need to make sure that you have access to the license server and that the server is running However there may be situat
309. lly downloaded The License is valid until 2008 08 01 If you experience any problems please contact The CLC Support Team Proxy Settings Previous Next Quit Workbench Figure 1 15 A license has been downloaded A progress for getting the license is shown and when the license is downloaded you will be able to click Next Go to license download web page Selecting the second option Go to license download web page opens the license web page as Shown in 1 16 Download a license Figure 1 16 The license web page where you can download a license Click the Request Evaluation License button and you will be able to save the license on your computer e g on the Desktop Back in the Workbench window you will now see the dialog shown in 1 17 Click the Choose License File button and browse to find the license file you saved before e g on your Desktop When you have selected the file click Next CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 24 License Wizard EJ p CLC Protein Workbench Import a license from a file Please click the button below and locate the file containing your license No file selected Choose License File If you experience any problems please contact The CLC Support Team Proxy Settings Previous Next Quit Workbench Figure 1 17 Importing the license downloaded from the web site Accepting the license agreement Regardless of whic
310. lumn CHAPTER 3 USER INTERFACE gt Cloning vecto E Rows BO 69 a a a ha 45 Column width Mame Modified Modifie Description Length Linear ma M13mp8 pLiCS Tue Jun 30 smoensted Mismp pUce 7229 Linear pose M13mp9 puca Tue Jun 30 smoensted Mi3mpa puCa 7599 Linear Show column Por p lio Tue Jun 30 smoensted Cloning vector 3941 Linear Type me pala Tue Jun 30 smoensted Cloning vector 4245 Circular En pasa p Ma4 Tue Jun 30 smoensted Cloning vector 6000 Linear oe p T153 Tue Jun 30 smoensted p T153 cloning 3658 Circular Modified ae p THi Tue Jun 30 smoensted Expression vec 3479 Linear Modified by ADC pAaTrTHiD Tue Jun 30 smoensted Cloning vector 3771 Circular NO p THII Tue Jun 30 smoensted Cloning vector a7 72 Linear Description ae p TH Tue Jun 30 smoensted Cloning vector 3 53 Linear Length poa p THS Tue Jun 30 smoensted Cloning vector 3763 Circular me pELCAT Tue Jun 30 smoensted Plasmid pELCA 4496 Linear _ Latin Name me pELTATS Tue Jun 30 smoensted Plasmid pBLCA 4344 Linear E Taxonomy JOE pBLCATS Tue Jun 30 smoensted Cloning vector 4404 Linear HO pELCATE Tue Jun 30 smoensted Cloning vector 4256 Linear Common Name HO pBR 322 Tue Jun 30 smoensted Cloning vector 4361 Circular Linear eo pBR 325 Tue Jun 30 smoensted pBR325 cloning 5996 Circular Select All Move to Recycle Bin Deselect A
311. ly advanced algorithm which has become very popular due to availability speed and accuracy In short a BLAST search identifies homologous sequences by searching one or more databases usually hosted by NCBI http www ncbi nlm nih gov on the query sequence of interest McGinnis and Madden 2004 BLAST is an open source program and anyone can download and change the program code This has also given rise to a number of BLAST derivatives WU BLAST is probably the most commonly used Altschul and Gish 1996 CHAPTER 12 BLAST SEARCH 177 BLAST is highly scalable and comes in a number of different computer platform configurations which makes usage on both small desktop computers and large computer clusters possible 12 5 1 Examples of BLAST usage BLAST can be used for a lot of different purposes A few of them are mentioned below e Looking for species If you are sequencing DNA from unknown species BLAST may help identify the correct species or homologous species e Looking for domains lf you BLAST a protein sequence or a translated nucleotide sequence BLAST will look for known domains in the query sequence e Looking at phylogeny You can use the BLAST web pages to generate a phylogenetic tree of the BLAST result e Mapping DNA to a known chromosome If you are sequencing a gene from a known species but have no idea of the chromosome location BLAST can help you BLAST will show you the position of the query sequence in relation
312. m DO NOT create symbolic links in the same location as the application Symbolic links should be installed in a location which is included in your environment PATH For a system wide installation you can choose for example usr local bin If you do not have root privileges you can create a bin directory in your home directory and install symbolic links there You can also choose not to create symbolic links e Wait for the installation process to complete and click Finish If you choose to create symbolic links in a location which is included in your PATH the program can be executed by running the command clcproteinwb5 Otherwise you start the application by navigating to the location where you choose to install it and running the command clcproteinwb5 CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 15 1 2 5 Installation on Linux with an RPM package Navigate to the directory containing the rom package and install it using the rpm tool by running a command similar to rpm ivh CLCProteinWorkbench 5 JRE rpm If you are installing from a CD the rpm packages are located in the RPMS directory Installation of RPM packages usually requires root privileges When the installation process is finished the program can be executed by running the command clcproteinwb5 1 3 System requirements The system requirements of CLC Protein Workbench are these e Windows XP Windows Vista or Windows 7 Windows Server 2003 or
313. m the Navigation Area directly without creating a database first To conduct a BLAST search or Toolbox BLAST Local BLAST 2 This opens the dialog seen in figure 12 5 Select one or more sequences of the same type DNA or protein and click Next CHAPTER 12 BLAST SEARCH 166 type Navigation Area Selected Elements 1 l CLC Data Se ATP8al 5 Protein orthologs XX ATP8al mRNA gt E Protein analyses BLAST program Program blastp Protein sequence and database Target Sequences BLAST database uniprotfun Protein uniprotfun A qu a ep vious gt Ne femme er ome lame a Previous gt Next X Cancel Figure 12 6 Choose a BLAST program and a target database This opens the dialog seen in figure 12 6 At the top you can choose between different BLAST programs See section 12 1 1 for information about these methods You then specify the target database to use e Sequences When you choose this option you can use sequence data from the Navigation Area as database by clicking the Browse and select icon a A temporary BLAST database will be created from these sequences and used for the BLAST search It is deleted afterwards If you want to be able to click in the BLAST result to retrieve the hit sequences from the BLAST database at a later point you should not use this option create a create a BLAST database first see section 12 3 3
314. m the selected elements Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish This will open a new view in the View Area displaying the new DNA sequence The new sequence is not saved automatically To save the sequence drag it into the Navigation Area or press Ctrl CHAPTER 15 NUCLEOTIDE ANALYSES 221 S S on Mac to activate a save dialog Note You can select multiple RNA sequences and sequence lists at a time If the sequence list contains DNA sequences as well they will not be converted 15 3 Reverse complements of sequences CLC Protein Workbench is able to create the reverse complement of a nucleotide sequence By doing that a new sequence is created which also has all the annotations reversed since they now occupy the opposite strand of their previous location To quickly obtain the reverse complement of a sequence or part of a sequence you may select a region on the negative strand and open it in a new view right click a selection on the negative strand Open selection in New View L By doing that the sequence will be reversed This is only possible when the double stranded view option is enabled It is possible to copy the selection and paste it in a word processing program or an e mail To obtain a reverse complement of an entire sequence select a sequence in the Navigation Area Toolbox in the Menu Bar Nucleotide Analyses GA Reverse Complement x or rig
315. most probable strongly influence on the outcome of the analysis In general a few rules apply to the selection of scoring matrices e For closely related sequences choose BLOSUM matrices created for highly similar align ments like BLOSUMSO You can also select low PAM matrices such as PAM1 e For distant related sequences select low BLOSUM matrices for example BLOSUM45 or high PAM matrices such as PAM250 The BLOSUM matrices with low numbers correspond to PAM matrices with high numbers See figure 14 13 for correlations between the PAM and BLOSUM matrices To summarize if you want to find distant related proteins to a sequence of interest using BLAST you could benefit of using BLOSUMA5 or similar matrices Other useful resources CHAPTER 14 GENERAL SEQUENCE ANALYSES 207 PAM 1 PAM 120 PAM250 BLOSUM80 BLOSUM62 BLOSUM45 dor Less divergent More divergent Figure 14 13 Relationship between scoring matrices The BLOSUM62 has become a de facto standard scoring matrix for a wide range of alignment programs It is the default matrix in BLAST Calculate your own PAM matrix AhELpPI Me DiGi note ice 1i tosols pam hem BLOKS database hte blocks Enero oro NCBI help site http www ncbi nim nih gov Education BLASTinfo Scoring2 html Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display a
316. mple motifs java regular expressions or PROSITE regular expression Read more in section 14 7 2 The motif list can contain a mix of different types of motifs This is practical because some motifs can be described with the simple syntax whereas others need the more advanced regular expression syntax Instead of manually adding motifs you can Import From Fasta File 55 This will show a dialog where you can select a fasta file on your computer and use this to create motifs This will automatically take the name description and sequence information from the fasta file and put it into the motif list The motif type will be simple CHAPTER 14 GENERAL SEQUENCE ANALYSES 224 Besides adding new motifs you can also edit and delete existing motifs in the list To edit a motif either double click the motif in the list or select and click the Edit 4 button at the bottom of the view To delete a motif select it and press the Delete key on the keyboard Alternatively click Delete 4 1 in the Tool bar Save the motif list in the Navigation Area and you will be able to use for Motif Search 40 see section 14 7 Chapter 15 Nucleotide analyses Contents 15 1 Convert DNA to RNA 3 cea tet ee cs oie a ew AAA 225 15 2 Convert RNA to DNA 2 2 2 ee eee 226 15 3 Reverse complements of sequences 00 0808 eee nnn nae 227 15 4 Reverse sequence 2 2 ee ee te 4 4 4 4 22
317. n 198433 home joeuser wrlupdate vrinew4 08 02 2011 40754 DNA 46691 home joeuser m Close Figure 12 14 Overview of available BLAST databases At the top of the dialog there is a list of the BLAST database locations These locations are folders where the Workbench will look for valid BLAST databases These can either be created from within the Workbench using the Create BLAST Database tool see section 12 3 3 or they can be pre formatted BLAST databases The list of locations can be modified using the Add Location and Remove Location buttons Once the Workbench has scanned the locations it will keep a cache of the databases in order CHAPTER 12 BLAST SEARCH 1 6 to improve performance If you have added new databases that are not listed you can press Refresh Locations to clear the cache and search the database locations again By default a BLAST database location will be added under your home area in a folder called CLCdatabases This folder is scanned recursively through all subfolders to look for valid databases All other folderlocations are scanned only at the top level Below the list of locations all the BLAST databases are listed with the following information e Name The name of the BLAST database e Description Detailed description of the contents of the database e Date The date the database was created e Sequences The number of sequences in the database Type The type can be either nucleoti
318. n Workbench is stored The data in the location can be organized into folders Create a folder File New Folder 7 or Ctrl Shift N 3 Shift N on Mac Name the folder My folder and press Enter 2 1 2 Import data Next we want to import a sequence called HUMDINUC fsa FASTA format from our own Desktop into the new My folder This file is chosen for demonstration purposes only you may have another file on your desktop which you can use to follow this tutorial You can import all kinds of files In order to import the HUMDINUC fsa file Select My folder Import E in the Toolbar navigate to HUMDINUC fsa on the desktop Select The sequence is imported into the folder that was selected in the Navigation Area before you clicked Import Double click the sequence in the Navigation Area to view it The final result looks like figure 2 2 2 2 Tutorial View sequence This brief tutorial will take you through some different ways to display a sequence in the program The tutorial introduces zooming on a sequence dragging tabs and opening selection in new view We will be working with the sequence called pcDNAS atp8a 1 located in the Cloning folder in the Example data Double click the sequence in the Navigation Area to open it The sequence is displayed with annotations above it See figure 2 3 As default CLC Protein Workbench displays a sequence with annotations colored arrows on the sequence
319. n function is to know the actual structure of the protein Many questions that are raised by molecular biologists are directly targeted at protein structure The alpha helix forms a coiled rodlike structure whereas a beta sheet show an extended sheet like structure Some proteins are almost devoid of alpha helices such as CHAPTER 16 PROTEIN ANALYSES 252 chymotrypsin PDB_ID 1AB9 whereas others like myoglobin PDB_ID 101M have a very high content of alpha helices With CLC Protein Workbench one can predict the secondary structure of proteins very fast Predicted elements are alpha helix beta sheet Same as beta strand and other regions Based on extracted protein sequences from the protein databank http www rcsb org pdb a hidden Makov model HMM was trained and evaluated for performance Machine learning methods have shown superior when it comes to prediction of secondary structure of proteins Rost 2001 By far the most common structures are Alpha helices and beta sheets which can be predicted and predicted structures are automatically added to the query as annotation which later can be edited In order to predict the secondary structure of proteins Select a protein sequence Toolbox in the Menu Bar Protein Analyses 4 Predict secondary structure Ww or right click a protein sequence Toolbox Protein Analyses ha Predict secondary structure ii This opens the dialog displayed in figure 16 18 E q Pre
320. n of subcellular localization use information within the mature protein and therefore they are more robust to N terminal truncation and gene finding errors CHAPTER 16 PROTEIN ANALYSES 238 bits Figure 10 4 Sequence logo of eukaryotic signal peptides showing conservation of amino acids in bits Schneider and Stephens 1990 Polar and hydrophobic residues are shown in green and black respectively while blue indicates positively charged residues and red negatively charged residues The logo is based on an ungapped sequence alignment fixed at the 1 position of the signal peptides The SignalP method One of the most cited and best methods for prediction of classical signal peptides is the SignalP method Nielsen et al 1997 Bendtsen et al 2004b In contrast to other methods SignalP also predicts the actual cleavage site thus the peptide which is cleaved off during translocation over the membrane Recently an independent research paper has rated SignalP version 3 0 to be the best standalone tool for signal peptide prediction It was shown that the D score which is reported by the SignalP method is the best measure for discriminating secretory from non secretory proteins Klee and Ellis 2005 SignalP is located at http www cbs dtu dk services SignalP What do the SignalP scores mean Many bioinformatics approaches or prediction tools do not give a yes no answer Often the user is facing an interpretation of the output which
321. n the Files of type box EM impor gt MA i EN File name Files of type All Files Options Automatic import Force import as type ACE files ace Force import as external file s Figure 7 1 The import dialog Next select one or more files or folders to import and click Select This allows you to select a place for saving the result files If you import one or more folders the contents of the folder is automatically imported and placed in that folder in the Navigation Area If the folder contains subfolders the whole folder structure iS imported In the import dialog figure 7 1 there are three import options Automatic import This will import the file and CLC Protein Workbench will try to determine the format of the file The format is determined based on the file extension e g SwissProt files have swp at the end of the file name in combination with a detection of elements in the file that are specific to the individual file formats If the file type is not recognized it will be imported as an external file In most cases automatic import will yield a successful result but if the import goes wrong the next option can be helpful CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 104 Force import as type This option should be used if CLC Protein Workbench cannot successfully determine the file format By forcing the import as a specific type the automatic determination of the file format is bypassed
322. n the circular sequence remember to switch to the Selection y tool in the tool bar and note that this selection is also reflected in the linear view above 2 3 Tutorial Side Panel Settings This brief tutorial will show you how to use the Side Panel to change the way your sequences alignments and other data are shown You will also see how to save the changes that you made in the Side Panel Open the protein alignment located under Protein orthologs in the Example data The initial view of the alignment has colored the residues according to the Rasmol color scheme and the alignment is automatically wrapped to fit the width of the view shown in figure 2 5 Now we are going to modify how this alignment is displayed For this we use the settings in the Side Panel to the right All the settings are organized into groups which can be expanded collapsed by clicking the name of the group The first group is Sequence Layout which is expanded by default CHAPTER 2 TUTORIALS EF ATPase protei 029449 MPTMRRTISE EE T 21 QONTI2 M _ See P39524 MN BORET PPKRKPG EOD THE DEE 094296 MARBUBNKON ARRESREREE DEBAcEsmiic GTRENPHEGH 4 2 P57792 MAT S GRRRKR Q9SX33 MGG a o ee Consensus MAT X XXRRXR 2222 2 eee eee eee 100 Conservation I gt 0 60 80 QUA AAA A AAA A PANTS e Ses RR DER NADA a CE E ene Se RD p39
323. n various databases and on the Internet using your computer s default browser You can look up a sequence in the databases of NCBI and UniProt search for a sequence on the Internet using Google and search for Pubmed references at NCBI This is useful for quickly obtaining updated and additional information about a sequence The functionality of these search functions depends on the information that the sequence contains You can see this information by viewing the sequence as text see section 10 5 In the following sections we will explain this in further detail The procedure for searching is identical for all four search options see also figure 11 6 Open a sequence or a sequence list Right click the name of the sequence Web Info p select the desired search function 20 PERH E Digest aa cesta Restriction Man CTGCCCATGGTTTCC Select Sequence T PERF Make Sequence Circular GAAATGGGAAGAGA 120 PERH Web Info 2 Google sequence TG 160 SS NCBI in PubMed References PERH3BC ACTCTCCACTCACA CTC Figure 11 6 Open webpages with information about this sequence This will open your computer s default browser searching for the sequence that you selected 11 4 1 Google sequence The Google search function uses the accession number of the sequence which is used as search term on http www google com The resulting web page is equivalent to typing the accession number of the sequence into the search fiel
324. nce is artificial and low complexity regions does not always show as a square Different scoring matrices PAM The first PAM matrix Point Accepted Mutation was published in 1978 by Dayhoff et al The PAM matrix was build through a global alignment of related sequences all having sequence similarity above 85 Dayhoff and Schwartz 1978 A PAM matrix shows the probability that any given amino acid will mutate into another in a given time interval As an example PAM1 gives that one amino acid out of a 100 will mutate in a given time interval In the other end of the scale a PAM256 matrix gives the probability of 256 mutations in a 100 amino acids see figure 14 13 There are some limitation to the PAM matrices which makes the BLOSUM matrices somewhat more attractive The dataset on which the initial PAM matrices were build is very old by now and the PAM matrices assume that all amino acids mutate at the same rate this is not a correct assumption BLOSUM In 1992 14 years after the PAM matrices were published the BLOSUM matrices BLOcks SUbstitution Matrix were developed and published Henikoff and Henikoff 1992 Henikoff et al wanted to model more divergent proteins thus they used locally aligned sequences where none of the aligned sequences share less than 62 identity This resulted CHAPTER 14 GENERAL SEQUENCE ANALYSES 206 I em i E foo to Mo ot Mo Mo Bo Bo E E Mo WONFNWARPRPWNHWNHNWNHDND BBW W ONVOPRPENER
325. nces You can also modify the layout of the view by zooming in or out Click Zoom in 545 or Zoom out in the Toolbar and click the view Finally you can modify the format of the text heading each lane in the Text format preferences in the Side Panel 17 4 Restriction enzyme lists CLC Protein Workbench includes all the restriction enzymes available in the REBASE database However when performing restriction site analyses it is often an advantage to use a customized list of enzymes In this case the user can create special lists containing e g all enzymes available in the laboratory freezer all enzymes used to create a given restriction map or all enzymes that are available form the preferred vendor In the example data see section 1 6 2 under Nucleotide gt Restriction analysis there are two enzyme lists one with the 50 most popular enzymes and another with all enzymes that are included in the CLC Protein Workbench This section describes how you can create an enzyme list and how you can modify it 17 4 1 Create enzyme list CLC Protein Workbench uses enzymes from the REBASE restriction enzyme database at http rebase neb com To create an enzyme list of a subset of these enzymes You can customize the enzyme database for your installation see section 2 You can customize the enzyme database for your installation see section 2 CHAPTER 17 RESTRICTION SITE ANALYSES 219 File New Enzyme list E E
326. nces found in the alignment If the sequences of the alignment differ the consensus sequence will reflect the most common sequences in the alignment Parameters for adjusting the consensus sequences are described below Limit This option determines how conserved the sequences must be in order to agree on a consensus Here you can also choose IUPAC which will display the ambiguity code when there are differences between the sequences E g an alignment with A and a G at the same position will display an R in the consensus line if the IUPAC option is selected The IUPAC codes can be found in section H and G No gaps Checking this option will not show gaps in the consensus Ambiguous symbol Select how ambiguities should be displayed in the consensus line as N or This option has now effect if IUPAC is selected in the Limit list above The Consensus Sequence can be opened in a new view simply by right clicking the Consensus Sequence and click Open Consensus in New View e Conservation Displays the level of conservation at each position in the alignment The conservation shows the conservation of all Sequence positions The height of the bar or the gradient of the color reflect how conserved that particular position is in the alignment If one position is 100 conserved the bar will be shown in full height and it is colored in the color specified at the right side of the gradient slider CHAPTER 18 SEQUENCE ALIGNMENT
327. nd choose not to connect to a license server in the dialog When you restart CLC Protein Workbench you will be asked for a license as described in section 1 4 1 4 6 Limited mode We have created the limited mode to prevent a situation where you are unable to access your data because you do not have a license When you run in limited mode a lot of the tools in the Workbench are not available but you still have access to your data also when stored in a CLC Bioinformatics Database When running in limited mode the functionality is equivalent to the CLC Sequence Viewer see section A To get out of the limited mode and run the Workbench normally restart the Workbench When you restart the Workbench will try to find a proper license and if it does it will start up normally If it can t find a license you will again have the option of running in limited mode 1 5 About CLC Workbenches In November 2005 CLC bio released two Workbenches CLC Free Workbench and CLC Protein Workbench CLC Protein Workbench is developed from the free version giving it the well tested user friendliness and look amp feel However the CLC Protein Workbench includes a range of more advanced analyses In March 2006 CLC DNA Workbench formerly CLC Gene Workbench and CLC Main Workbench were added to the product portfolio of CLC bio Like CLC Protein Workbench CLC DNA Workbench CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 28 builds on CLC Free Workbench I
328. nd color of the residues using a gradient in the same way as described above Graph Displays the gap fraction as a graph at the bottom of the alignment Learn how to export the data behind the graph in section 7 4 x Height Specifies the height of the graph x Type The type of the graph Line plot Displays the graph as a line plot Bar plot Displays the graph as a line plot Colors Displays the graph as a color bar using a gradient like the foreground and background colors x Color box Specifies the color of the graph for line and bar plots and specifies a gradient for colors e Color different residues Indicates differences in aligned residues Foreground color Colors the letter Background color Sets a background color of the residues e Sequence logo A sequence logo displays the frequencies of residues at each position in an alignment This is presented as the relative heights of letters along with the degree of sequence conservation as the total height of a stack of letters measured in bits of information The vertical scale is in bits with a maximum of 2 bits for nucleotides and approximately 4 32 bits for amino acid residues See section 18 2 1 for more details CHAPTER 18 SEQUENCE ALIGNMENT 290 Foreground color Color the residues using a gradient according to the information content of the alignment column Low values indicate columns with high variability whereas high values indicate columns wi
329. nd in UNIPROT Accession POZ679 ID FIBG HUMAN 114 432 EF E EP pal PLF l Disulfide bridge Eto E Disulfide bridge E to E e ri Parma lA de Lopo TE bm O t a Figure 13 4 Details shown by holding the mouse cursor on a subunit For each item in the hierarchy view you can apply special view settings Simply click an item in the hierarchy view and click the Settings button above This will display the settings dialog as shown in figure 13 5 Customize appearance Atoms Bonds Representations Models Display atoms Coloring options Default color Color Opacity 0 Atom Size Size by vd radius J 20 Figure 13 5 Customize appearance CHAPTER 13 3D MOLECULE VIEWING 191 There are four tabs at the top You can specify settings for each tab and then click OK to apply and close the dialog You can also click Apply which will keep the settings dialog visible You will then be able to select another item in the hierarchical view and apply settings for this also Atoms The Atoms tab is shown in figure 13 5 At the top you can choose to show atoms and below you can specify their appearance e Color Clicking the color box allows you to select a color e Opacity Determines the level of opacity e Atom size The size of the atoms measured by vdW radius Bonds The Bonds tab is shown in figure 13 6 Customize appearance Atoms Bonds Representations Models Disp
330. nd use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents 14 3 Local complexity plot In CLC Protein Workbench it is possible to calculate local complexity for both DNA and protein sequences The local complexity is a measure of the diversity in the composition of amino acids within a given range window of the sequence The K2 algorithm is used for calculating local complexity Wootton and Federhen 1993 To conduct a complexity calculation do the following Select sequences in Navigation Area Toolbox in Menu Bar General Sequence Analyses GA Create Complexity Plot l This opens a dialog In Step 1 you can change remove and add DNA and protein sequences When the relevant sequences are selected clicking Next takes you to Step 2 This step allows you to adjust the window size from which the complexity plot is calculated Default is set to 11 amino acids and the number should always be odd The higher the number the less volatile the graph Figure 14 14 shows an example of a local complexity plot CHAPTER 14 GENERAL SEQUENCE ANALYSES 208 Complexity plot of C
331. nd von Heijne G 1997 Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites Protein Eng 10 1 1 6 Purvis 1995 Purvis A 1995 A composite estimate of primate phylogeny Philos Trans R Soc Lond B Biol Sci 348 1326 405 421 Reinhardt and Hubbard 1998 Reinhardt A and Hubbard T 1998 Using neural networks for prediction of the subcellular location of proteins Nucleic Acids Res 26 9 2230 2236 Rose et al 1985 Rose G D Geselowitz A R Lesser G J Lee R H and Zehfus M H 1985 Hydrophobicity of amino acid residues in globular proteins Science 229 4 16 834 838 Rost 2001 Rost B 2001 Review protein secondary structure prediction continues to rise J Struct Biol 134 2 3 204 218 Saitou and Nei 1987 Saitou N and Nei M 1987 The neighbor joining method a new method for reconstructing phylogenetic trees Mol Biol Evol 4 4 406 425 Schechter and Berger 1967 Schechter I and Berger A 1967 On the size of the active site in proteases Papain Biochem Biophys Res Commun 27 2 157 162 Schechter and Berger 1968 Schechter and Berger A 1968 On the active site of pro teases 3 Mapping the active site of papain specific peptide inhibitors of papain Biochem Biophys Res Commun 32 5 898 902 Schneider and Stephens 1990 Schneider T D and Stephens R M 1990 Sequence logos a new way to display consensus seq
332. ne by selecting the appropriate zoom tool in the toolbar and clicking with the mouse on the view area Alternatively click and hold the left mouse button while using either zoom tool and move the mouse up or down to zoom out or in respectively The view can be restored to display the entire structure by clicking the Fit width 41 button on the toolbar read more about zooming in section 3 3 e Rotate mode The structure is rotated when the Pan mode is selected in the toolbar If the pan mode is not enabled on the first view of a structure a warning is shown e Zoom mode Use the zoom buttons on the toolbar to enable zoom mode A single click with the mouse will zoom slightly on the structure Moreover it is possible to zoom in and out on the structure by keeping the left mouse button pressed while moving the mouse up and down e Move mode It is possible to move the structure from side to side if the Ctrl key on Windows and 6 key on Mac is pressed while dragging with the mouse 13 3 Selections and display of the 3D structure The view of the structure can be changed in several ways All graphical changes are carried out through the Side Panel At the top you can change the default coloring Default colors and Settings and at the bottom you can change the representation of specific parts of the structure in order to high light e g an active site 13 3 1 Coloring of the 3D structure The default colors apply if nothing else has b
333. ned visually in figure 3 3 CLC Data File Edit View Favorites Tools Help ay Back S ya Search A A A O 2 2 2 2 SY Sa tas Y z e CLC Data Example data ta Extra Nucleotide ES Assembly ES Cloning ES More data l Primer design ES Restriction analysis EJ Sequences Protein ES 3D structures Address C Documents and Settings clcuserfCLC_Data Y Go Folders x B CLC Data a 3 Example data CD Extra O Nucleotide O Assembly O Cloning 3 More data 5 Primer design O Restriction analysis f a An ES More data ao Protein c Sequences O 3D structures README O More data ER Recycle bin 0 O Sequences E HHH E be ER Figure 3 3 In this example the location called CLC_Data points to the folder at C Documents and settings clcuser CLC_Data Adding locations Per default there is one location in the Navigation Area called CLC_Data It points to the following folder e On Windows C Documents and settings lt username gt CLC_Data e On Mac CLC Data e On Linux homefolder CLC Data You can easily add more locations to the Navigation Area File New Location 1 73 This will bring up a dialog where you can navigate to the folder you wish to use as your new location see figure 3 4 When you click Open the new location is added to the Navigation Area as shown in figure 3 5 The name of the new location will be the name of the folder select
334. netic tree parameters Distance based methods E BB Create Tree LES 1 Select alignments of See parameters same type 2 Set parameters Algorithm Neighbor Joining w Bootstrapping V Perform bootstrap analysis Replicates 100 S Previous et Y emm Xena Figure 19 2 Adjusting parameters for distance based methods Figure 19 2 shows the parameters that can be set for the distance based methods e Algorithms The UPGMA method assumes that evolution has occurred at a constant rate in the different lineages This means that a root of the tree is also estimated The neighbor joining method builds a tree where the evolutionary rates are free to differ in different lineages CLC Protein Workbench always draws trees with roots for practical reasons but with the neighbor joining method no particular biological hypothesis is postulated by the placement of the root Figure 19 3 shows the difference between the two methods CHAPTER 19 PHYLOGENETIC TREES 303 e To evaluate the reliability of the inferred trees CLC Protein Workbench allows the option of doing a bootstrap analysis A bootstrap value will be attached to each branch and this value is a measure of the confidence in this branch The number of replicates in the bootstrap analysis can be adjusted in the wizard The default value is 100 For a more detailed explanation see Bioinformatics explained in section 19 2 A
335. network drive y Search all your data y Assembly of sequencing data Viewer Protein Advanced contig assembly Importing and viewing trace data Trim sequences Assemble without use of reference sequence Map to reference sequence Assemble to existing contig Viewing and edit contigs Tabular view of an assembled contig eas data overview Secondary peak calling Multiplexing based on barcode or name 311 DNA RNA E E E E E DNA RNA E E E y E y E E E E Main Main E Genomics Genomics E APPENDIX A COMPARISON OF WORKBENCHES Next generation Sequencing Data Analysis Viewer Import of 454 Illumina Genome Analyzer SOLID and Helicos data Reference assembly of human size genomes De novo assembly SNP DIP detection Graphical display of large contigs Support for mixed data assembly Paired data support RNA Seq analysis Expression profiling by tags ChIP Seq analysis Expression Analysis Viewer Import of Illumina BeadChip Affymetrix GEO data Import of Gene Ontology annotation files Import of Custom expression data table and Custom annotation files Multigroup comparisons Advanced plots scatter plot volcano plot box plot and MA plot Hierarchical clustering Statistical analysis on count based and gaus Sian data Annotation tests Principal component analysis PCA Hierarchical clustering and heat maps Analysis of RNA Seq Tag profiling samples Molecular cloning Viewer Advanced molecular clo
336. ning Graphical display of in silico cloning Advanced sequence manipulation Database searches Viewer GenBank Entrez searches y UniProt searches Swiss Prot TrEMBL Web based sequence search using BLAST BLAST on local database Creation of local BLAST database PubMed lookup Web based lookup of sequence data Search for structures at NCBI Protein Protein Protein Protein E DNA RNA DNA RNA DNA RNA Ly E E DNA RNA y y E E E E y y E Main Main y Main Main E 312 Genomics E Genomics E Genomics Genomics Li APPENDIX A COMPARISON OF WORKBENCHES General sequence analyses Linear sequence view Circular sequence view Text based sequence view Editing sequences Adding and editing sequence annotations Advanced annotation table Join multiple sequences into one Sequence statistics Shuffle sequence Local complexity region analyses Advanced protein statistics Comprehensive protein characteristics repor Nucleotide analyses Basic gene finding Reverse complement without loss of annota tion Restriction site analysis Advanced interactive restriction site analysis Translation of sequences from DNA to pro teins Interactive translations of sequences anc alignments G C content analyses and graphs Protein analyses 3D molecule view Hydrophobicity analyses Antigenicity analysis Protein charge analysis Reverse translation from protein to DNA Proteolytic cleavage detec
337. nkar A semi empirical method for prediction of antigenic regions has been developed Kolaskar and Tongaonkar 1990 This method also includes information of surface accessibility and flexibility and at the time of publication the method was able to predict antigenic determinants with an accuracy of 75 Surface Probability Display of surface probability based on the algorithm by Emini et al 1985 This algorithm has been used to identify antigenic determinants on the surface of proteins Chain Flexibility isplay of backbone chain flexibility based on the algorithm by Karplus and Schulz 1985 It is known that chain flexibility is an indication of a putative antigenic determinant Many more scales have been published throughout the last three decades Even though more advanced methods have been developed for prediction of membrane spanning regions the simple and very fast calculations are still highly used Other useful resources AAindex Amino acid index database http www genome ad jp dbget aaindex html Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial p
338. nly cleaves at lysine or arginine residues but it does not matter with a few exceptions which amino acid is located at position P1 carboxyterminal of the cleavage site Another example is trombin which cleaves if an arginine is found in position P1 but not if a D or E is found in position P1 at the same time See figure 16 28 CHAPTER 16 PROTEIN ANALYSES 203 Cleavage site P4 P3 P2 P1 P1 P2 P3 Figure 16 27 Nomenclature of the peptide substrate The substrate is cleaved between position P1 P1 rydrolysis site for trypsin H Q H 0 N C CN C C H HOR Lysine or arginine y Mydrolysis site for trombin H O HO N C C N C C HI H Arginine Glycine Figure 16 28 Hydrolysis of the peptide bond between two amino acids Trypsin cleaves unspecifi cally at lysine or arginine residues whereas trombin cleaves at arginines if asparate or glutamate is absent Bioinformatics approaches are used to identify potential peptidase cleavage sites Fragments can be found by scanning the amino acid sequence for patterns which match the corresponding cleavage site for the protease When identifying cleaved fragments it is relatively important to know the calculated molecular weight and the isoelectric point Other useful resources The Peptidase Database http merops sanger ac uk Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDeriv
339. ny problems please contact The CLC Support Team Proxy Settings Previous Quit Workbench Figure 1 2 Choosing between direct download or download web page e Go to license download web page The workbench will open a Web Browser with the License Download web page when you click Next From there you will be able to download your license as a file and import it This option allows you to get a license even though the Workbench does not have direct access to the CLC Licenses Service If you select the first option and it turns out that you do not have internet access from the Workbench because of a firewall proxy server etc you will be able to click Previous and use the other option instead Direct download Selecting the first option takes you to the dialog shown in figure 1 3 License Wizard 53 B CLC Protein Workbench Requesting a license Requesting and downloading an evaluation license by establishing a direct connection to the CLC bio License Web Service An Evaluation License was successfully downloaded The License is valid until 2008 07 03 If you experience any problems please contact The CLC Support Team Proxy Settings Previous next Quit Workbench Figure 1 3 A license has been downloaded A progress for getting the license is shown and when the license is downloaded you will be able to click Next Go to license download web page Selecting the second option
340. nzyme for one second to display additional information see figure 17 21 or use the view of enzyme lists See 17 4 Click Finish to open the enzyme list The CLC Protein Workbench comes with a standard set of enzymes based on http www rebase neb com You can customize the enzyme database for your installation see section CHAPTER 17 RESTRICTION SITE ANALYSES 280 Restriction Site Analysis Select DNA RNA sequence s Enzyme list Enzymes to be considered TA 4 Le v Use existing enzyme list Popular enzymes v in calculation aes P Y E Enzymes in Popular en Enzymes to be used Filter g Filter Name Overhang Methylat Popul Name Overhang Methyla Pop PstI tgca N6 met te Kpnl gtac N met PRE Sacl aget i S meth SphI catg alo Apal ggce 5 meth Ball nnn N4 met Chal gate FokI lt NA gt 3 N6 met Hhal cg S meth NsiI tgca SacII gc S meth Figure 17 20 Selecting enzymes All enzymes Filter 3 Name Overh Methyl Pop PstI 3 N6 meth eer a KpnI 3 N6 meth Pet Sacl 3 S methyl ee SphI 3 lok Apal 3 S methyl pee Sacll 3 5 methyl pet Nsil Enzyme Sacll Chal Recognition site pattern CCGCGG Ball Suppliers GE Healthcare Hhal Qbiogene Xml American Allied Biochemical Inc Dralll Nippon Gene Co Ltd
341. o another CLC Workbench you can use the CLC format to export several elements in one file and you will preserve all the information Note CLC files can be exported from and imported into all the different CLC Workbenches Backup If you wish to secure your data from computer breakdowns it is advisable to perform regular backups of your data Backing up data in the CLC Protein Workbench is done in two ways e Making a backup of each of the folders represented by the locations in the Navigation Area e Selecting all locations in the Navigation Area and export E in zip format The resulting file will contain all the data stored in the Navigation Area and can be imported into CLC Protein Workbench if you wish to restore from the back up at some point CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 109 No matter which method is used for backup you may have to re define the locations in the Navigation Area if you restore your data from a computer breakdown 7 2 External files In order to help you organize your research projects CLC Protein Workbench lets you import all kinds of files E g if you have Word Excel or pdf files related to your project you can import them into the Navigation Area of CLC Protein Workbench Importing an external file creates a copy of the file which is stored at the location you have chosen for import The file can now be opened by double clicking the file in the Navigation Area The file is opened using the
342. o launch CLC Protein Workbench and click Next e Choose if CLC Protein Workbench should be used to open CLC files and click Next e Choose where you would like to create shortcuts for launching CLC Protein Workbench and click Next e Choose if you would like to associate clc files to CLC Protein Workbench If you check this option double clicking a file with a clc extension will open the CLC Protein Workbench e Wait for the installation process to complete choose whether you would like to launch CLC Protein Workbench right away and click Finish When the installation is complete the program can be launched from the Start Menu or from one of the shortcuts you chose to create 1 2 3 Installation on Mac OS X Starting the installation process is done in one of the following ways If you have downloaded an installer Locate the downloaded installer and double click the icon The default location for downloaded files is your desktop If you are installing from a CD Insert the CD into your CD ROM drive and open it by double clicking on the CD icon on your desktop Launch the installer by double clicking on the CLC Protein Workbench icon Installing the program is done in the following steps e On the welcome screen click Next e Read and accept the License agreement and click Next CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 14 e Choose where you would like to install the application and click Next e Choose if CLC
343. o the sequence the result will be similar to the sequence shown in figure 17 14 See section 10 3 for more information about viewing 5 acll T Ei ATPsal MRNA GGTGGGAGGCGCGGCCCCGCGGCAGCTGAGCCC Figure 17 14 The result of the restriction analysis shown as annotations annotations 17 2 5 Table of restriction sites The restriction map can be shown as a table of restriction sites see figure 17 15 Each row in the table represents a restriction enzyme The following information is available for each enzyme CHAPTER 17 RESTRICTION SITE ANALYSES 2 4 FA Restriction m E Rows 5 Restriction sites table Fiter O Segu Mame Pattern Owerhang Number Cut position s PERHSBC CjePI ccannnnnnntc 3 151 184 PERH o no Sh PERHGEC No maga o o A PERHOEC Tso rca Po o qa pERHGEC Jrhili arca Bo o foi EA ETTTT Figure 17 15 The result of the restriction analysis shown as annotations Sequence The name of the sequence which is relevant if you have performed restriction map analysis on more than one sequence Name The name of the enzyme Pattern The recognition sequence of the enzyme Overhang The overhang produced by cutting with the enzyme 3 5 or Blunt e Number of cut sites Cut position s The position of each cut If the enzyme cuts more than once the positions are separated by commas If the enzyme s recognition
344. ocesses are not deleted A Toolbo Search Database nucleotide NC 012671 Al O Create Alignment FA 5 mM Create Alignment Es PMR DD RRR A Search Database nucleotide NC 012671 8 8 0 0 0 A 100 Figure 3 17 A database search and an alignment calculation are running Clicking the small icon next to the process allow you to stop pause and resume processes Besides the options to stop pause and resume processes there are some extra options for a selected number of the tools running from the Toolbox e Show results If you have chosen to save the results see section 9 1 you will be able to open the results directly from the process by clicking this option e Find results If you have chosen to save the results see section 9 1 you will be able to high light the results in the Navigation Area e Show Log Information This will display a log file showing progress of the process The log file can also be shown by clicking Show Log in the handle results dialog where you choose between saving and opening the results e Show Messages Some analyses will give you a message when processing your data The messages are the black dialogs shown in the lower left corner of the Workbench that disappear after a few seconds You can reiterate the messages that have been shown by clicking this option The terminated processes can be removed by View Remove Terminated Processe
345. of a View E g if you are studying a sequence you can click anywhere in the sequence and hold the mouse button By moving the mouse you move the sequence in the View 3 3 6 Selection The Selection mode y is used for selecting in a View selecting a part of a sequence selecting nodes in a tree etc It is also used for moving e g branches in a tree or sequences in an alignment When you make a selection on a sequence or in an alignment the location is shown in the bottom right corner of the screen E g 23 24 means that the selection is between two residues 23 means that the residue at position 23 is selected and finally 23 25 means that 23 24 and 25 are selected By holding ctrl 38 you can make multiple selections 3 3 7 Changing compactness There is a shortcut way of changing the compactness setting for read mappings or Press and hold Alt key Scroll using your mouse wheel or touchpad CHAPTER 3 USER INTERFACE 18 3 4 Toolbox and Status Bar The Toolbox is placed in the left side of the user interface of CLC Protein Workbench below the Navigation Area The Toolbox shows a Processes tab and a Toolbox tab 3 4 1 Processes By clicking the Processes tab the Toolbox displays previous and running processes e g an NCBI search or a calculation of an alignment The running processes can be stopped paused and resumed by clicking the small icon next to the process see figure 3 17 Running and paused pr
346. of element The name is what is displayed in the Navigation Area per default e Length The length of the sequence e Organism Sequences which contain information about organism can be searched In this way you could search for e g Homo sapiens sequences e Database fields If your data is stored in a CLC Bioinformatics Database you will be able to search for custom defined information Read more in the database user manual 83 CHAPTER 4 SEARCHING YOUR DATA 84 Only the first item in the list Name is available for all kinds of data The rest is only relevant for sequences If you wish to perform a search for sequence similarity use Local BLAST see section 12 1 3 instead 4 2 Quick search At the bottom of the Navigation Area there is a text field as shown in figure 4 1 Y AA a ta o EA TLC Data S E Example Data EFA Extra amp F5 Nucleotide oa Protein E EADME a E Recycle bin 12 Ar center search term A Figure 4 1 Search simply by typing in the text field and press Enter To search simply enter a text to search for and press Enter 4 2 1 Quick search results To show the results the search pane is expanded as shown in figure 4 2 Li Lj Los ma L E H TLC Data Es Example Data Pa Extra PF Nucleotide EF Protein README E Recycle bin 14 Figure 4 2 Search results Showing 1 50 If there are many hits only the 50 first hits are immediately sho
347. older where the sequences are saved when they are downloaded Choosing Download and Open opens a new view for each of the selected sequences Definition Eni EE k Edit k View b _ Toolbox k Shows k a Download and Open lc HE Download and Save Open at NCBI PEI Figure 11 2 By right clicking a search result it is possible to choose how to handle the relevant sequence Copy paste from GenBank search results When using copy paste to bring the search results into the Navigation Area the actual files are downloaded from GenBank To copy paste files into the Navigation Area select one or more of the search results Ctrl C 36 C on Mac select a folder in the Navigation Area Ctrl V Note Search results are downloaded before they are saved Downloading and saving several files may take some time However since the process runs in the background displayed in the Status bar it is possible to continue other tasks in the program Like the search process the download process can be stopped This is done in the Toolbox in the Processes tab 11 1 3 Save GenBank search parameters The search view can be saved either using dragging the search tab and and dropping it in the Navigation Area or by clicking Save E When saving the search only the parameters are saved not the results of the search This is useful if you have a special search that you perform from time to time CHAPTER 11 ONLINE DATABASE SEARC
348. ometimes be faster and more reliable for big batch BLAST jobs Figure 12 8 shows an example of a BLAST result in the CLC Protein Workbench L ATP8al BLAST AtpBai ATP8a1 me a A A lM nan IMM 2QUIATBA1 HUMAN e Mm eee NTI2 ATBA2_H 8198 ATBB2_H sp Q9NTIZIATEA HUMAN Probable phospholipicttransporting ATPase IB ATPase class 2 ML 1 TF62 A18B4_H ae Se sity et 144 82 Gaps 291144 2 AT e da ie i 0 e OG3 AT11B HUMAN ee ee ee M Y IEA TA HUMAN me A a es e e 7 IB4S AT1I1C HUMAN O M PP o EA A A e id 0423 AT883_HUMAN 3110148 7P94_ HUMAN A A a DOCA ATOOD UIIRAARI e Tn ti e ii 4 Th p PEJE YE Figure 12 1 Display of the output of a BLAST search At the top is there a graphical representation of BLAST hits with tool tips showing additional information on individual hits Below is a tabular form of the BLAST results 12 1 Running BLAST searches With the CLC Protein Workbench there are two ways of performing BLAST searches You can either have the BLAST process run on NCBI s BLAST servers http www ncbi nlm nih gov or you can perform the BLAST search on your own computer The advantage of running the BLAST search on NCBI servers is that you have readily access to the popular and often very large BLAST databases without having to download them to your own computer The advantages of running BLAST on your own com
349. ommon name house mouse Latin name Musmusculus Type 20 DNA OC RNA S O Protein Description Probable phospholipid transporting ATPase IA Sequence required 180 1 mptmrrtvse irsraegyek tddvsektsl adqeevrtif ingpqltkfc nnh vstakyn 61 vitflprfly sgfrraansf flfiallqqi pdvsptgryt tlvpllfil a vaaikeiied 121 ikrhkadnav nkkqtqvlrn gaweivhwek vnvgdiviik gkeyipadt v llsssepqam EA Figure 10 14 Creating a sequence e Type Select between DNA RNA and protein e Circular Specifies whether the sequence is circular This will open the sequence in a circular view as default applies only to nucleotide sequences e Description A description of the sequence e Keywords A set of keywords separated by semicolons e Comments Your own comments to the sequence e Sequence Depending on the type chosen this field accepts nucleotides or amino acids Spaces and numbers can be entered but they are ignored when the sequence is created This allows you to paste Ctrl V on Windows and 8 V on Mac in a sequence directly from a different source even if the residue numbers are included Characters that are not part of the IUPAC codes cannot be entered At the top right corner of the field the number of residues are counted The counter does not count spaces or numbers Clicking Finish opens the sequence It can be saved by clicking Save E or by dragging the tab of the sequence view into the Navigation Area
350. ommons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents Chapter 19 Phylogenetic trees Contents 19 1 Inferring phylogenetic trees 2 ee eee te es 301 19 1 1 Phylogenetic tree parameters 00 pe eee eee ee 302 19 1 2 Tree View Preferences 304 19 2 Bioinformatics explained phylogenetics lt 2 enue 306 19 2 1 The phylogenetic tree lt 306 19 2 2 Modern usage of phylogenies eee ee 307 19 2 3 Reconstructing phylogenies from molecular data 307 19 2 4 Interpreting phylogenies lt a 309 CLC Protein Workbench offers different ways of inferring phylogenetic trees The first part of this chapter will briefly explain the different ways of inferring trees in CLC Protein Workbench The second part Bioinformatics explained will give a more general introduction to the concept of phylogeny and the associated
351. on Select the enzymes you want to use for detection When the relevant enzymes are chosen click Next In Step 3 you can set parameters for the detection This limits the number of detected cleavages Figure 16 25 shows an example of how parameters can be set e Min and max number of cleavage sites Certain proteolytic enzymes cleave at many positions in the amino acid sequence For instance proteinase K cleaves at nine different amino acids regardless of the surrounding residues Thus it can be very useful to limit the number of actual cleavage sites before running the analysis CHAPTER 16 PROTEIN ANALYSES E q Proteolytic Cleavage 1 Select protein sequences 2 Select enzymes lions ma Include Name Cyanogen bromide CNBr Asp N endopeptidase Chymotrypsin high spec Chymotrypsin low spec o Iodosobenzoate Thermolysin Post Pro Glu C Asp N Proteinase K Thrombin Factor Xa Granzyme B L ml msi AEE Da Rn Select all De select all Previous gt Next f Finish Figure 16 25 Setting parameters for proteolytic cleavage detection 261 e Min and max fragment length Likewise it is possible to limit the output to only display sequence fragments between a chosen length Both a lower and upper limit can be chosen e Min and max
352. ormation on bands fragments You can get information about the individual bands by hovering the mouse cursor on the band of interest This will display a tool tip with the following information e Fragment length e Fragment region on the original sequence e Enzymes cutting at the left and right ends respectively CHAPTER 17 RESTRICTION SITE ANALYSES 218 For gels comparing whole sequences you will see the sequence name and the length of the sequence Note You have to be in Selection or Pan 7 mode in order to get this information It can be useful to add markers to the gel which enables you to compare the sizes of the bands This is done by clicking Show marker ladder in the Side Panel Markers can be entered into the text field separated by commas Modifying the layout The background of the lane and the colors of the bands can be changed in the Side Panel Click the colored box to display a dialog for picking a color The slider Scale band spread can be used to adjust the effective time of separation on the gel i e how much the bands will be spread over the lane In a real electrophoresis experiment this property will be determined by several factors including time of separation voltage and gel density You can also choose how many lanes should be displayed e Sequences in separate lanes This simulates that a gel is run for each sequence e All sequences in one lane This simulates that one gel is run for all Seque
353. osing BLASTx or tBLASTx to conduct a search you get the option of selecting a translation table for the genetic code The standard genetic code is set as default This setting is particularly useful when working with organisms or organelles that have a genetic code different from the standard genetic code The following description of BLAST search parameters is based on information from http www ncbi nlm nih gov BLAST blastcgihelp shtml e Limit by Entrez query BLAST searches can be limited to the results of an Entrez query against the database chosen This can be used to limit searches to subsets of entries in the BLAST databases Any terms can be entered that would normally be allowed in an Entrez search CHAPTER 12 BLAST SEARCH 164 session More information about Entrez queries can be found at http www ncbi nlm nih gov books NBK3837 fEntrezHelp Entrez Searching Options The syntax described there is the same as would be accepted in the CLC interface Some commonly used Entrez queries are pre entered and can be chosen in the drop down menu e Choose filter Low complexity Mask off segments of the query sequence that have low compo sitional complexity Filtering can eliminate statistically significant but biologically uninteresting reports from the BLAST output e g hits against common acidic basic or proline rich regions leaving the more biologically interesting regions of the query sequence available for specific matching a
354. otation and see which databases are available Chapter 12 BLAST Search Contents 12 1 Running BLAST searches 008 ee eee eee es 161 211 BEAST UNGER cars Cone eee eee A we a 162 12 1 2 BLAST a partial sequence against NCBI 206 165 12 1 3 BLAST against local data lt lt ew we mas mm Dee e amp 165 12 1 4 BLAST a partial sequence against a local database 167 12 2 Output from BLAST searches 00 2 eee eee ee ee 167 12 2 1 Graphical overview for each query sequence 167 12 2 2 Overview BLAST table 167 12 2 3 BLASTEMODNI S esse seras 169 LO PLAST DO Ls nani oa RARAS A 170 12 3 Local BLAST databases ee 172 12 3 1 Make pre formatted BLAST databases available 173 12 3 2 Download NCBI pre formatted BLAST databases 173 12 3 3 Create local BLAST databases 0 1 174 12 4 Manage BLAST databases 008 eee eee ee 175 12 4 1 Migrating from a previous version of the Workbench 176 12 5 Bioinformatics explained BLAST 0 0088 ee ee een eens 176 12 5 1 Examples of BLAST Usage css we Ee A we 177 12 5 2 Searching for homology our ee wee ae ee eee ew a A 177 12 5 3 How does BLAST work 0 00 ee eee ee ee ee 177 12 5 4 Which BLAST program should use 179 12 5 5 Which BLAST options should I chan
355. ouse turns into an arrow pointing left and right Alter the preferences in the Side Panel for changing the presentation of the tree 19 2 Bioinformatics explained phylogenetics Phylogenetics describes the taxonomical classification of organisms based on their evolutionary history i e their phylogeny Phylogenetics is therefore an integral part of the science of systematics that aims to establish the phylogeny of organisms based on their characteristics Furthermore phylogenetics is central to evolutionary biology as a whole as it is the condensation of the overall paradigm of how life arose and developed on earth 19 2 1 The phylogenetic tree The evolutionary hypothesis of a phylogeny can be graphically represented by a phylogenetic tree Figure 19 5 shows a proposed phylogeny for the great apes Hominidae taken in part from Purvis Purvis 1995 The tree consists of a number of nodes also termed vertices and branches also termed edges These nodes can represent either an individual a species or a higher grouping and are thus broadly termed taxonomical units In this case the terminal nodes also called leaves or tips of the tree represent extant species of Hominidae and are the operational taxonomical units OTUs The internal nodes which here represent extinct common ancestors of the great apes are termed hypothetical taxonomical units since they are not directly observable Root node Branches edges Terminal nodes leaves
356. plained Sequence logo 290 18 3 Edit alignments lt lt lt 2 292 18 3 1 Move residues and gaps a 292 ro IMSS EAS cas eh DE ras eee 292 18 3 3 Delete residues and gaps ee 292 18 3 4 Copy annotations to other sequences 293 18 3 5 Move sequences up and down 293 18 3 6 Delete rename and add sequences 293 18 3 7 Realign selection 294 18 4 Join alignments lt 2 294 18 4 1 How alignments are joined 296 18 5 Pairwise comparison lt lt lt 4 4 4 296 18 5 1 Pairwise comparison on alignment selection 296 18 5 2 Pairwise comparison parameters 297 18 5 3 The pairwise comparison table 298 18 6 Bioinformatics explained Multiple alignments 299 18 6 1 Use of multiple alignments a wes 299 18 6 2 Constructing multiple alignments 299 CLC Protein Workbench can align nucleotides and proteins using a progressive alignment algorithm see section 18 6 or read the White paper on alignments in the Science section of http www clcbio com This chapter describes
357. ple of the Side Panel of a sequence view STS LS E Ja Tr rE Sequence layout Annotation layout F Annotation types k Restriction sites Residue coloring b Nucleotide info b Find Text Format Figure 5 8 The Side Panel of a sequence contains several groups Sequence layout Annotation types Annotation layout etc Several of these groups are present in more views E g Sequence layout is also in the Side Panel of alignment views By clicking the black triangles or the corresponding headings the groups can be expanded or collapsed An example is shown in figure 5 9 where the Sequence layout is expanded Sequence layout Fl Spaces every 10 residues No wrap Auto wrap Fixed wrap 10000 Double stranded Numbers on sequences Relative to Numbers on plus strand Follow selection Lock labels Sequence label Mame ma k Annotation layout H Annotation types k Restriction sites k Residue coloring Nucleotide info k Find k Text Format Figure 5 9 The Sequence layout is expanded CHAPTER 5 USER PREFERENCES AND SETTINGS 96 The content of the groups is described in the sections where the functionality is explained E g Sequence Layout for sequences is described in chapter 10 1 1 When you have adjusted a view of e g a sequence your settings in the Side Panel can be saved When you open other sequences which you want to display in a similar way the saved settings can be ap
358. plied The options for saving and applying are available in the top of the Side Panel see figure 5 10 Figure 5 10 At the top of the Side Panel you can Expand all groups Collapse all preferences Dock Undock preferences Help and Save Restore preferences To save and apply the saved settings click 35 seen in figure 5 10 This opens a menu where the following options are available e Save Settings This brings up a dialog as shown in figure 5 11 where you can enter a name for your settings Furthermore by clicking the checkbox Always apply these settings you can choose to use these settings every time you open a new view of this type If you wish to change which settings should be used per default open the Preferences dialog see section 5 2 e Delete Settings Opens a dialog to select which of the saved settings to delete e Apply Saved Settings This is a submenu containing the settings that you have previously saved By clicking one of the settings they will be applied to the current view You will also see a number of pre defined view settings in this submenu They are meant to be examples of how to use the Side Panel and provide quick ways of adjusting the view to common usages At the bottom of the list of settings you will see CLC Standard Settings which represent the way the program was set up when you first launched it q Save Settings a Xd Please enter a name For these user settings my settings v Alwa
359. prediction See section 16 for more about this topic e Pfam domain search See section 16 6 for more about this topic e Local BLAST See section 12 1 3 for more about this topic e NCBI BLAST See section 12 1 1 for more about this topic When you have selected the relevant analyses click Next Step 3 to Step 7 if you select all the analyses in Step 2 are adjustments of parameters for the different analyses The parameters CHAPTER 16 PROTEIN ANALYSES 254 are mentioned briefly in relation to the following steps and you can turn to the relevant chapters or sections mentioned above to learn more about the significance of the parameters In Step 3 you can adjust parameters for sequence statistics e Individual Statistics Layout Comparative is disabled because reports are generated for one protein at a time e Include Background Distribution of Amino Acids Includes distributions from different organisms Background distributions are calculated from UniProt www uniprot org version 6 0 dated September 13 2005 In Step 4 you can adjust parameters for hydrophobicity plots e Window size Width of window on sequence odd number e Hydrophobicity scales Lets you choose between different scales In Step 5 you can adjust a parameter for complexity plots e Window size Width of window on sequence must be oda In Step 6 you can adjust parameters for dot plots e Score model Different scoring matrices e Window size Width o
360. provides flexibility in the treatment of gaps at the ends of the sequences There are three possibilities Free end gaps Any number of gaps can be inserted in the ends of the sequences without any cost Cheap end gaps All end gaps are treated as gap extensions and any gaps past 10 are free End gaps as any other Gaps at the ends of sequences are treated like gaps in any other place in the sequences When aligning a long sequence with a short partial sequence it is ideal to use free end gaps since this will be the best approximation to the situation The many gaps inserted at the ends are not due to evolutionary events but rather to partial data Many homologous proteins have quite different ends often with large insertions or deletions This confuses alignment algorithms but using the Cheap end gaps option large gaps will generally be tolerated at the sequence ends improving the overall alignment This is the default setting of the algorithm Finally treating end gaps like any other gaps is the best option when you know that there are no biologically distinct effects at the ends of the sequences Figures 18 3 and 18 4 illustrate the differences between the different gap scores at the sequence ends 18 1 2 Fast or accurate alignment algorithm CLC Protein Workbench has two algorithms for calculating alignments e Fast less accurate This allows for use of an optimized alignment algorithm which is very fast The
361. ption is only available in Gen Bank when searching for nucleotide structures For more information about how to use this syntax see http www ncbi nlm nih gov entrez query static help Summary_ Matrices html Search Fields and Ouali fiers When you are satisfied with the parameters you have entered click Start search Note When conducting a search no files are downloaded Instead the program produces a list of links to the files in the NCBI database This ensures a much faster search 11 3 2 Handling of NCBI structure search results The search result is presented as a list of links to the files in the NCBI database The View displays 50 hits at a time can be changed in the Preferences see chapter 5 More hits can be displayed by clicking the More button at the bottom right of the View Each structure hit is represented by text in three columns e Accession e Description e Resolution e Method Protein chains e Release date It is possible to exclude one or more of these columns by adjust the View preferences for the database search view Furthermore your changes in the View preferences can be saved See section 5 5 Several structures can be selected and by clicking the buttons in the bottom of the search view you can do the following e Download and open Download and open immediately e Download and save Download and save lets you choose location for saving structure e Open at NCBI Open additional informat
362. put button at the bottom of the view Clicking the Open Query Sequence will open a sequence CHAPTER 12 BLAST SEARCH ATPBa1 2QU AT8BA1 HUMAN NTI2 ATBA2_H 8198 ATBB2_H Score 1567 8 bits 4058 TFGZATOBA M identities 779 1144 68 Positives 933 1144 82 3920 478B1_ HUMAN 2GIAT11B_ HUMAN B196 AT114 HUMAN IB4S AT11C HUMAN CA23JATOBS HUMAN 0423 478B3_ HUMAN J1TO ATPSA HUMAN BOC dA TAC LITIR A RI 4 HE F EE EMO Figure 12 8 Default display of the Expect DEDO Gaps 22 1 O 144 2 168 E sp Q9NTIZIATEA HUMAN Probable phospholipicttransporting ATPase IB ATPase class 2 ML 1 output of a BLAST search for one query sequence At the top is there a graphical representation of BLAST hits with tool tips showing additional information on individual hits 8 Multi BLAST Rows 6 Filter 0 O num 7 gt 5 Column width Query Number of hits Lowest E value Accession E value 094296 101 0 00 NP 596406 as P39524 101 0 00 P39524 Ed P57792 101 0 00 NP 173938 m7 Q29449 113 0 00 NP_777263 i QONTIZ 111 0 00 NP 057613 Y Q95x33 102 0 00 NP 177038 Open BLAST Output Open Query Sequence Automatic w Show column Query Number of hits Lowest E value Accession E value Description E value Greatest identity Accession identity Description identity Greatest positive Accession positive
363. puter include that you can use your own sequence collections as blast databases and that running big batch BLAST jobs can be faster and more reliable when done locally CHAPTER 12 BLAST SEARCH 162 12 1 1 BLAST at NCBI When running a BLAST search at the NCBI the Workbench sends the sequences you select to the NCBI s BLAST servers When the results are ready they will be automatically downloaded and displayed in the Workbench When you enter a large number of sequences for searching with BLAST the Workbench automatically splits the sequences up into smaller subsets and sends one subset at the time to NCBI This is to avoid exceeding any internal limits the NCBI places on the number of sequences that can be submitted to them for BLAST searching The size of the Subset created in the CLC software depends both on the number and size of the sequences To start a BLAST job to search your sequences against databases held at the NCBI Toolbox BLAST 5 NCBI BLAST i Alternatively use the keyboard shortcut Ctrl Shift B for Windows and 3 Shift B on Mac OS This opens the dialog seen in figure 12 2 e BLAST at NCBI 1 Select sequences of same select sequences oF S type Navigation Area Selected Elements 1 EB CLC Data As ATP8al 9 E3 Example Data A gt E Protein orthologs 2 ATP8al MRNA gt 5 Protein analyses gt EG Cloning X lt ATP8al genomic sequence gt 5 Sequencing data o E Primers o fq RNA secondary str
364. quence has a gap and the other does not e Identities Calculates the number of identical alignment positions to overlapping alignment positions between the two sequences e Differences Calculates the number of alignment positions where one sequence is different from the other This includes gap differences as in the Gaps comparison e Distance Calculates the Jukes Cantor distance between the two sequences This number is given as the Jukes Cantor correction of the proportion between identical and overlapping alignment positions between the two sequences e Percent identity Calculates the percentage of identical residues in alignment positions to overlapping alignment positions between the two sequences CHAPTER 18 SEQUENCE ALIGNMENT Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish 18 5 3 The pairwise comparison table The table shows the results of selected comparisons see an example in figure 18 15 Since comparisons are often symmetric the table can show the results of two comparisons at the same time one in the upper right and one in the lower left triangle ER TDPZ BOMMD al TOP2 BOMMO TOP2 DROME TOP2 PEA TOP2 ARATH TOP2 PLAFK TOP2 CANGA TOP2 YEAST TOP2 CANAL TOP2 SCHPO TOP2 LEICH TOP2 CRIFA TOP2_TRYBB TOP2_TRYCR TOP2 ASFM2 TOP2 ASFB7 ES Op E n os voa eu or o E sm ais we ral so aval o 1145 1089 1109 1124 3 m ae
365. r Active site BINDING SITE FOR RESIDUE MAG E 3 Active site BINDING SITE FOR RESIDUE NAGE 3 Active site BINDING SITE FOR RESIDUE MAG E 462 Active site BINDING SITE FOR RESIDUE CA E 1 Active site BINDING SITE FOR RESIDUE CA E 2 Active site BINDING SITE FOR RESIDUE CAC 1 Active site BINDING SITE FOR RESIDUE CAE 1 Active site BINDING SITE FOR RESIDUE CAE 2 Active site BINDING SITE FOR RESIDUE CAF 1 Active site BINDING SITE FOR RESIDUE CAC 2 Disulfide bridge F to D Disulfide bridge E to E m Figure 13 3 Hierarchy view in the Side Panel The hierarchical view shows the structure in a detailed manner Individual structure subunits residues active sites disulfide bridges or even down to the atom level can be selected individually and colored accordingly CHAPTER 13 3D MOLECULE VIEWING 190 You can show additional information from the hierarchical view by holding your mouse still for one second see an example in figure 13 4 Molecule ERA A Subunit protein a E Subunit protein fw C Subunit protein e Pe O Subunit Subunit C protein Compound Name FIBRINOGEN GAMMA CHAIM Fragment M CHAIM Engineered YES SOUrCE Organism sci HOMO SAPIENS Organism cora HUMAN Strain HUMAN Gene Flas PROZO61 Expression system CRICETULUS GRISEUS Expression system strain HUMAN Expression system vector type CHO Expression system plasmid PMLP Sequence POB sequence 88 406 Fou
366. r circular molecules The units are monophosphates Both the weight for single and double stranded molecules are includes The atomic composition is defined the same way CHAPTER 14 GENERAL SEQUENCE ANALYSES 211 e Atomic composition Nucleotide distribution table Nucleotide distribution histogram e Annotation table e Counts of di nucleotides Frequency of di nucleotides A short description of the different areas of the statistical output is given in section 14 4 1 14 4 1 Bioinformatics explained Protein statistics Every protein holds specific and individual features which are unique to that particular protein Features such as isoelectric point or amino acid composition can reveal important information of a novel protein Many of the features described below are calculated in a simple way Molecular weight The molecular weight is the mass of a protein or molecule The molecular weight is simply calculated as the sum of the atomic mass of all the atoms in the molecule The weight of a protein is usually represented in Daltons Da A calculation of the molecular weight of a protein does not usually include additional posttransla tional modifications For native and unknown proteins it tends to be difficult to assess whether posttranslational modifications such as glycosylations are present on the protein making a calculation based solely on the amino acid sequence inaccurate The molecular weight can be determined very accur
367. r very accurate results you should consider using other algorithms such as Smith Waterman You can read Bioinformatics explained BLAST versus Smith Waterman here http www clcbio com BE CHAPTER 2 TUTORIALS 52 EB Local BLAST 52 1 Select sequences of same EIA type 2 Set program parameters 3 Set input parameters Choose parameters Low Complexity Choose filter Mask lower case Expect 20000 Word size 2 No of processors 2 Matrix PAM3O Gap cost Existence 9 Extension 1 v Command line options Figure 2 20 Settings for searching for remote homologues 2 7 Tutorial Proteolytic cleavage detection This tutorial shows you how to find cut sites and see an overview of fragments when cleaving proteins with proteolytic cleavage enzymes Suppose you are working with protein ATP8a1 from the example data and you wish to see where the enzyme trypsin will cleave the protein Furthermore you want to see details for the resulting fragments which are between 10 and 15 amino acids long select protein ATP8a1 Toolbox Protein Analyses ia Proteolytic Cleavage This opens Step 1 of the Proteolytic Cleavage dialog In this step you can choose which sequences to include in the analysis Since you have already chosen ATP8a1 click Next In this step you should select Trypsin This is illustrated in figure 2 21 o BB Proteolytic Cleavage 88 1 Select protein sequences A AA 2 Se
368. r view It will place the restriction site labels as close to the cut site as possible see an example in figure 17 4 e Stacked This is similar to the flag option for linear sequence views but it will stack the labels so that all enzymes are shown For circular views it will align all the labels on each side of the circle This can be useful for clearly seeing the order of the cut sites when they are located closely together see an example in figure 17 3 o T Figure 17 4 Restriction site labels in radial layout Note that in a circular view the Stacked and Radial options also affect the layout of annotations 17 1 1 Sort enzymes Just above the list of enzymes there are three buttons to be used for sorting the list see figure 17 5 Sorting Aa LI Figure 17 5 Buttons to sort restriction enzymes e Sort enzymes alphabetically Aa Clicking this button will sort the list of enzymes alphabetically e Sort enzymes by number of restriction sites T This will divide the enzymes into four groups Non cutters Single cutters Double cutters Multiple cutters CHAPTER 17 RESTRICTION SITE ANALYSES 268 There is a checkbox for each group which can be used to hide show all the enzymes in a group O e Sort enzymes by overhang T T This will divide the enzymes into three groups Blunt Enzymes cutting both strands at the same position 3 Enzymes producing an overhang at the 3
369. rabidopsis thaliana Arabidopsis thaliana Saccharomyces cerevisiae Schizosaccharomyces pombe 100 Mus musculus Bos taurus Homo sapiens soot Mus musculus Bos taurus Homo sapiens Saccharomyces cerevisiae Schizosaccharomyces pombe Arabidopsis thaliana Arabidopsis thaliana Figure 19 3 Method choices for phylogenetic inference The bottom shows a tree found by neighbor joining while the top shows a tree found by UPGMA The latter method assumes that the evolution occurs at a constant rate in different lineages Maximum likelihood phylogeny fr E Maximum Likelihood Phylogeny 53 1 Select one nucleotide Setparancers eee Set starting tree 2 Set parameters 9 Neighbor Joining UPGMA Use tree from file Select substitution model Jukes Cantor v Transition transversion ratio 2 Rate variation 7 Include rate variation Number of substitution rate categories 4 Gamma distribution parameter 1 Estimation V Estimate substitution rate parameter s 4 Estimate topology Estimate Gamma distribution parameter A a erevous J Quer Semen Xena Figure 19 4 Adjusting parameters for ML phylogeny Figure 19 4 shows the parameters that can be set for the ML phylogenetic tree reconstruction e Starting tree the user is asked to specify a starting tree for the tree reconstruction There are three possibilities Neighbor joining UPGMA CHAPTER 19 PHYLOGENETIC TREES 30
370. raction of identity between the subsequence and the simple motif If you use a list of motifs the accuracy applies only to the simple motifs in the list e Search for reverse motif This enables searching on the negative strand on nucleotide sequences e Exclude unknown regions Genome sequence often have large regions with unknown sequence These regions are very often padded with N s Ticking this checkbox will not display hits found in N regions Motif search handles ambiguous characters in the way that two residues are different if they do not have any residues in common For example For nucleotides N matches any character and R matches A G For proteins X matches any character and Z matches E Q Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish There are two types of results that can be produced e Add annotations This will add an annotation to the sequence when a motif is found an example is shown in figure 14 27 e Create table This will create an overview table of all the motifs found for all the input sequences TTAGCTGTGGCTGCTATTASAGAGATAATAGAAGATATTAAACGA Figure 14 27 Sequence view displaying the pattern found The search string was tataaa 14 7 3 Java regular expressions A regular expressions is a string that describes or matches a set of strings according to certain syntax rules They are usually used to give a concise description of a set without CHAPTER
371. ral Sequence Analyses A Nucleotide Analyses af Restriction Sites jaj Protein Analyses sa E Database Search Processes Toolbox mem E Idle 1 element s are selected Figure 3 18 An empty Workspace Workspace E in the Toolbar Select the Workspace to activate or Workspace in the Menu Bar Select Workspace E choose which Workspace to activate OK The name of the selected Workspace is shown after CLC Protein Workbench at the top left corner of the main window in figure 3 18 it says default 3 5 3 Delete Workspace Deleting a Workspace can be done in the following way Workspace in the Menu Bar Delete Workspace choose which Workspace to delete OK Note Be careful to select the right Workspace when deleting The delete action cannot be undone However no data is lost because a workspace is only a representation of data It is not possible to delete the default workspace 3 6 List of shortcuts The keyboard shortcuts in CLC Protein Workbench are listed below CHAPTER 3 USER INTERFACE Action Adjust selection Change between tabs Close Close all views Copy Cut Delete Exit Export Export graphics Find Next Conflict Find Previous Conflict Help Import Maximize restore size of View Move gaps in alignment Navigate sequence views New Folder New Sequence View Paste Print Redo Rename Save Search local data Search within a sequence Sear
372. residues The wider the window the less fluctuations in the antigenicity scores 16 4 2 Antigenicity graphs along sequence Antigenicity graphs along the sequence can be displayed using the Side Panel The functionality is similar to hydrophobicity see section 16 5 2 16 5 Hydrophobicity CLC Protein Workbench can calculate the hydrophobicity of protein sequences in different ways using different algorithms See section 16 5 3 Furthermore hydrophobicity of sequences can be displayed as hydrophobicity plots and as graphs along sequences In addition CLC Protein Workbench can calculate hydrophobicity for several sequences at the same time and for CHAPTER 16 PROTEIN ANALYSES 245 alignments 16 5 1 Hydrophobicity plot Displaying the hydrophobicity for a protein sequence in a plot is done in the following way select a protein sequence in Navigation Area Toolbox in the Menu Bar Protein Analyses ia Create Hydrophobicity Plot l2 This opens a dialog The first step allows you to add or remove sequences Clicking Next takes you through to Step 2 which is displayed in figure 16 11 E y Create Hydrophobicity Plot 1 Select protein sequences les erel 2 Set parameters Hydrophobicity scale V Kyte Doolittle Eisenberg v Engelman Hopp Woods Janin Rose Cornette Window size Number of residues must be odd 11 IS Previous gt Next Weinish Xana Figure 16 11
373. restriction sites Suppose you are working with sequence ATP8a1 MRNA from the example data and you wish to know which restriction enzymes will cut this sequence exactly once and create a 3 overhang Do the following select the ATP8a1 mRNA Toolbox in the Menu Bar Restriction Sites 3 Restriction Site Analysis ck Click Next to set parameters for the restriction map analysis In this step first select Use existing enzyme list and click the Browse for enzyme list button acy Select the Popular enzymes in the Cloning folder under Enzyme lists Then write 3 into the filter below to the left Select all the enzymes and click the Add button The result should be like in figure 2 30 Restriction Site Analysis 1 Select DNA RNA Mes 9 DE CONSIDENEO IN COICUIaci n sequence s Enzyme list 2 Enzymes to be considered Use existing enzyme list Popular enzymes v O in calculation A Enzymes in Popular en Enzymes to be used Filter 3 Filter Name Overhang Methylat Popul Name Overhang Methyla Pop PstI 3 tgca 5 N6 met KpnI 3 gtac 5 N6 met Sacl 3 agct 5 S meth Pee SphI 3 catg Apal 3 ggcc 5 S meth Ball 3 nnn 5 N4 met Chal 3 gate etek FokI 5 lt NA gt 3 N met Hhal 3 cg 5 S meth Nsil 3 tgca Sacll 3 gc 5 S meth Figure 2 30 Selecting enzymes Click Next In this step you specify that yo
374. riction enzymes based on 269 271 279 pa4 file format 329 Page heading 101 Page number 101 Page setup 100 Pairwise comparison 296 PAM scoring matrices 204 Parameters search 149 152 155 Partition function 314 Paste text to create a new sequence 104 Paste copy 115 Pattern Discovery 215 Pattern discovery 314 Pattern Search 217 PCR primers 314 pdb file format 186 329 seg file format 329 PDB file format 329 pdf format export 111 Peptidase 259 Peptide sequence databases 321 Percent identity pairwise comparison of se quences in alignments 298 Personal information 28 Pfam domain search 249 313 phr file format 329 INDEX PHR file format 329 Phred file format 327 phy file format 329 Phylip file format 328 Phylogenetic tree 301 314 tutorial DO Phylogenetics Bioinformatics explained 306 pir file format 329 PIR NBRP file format 327 Plot dot plot 197 local complexity 207 Plug ins 30 png format export 111 Polarity colors 125 Portrait Print orientation 100 Positively charged residues 213 PostScript export 111 Preference group 95 Preferences 89 advanced 93 export 94 General 89 import 94 style sheet 95 toolbar 91 View 90 view 5 Primer design 314 design from alignments 314 Print 98 3D molecule view 193 dot plots 199 preview 101 visible area 99 whole view 99 pro file format 329 Problems when starting up 29
375. righter annotations are the ORFs with a length of at least 100 amino acids On the positive strand around position 11 000 a gene starts before the ORF This is due to the use of the standard genetic code rather than the bacterial code This particular gene starts with CTG which is a start codon in bacteria Two short genes are entirely missing while a handful of open reading frames do not correspond to any of the annotated genes NC 000913 selection NC 000913 selection Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish Finding open reading frames is often a good first step in annotating sequences such as cloning vectors or bacterial genomes For eukaryotic genes ORF determination may not always be very helpful since the intron exon structure is not part of the algorithm Chapter 16 Protein analyses Contents 16 1 Signal peptide prediction 0 008 eee ee ee 234 16 1 1 Signal peptide prediction parameter settings 234 16 1 2 Signal peptide prediction output 0 00 ee eee eee 235 16 1 3 Bioinformatics explained Prediction of signal peptides 235 16 2 Protein Charge 0 2 4 4 240 16 2 1 Modifying the layout sk wee a ea ee a ae 241 16 3 Transmembrane helix prediction 2 0 0 ee eee ee ee 0 241 16 4 Antigenicity 648 ee Ree eae ee AAA 242 16 4 1 Plot of antigenicity 2
376. rline prediction but closer inspection of the sequence revealed an internal methionine at position 12 which could indicate a erroneously annotated start of the protein Later this protein was re annotated by Swiss Prot to start at the M in position 12 See the text for description of the scores The C score is the cleavage site score For each position in the submitted sequence a C score is reported which should only be significantly high at the cleavage site Confusion is often seen with the position numbering of the cleavage site When a cleavage site position is referred to by a single number the number indicates the first residue in the mature protein This means that a reported cleavage site between amino acid 26 27 corresponds to the mature protein starting at and include position 27 Y max is a derivative of the C score combined with the S score resulting in a better cleavage site prediction than the raw C score alone This is due to the fact that multiple high peaking C scores can be found in one sequence where only one is the true cleavage site The cleavage site is assigned from the Y score where the slope of the S score is steep and a significant C score is found The S mean is the average of the S score ranging from the N terminal amino acid to the amino acid assigned with the highest Y max score thus the S mean score is calculated for the length of the predicted signal peptide The S mean score was in SignalP version 2 0 used as
377. rm Enter the text to search for The search function does not discriminate between lower and upper case characters e Sequence search Search the nucleotides or amino acids For amino acids the single letter abbreviations should be used for searching The sequence search also has a set of advanced search parameters Include negative strand This will search on the negative strand as well Treat ambiguous characters as wildcards in search term If you search for e g ATN you will find both ATG and ATC If you wish to find literally exact matches for ATN i e only find ATN not ATG this option should not be selected Treat ambiguous characters as wildcards in sequence If you search for e g ATG you will find both ATG and ATN If you have large regions of Ns this option should not be selected Note that if you enter a position instead of a sequence it will automatically switch to position search e Annotation search Searches the annotations on the sequence The search is performed both on the labels of the annotations but also on the text appearing in the tooltip that you see when you keep the mouse cursor fixed If the search term is found the part of the sequence corresponding to the matching annotation is selected Below this option you can choose to search for translations as well Sequences annotated with coding regions often have the translation specified which can lead to undesired results e Position search Fin
378. rmat clc will export the history too In this way you can share folders and files with others while preserving the history If an element s history includes source elements i e if there are elements listed in Origins from they must also be exported in order to see the full history Otherwise the history will have entries named Element deleted An easy way to export an element with all its source elements is to use the Export Dependent Elements function described in section 7 1 3 The history view can be printed To do so click the Print icon E The history can also be exported as a pdf file Select the element in the Navigation Area Export ES in File of type choose History PDF Save Chapter 9 Batching and result handling Contents 9 1 Howto handle results of analyses 2 2 ee ee te ee 0 01 118 9 1 1 Table outputs 42 6554 errada 119 ele BAN ecos aora AA 120 9 1 How to handle results of analyses This section will explain how results generated from tools in the Toolbox are handled by CLC Protein Workbench Note that this also applies to tools not running in batch mode see above All the analyses in the Toolbox are performed in a step by step procedure First you select elements for analyses and then there are a number of steps where you can specify parameters some of the analyses have no parameters e g when translating DNA to RNA The final step concerns the handling of the results of the anal
379. rom the search If the desired sequence is not shown you can click the More button below the list to see more hits 2 4 2 Saving the sequence The sequences which are found during the search can be displayed by double clicking in the list of hits However this does not save the sequence You can save one or more sequence by selecting them and click Download and Save or drag the sequences into the Navigation Area 2 5 Tutorial BLAST search BLAST is an invaluable tool in bioinformatics It has become central to identification of homologues and similar sequences and can also be used for many other different purposes This tutorial takes you through the steps of running a blast search in CLC Workbenches If you plan to use blast for your research we highly recommend that you read further about it Understanding how blast works is key to setting up meaningful and efficient searches CHAPTER 2 TUTORIALS 45 Suppose you are working with the ATP8a1 protein sequence which is a phospholipid transporting ATPase expressed in the adult house mouse Mus musculus To obtain more information about this molecule you wish to query the peptides held in the Swiss Prot database to find homologous proteins in humans Homo sapiens using the Basic Local Alignment Search Tool BLAST algorithm This tutorial involves running BLAST remotely using databases housed at the NCBI Your computer must be connected to the internet to complete this tutoria
380. roteins are integral membrane proteins Most membrane proteins have hydrophobic regions which span the hydrophobic core of the membrane bi layer and hydrophilic regions located on the outside or the inside of the membrane Many receptor proteins have several transmembrane helices spanning the cellular membrane For prediction of transmembrane helices CLC Protein Workbench uses TMHMM version 2 0 Krogh et al 2001 located at http www cbs dtu dk services TMHMM thus an active internet connection is required to run the transmembrane helix prediction Additional information on THMHH and Center for Biological Sequence analysis CBS can be found at CHAPTER 16 PROTEIN ANALYSES 242 http www cbs dtu dk and in the original research paper Krogh et al 2001 In order to use the transmembrane helix prediction you need to download the plug in using the plug in manager see section 1 7 1 When the plug in is downloaded and installed you can use it to predict transmembrane helices Select a protein sequence Toolbox in the Menu Bar Protein Analyses Ga Transmembrane Helix Prediction 8 or right click a protein sequence Toolbox Protein Analyses Ab Transmembrane Helix Prediction it If a sequence was selected before choosing the Toolbox action this sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements The predictions obta
381. rthermore you can also right click an empty part of the view of the graphical view of sequence lists and choose Digest All Sequences with Selected Enzymes and Run on Gel Note When using the right click options the sequence will be digested with the enzymes that are selected in the Side Panel This is explained in section 10 1 2 The view of the gel is explained in section 17 3 3 17 3 2 Separate sequences on gel To separate sequences without restriction enzyme digestion first create a sequence list of the sequences in question see section 10 7 Then click the Gel button Z at the bottom of the view of the sequence list For more information about the view of the gel see the next section 17 3 3 Gel view In figure 17 18 you can see a simulation of a gel with ts Side Panel to the right This view will be explained in this section CHAPTER 17 RESTRICTION SITE ANALYSES 211 Separated sequences HUMDINUC pBR322 HUMHBB Figure 17 17 A sequence list shown as a gel Restriction nv A Gesetna Y e 2 amp o a ama e O 8 E ET E I E E F w Gel options ia a 14 14 Joel background wi wi wi TT wi 2 oa 0 oa 0 Meana Scale band spread E Show marker ladder 3 5 10 20 50 200 2 Sequences in separate lanes All sequences in one lane Text Format RASA Figure 17 18 Five lanes showing fragments of five sequences cut with restriction enzymes Inf
382. s 0 If you close the program while there are running processes a dialog will ask if you are sure that you want to close the program Closing the program will stop the process and it cannot be restarted when you open the program again CHAPTER 3 USER INTERFACE 19 3 4 2 Toolbox The content of the Toolbox tab in the Toolbox corresponds to Toolbox in the Menu Bar The Toolbox can be hidden so that the Navigation Area is enlarged and thereby displays more elements View Show Hide Toolbox The tools in the toolbox can be accessed by double clicking or by dragging elements from the Navigation Area to an item in the Toolbox 3 4 3 Status Bar As can be seen from figure 3 1 the Status Bar is located at the bottom of the window In the left side of the bar is an indication of whether the computer is making calculations or whether it is idle The right side of the Status Bar indicates the range of the selection of a sequence See chapter 3 3 6 for more about the Selection mode button 3 5 Workspace If you are working on a project and have arranged the views for this project you can save this arrangement using Workspaces A Workspace remembers the way you have arranged the views and you can switch between different workspaces The Navigation Area always contains the same data across Workspaces It is however possible to open different folders in the different Workspaces Consequently the program allows you to display different clu
383. s Click Next to adjust alignment algorithm parameters Clicking Next opens the dialog shown in figure 18 2 a BN Create Alignment 3 1 Select sequences of same See parameter type 2 Set parameters Gap settings Gap open cost 10 Gap extension cost 1 End gap cost As any other w Alignment Fast less accurate Slow very accurate Redo alignments Use fixpoints GOS Eev due la en Figure 18 2 Adjusting alignment algorithm parameters CHAPTER 18 SEQUENCE ALIGNMENT 284 18 1 1 Gap costs The alignment algorithm has three parameters concerning gap costs Gap open cost Gap extension cost and End gap cost The precision of these parameters is to one place of decimal e Gap open cost The price for introducing gaps in an alignment e Gap extension cost The price for every extension past the initial gap If you expect a lot of small gaps in your alignment the Gap open cost should equal the Gap extension cost On the other hand if you expect few but large gaps the Gap open cost should be set significantly higher than the Gap extension cost However for most alignments it is a good idea to make the Gap open cost quite a bit higher than the Gap extension cost The default values are 10 0 and 1 0 for the two parameters respectively e End gap cost The price of gaps at the beginning or the end of the alignment One of the advantages of the CLC Protein Workbench alignment method is that it
384. s 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work CHAPTER 16 PROTEIN ANALYSES 264 SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents Chapter 17 Restriction site analyses Contents 17 1 Dynamic restriction sites 0 eee 266 17 1 1 Sort enzymes 6 we o a AR A Oe ee 267 17 1 2 Manage enzymes ten osorno ra A 208 17 2 Restriction site analysis from the Toolbox lt lt 270 17 2 1 Selecting sorting and filtering enzymes 270 17 2 2 Number of cut sites y ua e E we a E ee we ee 2 1 17 2 3 Output of restriction map analysis 0 0 eee ee es 2 2 17 2 4 Restriction sites as annotation on the sequence 213 17 2 5 Table of restriction sites 213 17 2 6 Table of restriction fragments 2 02 214 Let Clans sara dA MASSA ERR 275 17 3 Gelelectrophoresis 0 0 ee ee et te aa 275 17 3 1 Separate fragments of sequences on gel 276 17 3 2 Separate sequences on gel 2 6 diet G
385. s and see the actual sequence alignments returned from the BLAST server There are several settings available in the BLAST Graphics view e BLAST Layout You can choose to Gather sequences at top Enabling this option affects the view that is shown when scrolling horizontally along a BLAST result If selected the sequence hits which did not contribute to the visible part of the BLAST graphics will be omitted whereas the found BLAST hits will automatically be placed right below the query sequence e Compactness You can control the level of sequence detail to be displayed Not compact Full detail and spaces between the sequences Low The normal settings where the residues are visible when zoomed in but with no extra Spaces between Medium The sequences are represented as lines and the residues are not visible There is some space between the sequences Compact Even less space between the sequences e BLAST hit coloring You can choose whether to color hit sequences and you can adjust the coloring e Coverage In the Alignment info in the Side Panel you can visualize the number of hit sequences at a given position on the query sequence The level of coverage is relative to the overall number of hits included in the result Foreground color Colors the letters using a gradient where the left side color is used for low coverage and the right side is used for maximum coverage Background color Colors the b
386. s consist of combinations of only five different atoms The atoms which can be found in these simple structures are Carbon Nitrogen Hydrogen Sulfur Oxygen The atomic composition of a protein can for example be used to calculate the precise molecular weight of the entire protein Total number of negatively charged residues Asp Glu At neutral pH the fraction of negatively charged residues provides information about the location of the protein Intracellular proteins tend to have a higher fraction of negatively charged residues than extracellular proteins Total number of positively charged residues Arg Lys At neutral pH nuclear proteins have a high relative percentage of positively charged amino acids Nuclear proteins often bind to the negatively charged DNA which may regulate gene expression or help to fold the DNA Nuclear proteins often have a low percentage of aromatic residues Andrade et al 1998 Amino acid distribution Amino acids are the basic components of proteins The amino acid distribution in a protein is simply the percentage of the different amino acids represented in a particular protein of interest Amino acid composition is generally conserved through family classes in different organisms which can be useful when studying a particular protein or enzymes across species borders Another interesting observation is that amino acid composition variate slightly between CHAPTER 14 GENERAL SEQUENCE ANALYSES 214 pro
387. s not wrapped e Lock labels When you scroll horizontally the label of the sequence remains visible e Sequence label Defines the label to the left of the sequence Name this is the default information to be shown Accession Sequences downloaded from databases like GenBank have an accession number Latin name Latin name accession Common name Common name accession Annotation Layout and Annotation Types See section 10 3 1 Restriction sites See section 10 1 2 CHAPTER 10 VIEWING AND EDITING SEQUENCES 125 Motifs See section 14 7 1 Residue coloring These preferences make it possible to color both the residue letter and set a background color for the residue e Non standard residues For nucleotide sequences this will color the residues that are not C G A Tor U For amino acids only B Z and X are colored as non standard residues Foreground color Sets the color of the letter Click the color box to change the color Background color Sets the background color of the residues Click the color box to change the color e Rasmol colors Colors the residues according to the Rasmol color scheme See http www openrasmol org doc rasmol html Foreground color Sets the color of the letter Click the color box to change the color Background color Sets the background color of the residues Click the color box to change the color e Polarity colors only protein Colors
388. s possible to exclude one or more of these columns by adjust the View preferences for the database search view Furthermore your changes in the View preferences can be saved See section 5 5 Several sequences can be selected and by clicking the buttons in the bottom of the search view you can do the following e Download and open doesn t save the sequence e Download and save lets you choose location for saving sequence e Open at NCBI searches the sequence at NCBI s web page Double clicking a hit will download and open the sequence The hits can also be copied into the View Area or the Navigation Area from the search results by drag and drop copy paste or by using the right click menu as described below CHAPTER 11 ONLINE DATABASE SEARCH 151 Drag and drop from GenBank search results The sequences from the search results can be opened by dragging them into a position in the View Area Note A sequence is not saved until the View displaying the sequence is closed When that happens a dialog opens Save changes of sequence x Yes or No The sequence can also be saved by dragging it into the Navigation Area It is possible to select more sequences and drag all of them into the Navigation Area at the same time Download GenBank search results using right click menu You may also select one or more sequences from the list and download using the right click menu see figure 11 2 Choosing Download and Save lets you select a f
389. s searched according to the PHYML method Guindon and Gascuel 2003 allowing efficient search and estimation of large phylogenies Branch lengths are given in terms of expected numbers of substitutions per nucleotide site 19 1 2 Tree View Preferences The Tree View preferences are these e Text format Changes the text format for all of the nodes the tree contains CHAPTER 19 PHYLOGENETIC TREES 305 Text size The size of the text representing the nodes can be modified in tiny small medium large or huge Font Sets the font of the text of all nodes Bold Sets the text bold if enabled e Tree Layout Different layouts for the tree Node symbol Changes the symbol of nodes into box dot circle or none if you don t want a node symbol Layout Displays the tree layout as standard or topology Show internal node labels This allows you to see labels for the internal nodes Initially there are no labels but right clicking a node allows you to type a label Label color Changes the color of the labels on the tree nodes Branch label color Modifies the color of the labels on the branches Node color Sets the color of all nodes Line color Alters the color of all lines in the tree e Labels Specifies the text to be displayed in the tree Nodes Sets the annotation of all nodes either to name or to species Branches Changes the annotation of the branches to bootstrap length or none if you don t want annotation on branc
390. s with No restriction site 0 One restriction site 1 Two restriction sites 2 Three restriction sites 3 N restriction sites Minimum 1 laximum Any number of restriction sites gt 0 A Previous gt Next Emis XX Cancel Figure 17 12 Selecting number of cut sites If you wish the output of the restriction map analysis only to include restriction enzymes which cut the sequence a specific number of times use the checkboxes in this dialog e No restriction site 0 e One restriction site 1 e Two restriction sites 2 e Three restriction site 3 N restriction sites Minimum Maximum Any number of restriction sites gt O The default setting is to include the enzymes which cut the sequence one or two times You can use the checkboxes to perform very specific searches for restriction sites e g if you wish to find enzymes which do not cut the sequence or enzymes cutting exactly twice 17 2 3 Output of restriction map analysis Clicking next shows the dialog in figure 17 13 This dialog lets you specify how the result of the restriction map analysis should be presented e Add restriction sites as annotations to sequence s This option makes it possible to see the restriction sites on the sequence see figure 1 14 and save the annotations for later use e Create restriction map When a restriction map is created it can be shown in three different ways
391. scoring matrix for blastp is BLOSUM62 12 5 6 Explanation of the BLAST output The BLAST output comes in different flavors On the NCBI web page the default output is html and the following description will use the html output as example Ordinary text and xml output for easy computational parsing is also available The default layout of the NCBI BLAST result is a graphical representation of the hits found a table of sequence identifiers of the hits together with scoring information and alignments of the query sequence and the hits The graphical output Shown in figure 12 19 gives a quick overview of the query Sequence and the resulting hit sequences The hits are colored according to the obtained alignment scores The table view shown in figure 12 20 provides more detailed information on each hit and furthermore acts as a hyperlink to the corresponding sequence in GenBank In the alignment view one can manually inspect the individual alignments generated by the BLAST algorithm This is particularly useful for detailed inspection of the sequence hit found sbjct and the corresponding alignment In the alignment view all Scores are described for each alignment CHAPTER 12 BLAST SEARCH 182 Color key for alignment scores lt 40 40 50 50 80 30 200 gt 200 Query EE EE EE EE EE EE FO 140 210 200 350 a ee a CU OU A gt EA eT nn oo A ooo o aa To jo TE IE Sau E
392. scribed in section 17 3 1 17 3 Gel electrophoresis CLC Protein Workbench enables the user to simulate the separation of nucleotide sequences on a gel This feature is useful when e g designing an experiment which will allow the differentiation CHAPTER 17 RESTRICTION SITE ANALYSES 2 6 of a successful and an unsuccessful cloning experiment on the basis of a restriction map There are two main ways to simulate gel separation of nucleotide sequences e One or more sequences can be digested with restriction enzymes and the resulting fragments can be separated on a gel e A number of existing sequences can be separated on a gel There are several ways to apply these functionalities as described below 17 3 1 Separate fragments of sequences on gel This section explains how to simulate a gel electrophoresis of one or more sequences which are digested with restriction enzymes There are two ways to do this e When performing the Restriction Site Analysis from the Toolbox you can choose to create a restriction map which can be shown as a gel This is explained in section 17 2 e From all the graphical views of sequences you can right click the name of the sequence and choose Digest Sequence with Selected Enzymes and Run on Gel El The views where this option is available are listed below Circular view see section 10 2 Ordinary sequence view see section 10 1 Graphical view of sequence lists see section 10 7 Fu
393. se IM ATPase class I type GB me 0 00 2 109 00 Probable phospholipid transporting ATPase IC Familial intrahepatic cholest 0 00 2 078 00 Probable phospholipid transporting ATPase IF ATPase class I type 118 A 0 00 1 732 00 Probable phospholipid transporting ATPase IH ATPase class I type 11A 0 00 1 711 00 Probable phospholipid transporting ATPase IG ATPase class I type 11C C 0 00 1 670 00 Probable a fi nk ATPase IK ll EEES I type SB me 2 93E 151 1 372 00 ATP Aa 499 00 Download and Open Download and Save Open at NCBI Open Structure 2 amp 2 OG Figure 2 15 Output of a BLAST search shown in a table This view provides more statistics about the hits and you can use the filter to search for e g a specific type of protein etc If you wish to download several of the hit sequences this is easily done in this view Simply select the relevant sequences and drag them into a folder in the Navigation Area 2 6 Tutorial Tips for specialized BLAST searches Here you will learn how to e Use BLAST to find the gene coding for a protein in a genomic sequence e Find primer binding sites on genomic sequences e Identify remote protein homologues Following through these sections of the tutorial requires some experience using the Workbench so if you get stuck at some point we recommend going through the more basic tutorials first CHAPTER 2 TUTORIALS 48 2 6 1 Locate a protein sequence on the chromosome
394. selection When looking for a specific enzyme it is easier to use the Filter If you wish to find e g Hindlll sites simply type Hindlll into the filter and the list of enzymes will shrink automatically to only include the Hindlll enzyme This can also be used to only show enzymes producing e g a 3 overhang as shown in figure 17 20 Restriction Site Analysis 1 Select DNA RNA _ enzymes to be conside ahammar sequence s Enzyme list 2 Enzymes to be considered 3 Use existing enzyme list Popular enzymes v in calculation l Enzymes in Popular en Enzymes to be used Filter E Filter Name Overhang Methylat Popul Name Overhang Methyla Pop PstI Egca 5 N6 met Pee KpnI gtac S N met Peer Sacl agct 5 S meth SphI cata FAN Apal ggcc 5 S meth Ball Ann 5 N4 met Chal gatc FokI lt NA gt 3 N6 met cg 5 S meth toca ge 5 5 meth Hhal NsiI Sacll 03 a 02 O 0 0 0 0 A ow Figure 17 7 Selecting enzymes If you need more detailed information and filtering of the enzymes either place your mouse cursor on an enzyme for one second to display additional information see figure 17 21 or use the view of enzyme lists see 17 4 All enzymes Filter 3 Name Overh Methyl Pop PstI 3 N6 meth Poe ES KpnI 3 N6 meth pro Sacl 3 S methyl Pete Sphl 3 si
395. selection while holding the button release the mouse button Alternatively you can search for a specific interval using the find function described above If you have made a selection and wish to adjust it drag the edge of the selection you can see the mouse cursor change to a horizontal arrow or press and hold the Shift key while using the right and left arrow keys to adjust the right side of the selection If you wish to select the entire sequence double click the sequence name to the left Selecting several parts at the same time multiselect You can select several parts of sequence by holding down the Ctrl button while making selections Holding down the Shift button lets you extend or reduce an existing selection to the position you clicked To select a part of a Sequence covered by an annotation right click the annotation Select annotation or double click the annotation To select a fragment between two restriction sites that are shown on the sequence double click the sequence between the two restriction sites Read more about restriction sites in section 10 1 2 Open a selection in a new view A selection can be opened in a new view and saved as a new sequence right click the selection Open selection in New View L This opens the annotated part of the sequence in a new view The new sequence can be saved by dragging the tab of the sequence view into the Navigation Area The process described above is also th
396. sentations where a product specialist from CLC bio demon strates our software This is a very easy way to get started using the program Read more about online presentations here http clcbio com presentation 1 6 1 Quick start When the program opens for the first time the background of the workspace is visible In the background are three quick start shortcuts which will help you getting started These can be seen in figure 1 23 Figure 1 23 Three available Quick start short cuts available in the background of the workspace CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 30 The function of the three quick start shortcuts is explained here e Import data Opens the Import dialog which you let you browse for and import data from your file system e New sequence Opens a dialog which allows you to enter your own sequence e Read tutorials Opens the tutorials menu with a number of tutorials These are also available from the Help menu in the Menu bar 1 6 2 Import of example data It might be easier to understand the logic of the program by trying to do simple operations on existing data Therefore CLC Protein Workbench includes an example data set When downloading CLC Protein Workbench you are asked if you would like to import the example data set If you accept the data is downloaded automatically and saved in the program If you didn t download the data or for some other reason need to download the data again you have
397. sequence Hit length The length of the hit Query start Shows the start position in the query sequence Query end Shows the end position in the query sequence Overlap Display a percentage value for the overlap of the query sequence and hit sequence Only the length of the local alignment is taken into account and not the full length query sequence Identity Shows the number of identical residues in the query and hit sequence Yldentity Shows the percentage of identical residues in the query and hit sequence CHAPTER 12 BLAST SEARCH 1 2 e Positive Shows the number of similar but not necessarily identical residues in the query and hit sequence e Positive Shows the percentage of similar but not necessarily identical residues in the query and hit sequence e Gaps Shows the number of gaps in the query and hit sequence e Gaps Shows the percentage of gaps in the query and hit sequence e Query Frame Strand Shows the frame or strand of the query sequence e Hit Frame Strand Shows the frame or strand of the hit sequence In the BLAST table view you can handle the hit sequences Select one or more sequences from the table and apply one of the following functions e Download and Open Download the full sequence from NCBI and opens it If multiple sequences are selected they will all open if the same sequence is listed several times only one copy of the sequence is downloaded and opened e Download and Save Download t
398. sequence is on the negative strand the cut position is put in brackets as the enzyme Tsol in figure 17 15 whose cut position is 134 Some enzymes cut the sequence twice for each recognition site and in this case the two cut positions are surrounded by parentheses 17 2 6 Table of restriction fragments The restriction map can be shown as a table of fragments produced by cutting the sequence with the enzymes Click the Fragments button at the bottom of the view The table is shown in see figure 17 16 Each row in the table represents a fragment If more than one enzyme cuts in the same region or if an enzyme s recognition site is cut by another enzyme there will be a fragment for each of the possible cut combinations gt The following information is available for each fragment e Sequence The name of the sequence which is relevant if you have performed restriction map analysis on more than one sequence e Length The length of the fragment If there are overhangs of the fragment these are included in the length both 3 and 5 overhangs e Region The fragment s region on the original sequence SFurthermore if this is the case you will see the names of the other enzymes in the Conflicting Enzymes column CHAPTER 17 RESTRICTION SITE ANALYSES 2 5 EH Restriction m E Rows 9 Restriction Fragment table Filter Sequence Length Region overhangs Leftend Rightend Conflicting enzymes
399. sequences in GenBank the NCBI Entrez database The NCBI search view is opened in this way figure 11 1 Search Search for Sequences at NCBI 8 or Ctrl B 3 Bon Mac This opens the following view 148 CHAPTER 11 ONLINE DATABASE SEARCH NCBI search O Choose database Nucleotide O Protein All Fields v human All Fields v hemoglobin al Fields v complete 7 7 Rows 50 Search results Filter Add search parameters Append wildcard to search words 8 Start search Accession l Definition Modification Date AM270L66 Aspergillus niger contig 4n08c0110 complete genome 2007 03 24 AM711867 Clavibacter michiganensis subsp michiganensis NCPPB 2007 05 18 AP008209 B4000016 Clostridium perfringens str 13 DNA complete genome Oryza sativa japonica cultivar group genomic DNA c 2007 05 19 2007 05 19 BCO29387 BC130457 Homo sapiens hemoglobin gamma G mRNA cDNA clon Homo sapiens hemoglobin gamma G MRNA cDNA clon 2007 02 08 2007 01 04 BC130459 BC139602 Homo sapiens hemoglobin gamma G mRNA cDNA clon Danio rerio hemoglobin beta embryonic 2 mRNA cDNA 2007 01 04 2007 04 18 BC142787 Danio rerio hemoglobin beta embryonic 1 mRNA cDNA 2007 06 11 BX842577 Mycobacterium tuberculosis H37Rw complete genome 2006 11 14
400. ses the last step looks like figure 9 3 g Find Open Reading Frames x 1 Select nucleotide sequences 2 Set parameters 3 Result handling Output options V Add annotation to sequence Y Create table Result handling o Open Save Log handling Make log D Gennes om Figure 9 3 Analyses which also generate tables In addition to the Open and Save options you can also choose whether the result of the analysis should be added as annotations on the sequence or shown on a table If both options are selected you will be able to click the results in the table and the corresponding region on the sequence will be selected CHAPTER 9 BATCHING AND RESULT HANDLING 120 If you choose to add annotations to the sequence they can be removed afterwards by clicking Undo in the Toolbar 9 1 2 Batch log For some analyses there is an extra option in the final step to create a log of the batch process see e g figure 9 3 This log will be created in the beginning of the process and continually updated with information about the results See an example of a log in figure 9 4 In this example the log displays information about how many open reading frames were found EX log Rows 9 Log Filter Name Description Type Time AY738615 Found 10 reading frames Fri Nov 17 HUMDINUC Found 5 reading Frames Fri Now 17 PERHIBA Found 5 reading Frames Fri Now 17 PERH1BB
401. should be used as a rule of thumb and deviations from the rule may occur e Cornette Cornette et al computed an optimal hydrophobicity scale based on 28 published scales Cornette et al 1987 This optimized scale is also suitable for prediction of alpha helices in proteins e Engelman The Engelman hydrophobicity scale also known as the GES scale is another scale which can be used for prediction of protein hydrophobicity Engelman et al 1986 As the Kyte Doolittle scale this scale is useful for predicting transmembrane regions in proteins e Eisenberg The Eisenberg scale is a normalized consensus hydrophobicity scale which shares many features with the other hydrophobicity scales Eisenberg et al 1984 e Rose The hydrophobicity scale by Rose et al is correlated to the average area of buried amino acids in globular proteins Rose et al 1985 This results in a scale which is not showing the helices of a protein but rather the surface accessibility e Janin This scale also provides information about the accessible and buried amino acid residues of globular proteins Janin 1979 e Hopp Woods Hopp and Woods developed their hydrophobicity scale for identification of potentially antigenic sites in proteins This scale is basically a hydrophilic index where apolar residues have been assigned negative values Antigenic sites are likely to be predicted when using a window size of 7 Hopp and Woods 1983 e Welling Welling et
402. st layout Gather sequences ak top ill Blast hit coloring Sequence color Identity 407 100 Sequence layout Numbers on sequences br tE Descrip Ion A Query start Query end Identity Positive PR E o 5204607 31 106 99 PF Score 5203539 5204827 E 147 98 C Btt score 5212021 31 106 95 5210901 105 147 88 Hit start 5212239 1 33 sa Hit end 5232322 31 106 78 5247484 31 106 75 LJ Hit length E ia 5 E Query start Query end C Identity hd Figure 2 16 Placement of translated nucleotide sequence hits on the Human beta globin Verify the result Open NC_000011 in a view and go to the Hit start position 5 204 729 and zoom to see the blue gene annotation You can now see the exon structure of the Human beta globin gene showing the three exons on the reverse strand see figure 2 17 NC 000011 select 1 000 5 203 500 5 204 000 5 204 500 3 205 000 HBB HBB HBB a Figure 2 17 Human beta globin exon view If you wish to verify the result make a selection covering the gene region and open it in a new view right click Open Selection in New View L Save 5 Save the sequence and perform a new BLAST search CHAPTER 2 TUTORIALS 50 e Use the new sequence as query e Use BLASTx e Use the protein sequence AAA16334 as database Using the genomic sequence as query the mapping of the protein sequence to the exons is visually very clear as shown
403. sters of the data in separate Workspaces All Workspaces are automatically saved when closing down CLC Protein Workbench The next time you run the program the Workspaces are reopened exactly as you left them Note It is not possible to run more than one version of CLC Protein Workbench at a time Use two or more Workspaces instead 3 5 1 Create Workspace When working with large amounts of data it might be a good idea to split the work into two or more Workspaces As default the CLC Protein Workbench opens one Workspace Additional Workspaces are created in the following way Workspace in the Menu Bar Create Workspace enter name of Workspace OK When the new Workspace is created the heading of the program frame displays the name of the new Workspace Initially the selected elements in the Navigation Area is collapsed and the View Area is empty and ready to work with See figure 3 18 3 5 2 Select Workspace When there is more than one Workspace in the CLC Protein Workbench there are two ways to switch between them CHAPTER 3 USER INTERFACE 80 CLC Protein Workbench 3 0 Current workspace Default File Edit Search View Toolbox Workspace Help ls g de eS Sl Se YO Em cido PP Show New Import Export Cut Copy Paste Delete Workspace Search Selection Zoom In Zoom Out faja CLC Data Example data Nucleotide H E protein w Extra README E Recycle bin 1 E ES Alignments and Trees A Gene
404. store deleted elements see section 3 1 7 You can set the number of possible undo actions in the Preferences dialog see section 5 3 2 6 Arrange views in View Area Views are arranged in the View Area by their tabs The order of the views can be changed using drag and drop E g drag the tab of one view onto the tab of a another The tab of the first view is now placed at the right side of the other tab lf a tab is dragged into a view an area of the view is made gray see fig 3 12 illustrating that the view will be placed in this part of the View Area The results of this action is illustrated in figure 3 13 You can also split a View Area horizontally or vertically using the menus Splitting horisontally may be done this way right click a tab of the view View Split Horizontally 3 This action opens the chosen view below the existing view See figure 3 14 When the split is made vertically the new view opens to the right of the existing view Splitting the View Area can be undone by dragging e g the tab of the bottom view to the tab of the top view This is marked by a gray area on the top of the view CHAPTER 3 USER INTERFACE 4 act P68046 O ar Pos053 O i PF68225 RLLVVYPWTQRFFESFGDLSSPDAVMGNPK P6s225 VKAHGKKVLGAFSDGLNHLDNLKGTFAQLS PF68225 ELHCDKLHVDPENFKLLGNVLVCVLAHHFG Figure 3 12 When dragging a view a gray area indicates where the view will be shown ast POSO46 O per Pagosa O act P6S0
405. sult of the analysis can be seen in figure 2 23 ac ATP8a1 O ha 4S 411 mi Spacing Trypsin Trypsin E pe pee po Trypsin Sequence layout No spacing No wrap ATP8al ETNLKIRQGLPATSDIKDIDSLMRISGRIECESPNRHL Auto wrap EE ATP8a1 proteo o able Settings x Rows 26 Table of remaining fragments based on parameter settings Filter All v ES 2 Automatic w Start End posi Length Mass pI C end Name Fragment N end Name Show column 28 37 10 1 147 19 4 27 K START TSLADQEEVR T Trypsin 7 Start position 38 48 11 1 302 52 9 75 R Trypsin TIFINQPQLTK E Trypsin ESA a o Y End position 49 58 10 1 120 24 9 22 K Trypsin FCNNHVSTAK Y Trypsin 140 150 11 1 368 50 5 58 R Trypsin NGAWEIVHWEK Vv Trypsin Z Length 151 160 10 1 069 30 7 07 K Trypsin VNVGDIVIIK G Trypsin 7 Mass 198 207 10 1 029 15 6 80 R Trypsin QGLPATSDIK D Trypsin ax Figure 2 23 The output of the proteolytic cleavage shows the cleavage sites as annontations in the protein sequence The accompanying table lists all the fragments which are between 10 and 15 amino acids long Note The output of proteolytic cleavage is two related views The sequence view displays annotations where the sequence is cleaved The table view shows information about the fragments satisfying the parameters set in the dialog Subsequently if you have restricted the fragment parameters you might have more annotations on th
406. t a result that looks like figure 6 4 This means that you also print the part of the sequence which is not visible when you have zoomed in CHAPTER 6 PRINTING 100 Figure 6 4 A print of the sequence selecting Print whole view The whole sequence is shown even though the view is zoomed in on a part of the sequence 6 2 Page setup No matter whether you have chosen to print the visible area or the whole view you can adjust page setup of the print An example of this can be seen in figure 6 5 E Page Setup O Portrait Landscape Paper Size A4 X Fit to pages Horizontal pages Vertical pages wf ok X Cancel Help Figure 6 5 Page Setup In this dialog you can adjust both the setup of the pages and specify a header and a footer by clicking the tab at the top of the dialog You can modify the layout of the page using the following options e Orientation Portrait Will print with the paper oriented vertically Landscape Will print with the paper oriented horizontally e Paper size Adjust the size to match the paper in your printer e Fit to pages Can be used to control how the graphics should be split across pages see figure 6 6 for an example Horizontal pages If you set the value to e g 2 the printed content will be broken up horizontally and split across 2 pages This is useful for Sequences that are not wrapped Vertical pages If you set the value to e g 2 th
407. t shares some of the advanced product features of CLC Protein Workbench and it has additional advanced features CLC Main Workbench holds all basic and advanced features of the CLC Workbenches In June 2007 CLC RNA Workbench was released as a sister product of CLC Protein Workbench and CLC DNA Workbench CLC Main Workbench now also includes all the features of CLC RNA Workbench In March 2008 the CLC Free Workbench changed name to CLC Sequence Viewer In June 2008 the first version of the CLC Genomics Workbench was released due to an extraordinary demand for software capable of handling sequencing data from the new high throughput sequencing systems like 454 Illumina Genome Analyzer and SOLID For an overview of which features all the applications include see http www clcbio com features In December 2006 CLC bio released a Software Developer Kit which makes it possible for anybody with a knowledge of programming in Java to develop plug ins The plug ins are fully integrated with the CLC Workbenches and the Viewer and provide an easy way to customize and extend their functionalities All our software will be improved continuously If you are interested in receiving news about updates you should register your e mail and contact data on http www clcbio com if you haven t already registered when you downloaded the program 1 5 1 New program feature request The CLC team is continuously improving the CLC Protein Workbench with o
408. taneously process data from multiple species Siepel and Haussler 2004 Through the comparative approach valuable evolutionary information can be obtained about which amino acid substitutions are functionally tolerant to the organism and which are not This information can be used to identify substitutions that affect protein function and stability and is of major importance to the study of proteins Knudsen and Miyamoto 2001 Knowledge of the underlying phylogeny is however paramount to comparative methods of inference as the phylogeny describes the underlying correlation from shared history that exists between data from different species In molecular epidemiology of infectious diseases phylogenetic inference is also an important tool The very fast substitution rate of microorganisms especially the RNA viruses means that these show substantial genetic divergence over the time scale of months and years Therefore the phylogenetic relationship between the pathogens from individuals in an epidemic can be resolved and contribute valuable epidemiological information about transmission chains and epidemiologically significant events Leitner and Albert 1999 Forsberg et al 2001 19 2 3 Reconstructing phylogenies from molecular data Traditionally phylogenies have been constructed from morphological data but following the growth of genetic information it has become common practice to construct phylogenies based on molecular data known as
409. te the file containing your license No file selected Choose License File If you experience any problems please contact The CLC Support Team Proxy Settings Previous Next Quit Workbench Figure 1 10 Importing the license downloaded from the web site Click the Choose License File button and browse to find the license file you saved before e g on your Desktop When you have selected the file click Next Accepting the license agreement Regardless of which option you chose above you will now see the dialog shown in figure 1 11 Please read the License agreement carefully before clicking accept these terms and Finish CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 21 License Wizard E p CLC Protein Workbench License Agreement Please read and accept the license agreement below to begin using you license END USER LICENSE AGREEMENT FOR CLC BIO SOFTWARE 4 CLC Genomics Workbench 1 0 E 1 Recitals 1 1 This End User License Agreement EULA is a legal agreement between you either an individual person or a single legal entity who will be referred to in this EULA as You and CLC bio A S CVR no 28 30 50 87 for the software products that accompanies this EULA including any associated media printed materials and electronic documentation the Software Product I accept these terms If you experience any problems please contact The CLC Support Team Figure 1 11 Read t
410. tein sequences set parameters 2 Set parameters Codons Use random codon Use only the most frequent codon Use codon based on frequency distribution A Transfer annotations Map annotations to reverse translated sequence COI Ctro ome vee Xena Figure 16 22 Choosing parameters for the reverse translation e Use random codon This will randomly back translate an amino acid to a codon without using the translation tables Every time you perform the analysis you will get a different result e Use only the most frequent codon On the basis of the selected translation table this parameter option will assign the codon that occurs most often When choosing this option the results of performing several reverse translations will always be the same contrary to the other two options e Use codon based on frequency distribution This option is a mix of the other two options The selected translation table is used to attach weights to each codon based on its frequency The codons are assigned randomly with a probability given by the weights A more frequent codon has a higher probability of being selected Every time you perform the analysis you will get a different result This option yields a result that is closer to the translation behavior of the organism assuming you choose an appropriate codon frequency table e Map annotations to reverse translated sequence If this checkbox is checked then all annot
411. teins from different subcellular localizations This fact has been used in several computational methods used for prediction of subcellular localization Annotation table This table provides an overview of all the different annotations associated with the sequence and their incidence Dipeptide distribution This measure is simply a count or frequency of all the observed adjacent pairs of amino acids dipeptides found in the protein It is only possible to report neighboring amino acids Knowledge on dipeptide composition have previously been used for prediction of subcellular localization Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents 14 5 Join sequences CLC Protein Workbench can join several nucleotide or protein sequences into one sequence This feature can for example be used to construct supergenes for phylogenetic inference by joining several disjoint genes
412. ter the protein has reached the correct and final destination Some of the best characterized signal peptides are depicted in figure 16 3 Numerous methods for prediction of protein targeting and signal peptides have been developed some of them are mentioned and cited in the introduction of the SignalP research paper Bendtsen et al 2004b However no prediction method will be able to cover all the different types of signal peptides Most methods predicts classical signal peptides targeting to the general secretory pathway in bacteria or classical secretory pathway in eukaryotes Furthermore a few methods for prediction of non classically secreted proteins have emerged Bendtsen et al 2004a Bendtsen et al 2005 Prediction of signal peptides and subcellular localization In the search for accurate prediction of signal peptides many approaches have been investigated Almost 20 years ago the first method for prediction of classical signal peptides was published von Heijne 1986 Nowadays more sophisticated machine learning methods such as neural networks support vector machines and hidden Markov models have arrived along with the increasing computational power and they all perform superior to the old weight matrix based methods Menne et al 2000 Also many other classical statistical approaches have been carried out often in conjunction with machine learning methods In the following sections a wide range of different signal pepti
413. th similar residues Background color Sets a background color of the residues using a gradient in the same way as described above Logo Displays sequence logo at the bottom of the alignment x Height Specifies the height of the sequence logo graph Color The sequence logo can be displayed in black or Rasmol colors For protein alignments a polarity color scheme is also available wnere hydrophobic residues are shown in black color hydrophilic residues as green acidic residues as red and basic residues as blue 18 2 1 Bioinformatics explained Sequence logo In the search for homologous sequences researchers are often interested in conserved sites residues or positions in a sequence which tend to differ a lot Most researches use alignments see Bioinformatics explained multiple alignments for visualization of homology on a given set of either DNA or protein sequences In proteins active sites in a given protein family are often highly conserved Thus in an alignment these positions which are not necessarily located in proximity are fully or nearly fully conserved On the other hand antigen binding sites in the Fap unit of immunoglobulins tend to differ quite a lot whereas the rest of the protein remains relatively unchanged In DNA promoter sites or other DNA binding sites are highly conserved see figure 18 8 This is also the case for repressor sites as seen for the Cro repressor of bacteriophage A When aligning
414. that protein sequences are evolutionarily more conserved than nucleotide sequences Another good reason for translating the query sequence before the search is that you get protein hits which are likely to be annotated Thus you can directly see the protein function of the sequenced gene 12 5 5 Which BLAST options should change The NCBI BLAST web pages and the BLAST command line tool offer a number of different options which can be changed in order to obtain the best possible result Changing these parameters can have a great impact on the search result It is not the scope of this document to comment on all of the options available but merely the options which can be changed with a direct impact on the search result The E value The expect value E value can be changed in order to limit the number of hits to the most significant ones The lower the E value the better the hit The E value is dependent on the length of the query sequence and the size of the database For example an alignment obtaining an E value of 0 05 means that there is a 5 in 100 chance of occurring by chance alone E values are very dependent on the query sequence length and the database size Short identical sequence may have a high E value and may be regarded as false positive hits This is often seen if one searches for short primer regions small domain regions etc The default threshold for the E value on the BLAST web page is 10 Increasing this value will most li
415. the contents 16 6 Pfam domain search With CLC Protein Workbench you can perform a search for Pfam domains on protein se quences The Pfam database at http pfam sanger ac uk is a large collection of multiple sequence alignments that covers approximately 9318 protein domains and protein families Bateman et al 2004 Based on the individual domain alignments profile HMMs have been developed These profile HMMs can be used to search for domains in unknown sequences Many proteins have a unique combination of domains which can be responsible for instance for the catalytic activities of enzymes Pfam was initially developed to aid the annotation of the C elegans genome Annotating unknown sequences based on pairwise alignment methods by simply transferring annotation from a known protein to the unknown partner does not take domain organization into account Galperin and Koonin 1998 An unknown protein may be annotated wrongly for instance as an enzyme if the pairwise alignment only finds a regulatory domain Using the Pfam search option in CLC Protein Workbench you can search for domains in sequence data which otherwise do not carry any annotation information The Pfam search option adds all found domains onto the protein sequence which was used for the search If domains of no relevance are found they can easily be removed as described in section 10 3 4 Setting a lower cutoff value will result in fewer domains CHAPTER 16 PROTEIN ANA
416. the corresponding codon Four different codons can be used for this reverse translation GCU GCC GCA or GCG By picking either one by random choice we will get an alanine The most frequent codon coding for an alanine in E coli is GCG encoding 33 7 of all alanines Then comes GCC 25 5 GCA 20 3 and finally GCU 15 3 The data are retrieved from the Codon usage database see below Always picking the most frequent codon does not necessarily give the best answer By selecting codons from a distribution of calculated codon frequencies the DNA sequence obtained after the reverse translation holds the correct or nearly correct codon distribution It CHAPTER 16 PROTEIN ANALYSES 259 should be kept in mind that the obtained DNA sequence is not necessarily identical to the original one encoding the protein in the first place due to the degeneracy of the genetic code In order to obtain the best possible result of the reverse translation one should use the codon frequency table from the correct organism or a closely related species The codon usage of the mitochondrial chromosome are often different from the native chromosome s thus mitochondrial codon frequency tables should only be used when working specifically with mitochondria Other useful resources The Genetic Code at NCBI http www ncbi nlm nih gov Taxonomy Utils wprintgc cgi mode c Codon usage database http www kazusa or jp codon Wikipedia on the genetic cod
417. the data Bayesian inference The objective of Bayesian phylogenetic inference is not to infer a single correct phylogeny but rather to obtain the full posterior probability distribution of all possible phylogenies This is obtained by combining the likelihood and the prior probability distribution of evolutionary parameters The vast number of possible trees means that bayesian phylogenetics must be performed by approximative Monte Carlo based methods Larget and Simon 1999 Yang and Rannala 1997 19 2 4 Interpreting phylogenies Bootstrap values A popular way of evaluating the reliability of an inferred phylogenetic tree is bootstrap analysis The first step in a bootstrap analysis is to re sample the alignment columns with replacement l e In the re sampled alignment a given column in the original alignment may occur two or more times while some columns may not be represented in the new alignment at all The re sampled alignment represents an estimate of how a different set of sequences from the same genes and the same species may have evolved on the same tree If a new tree reconstruction on the re sampled alignment results in a tree similar to the original one this increases the confidence in the original tree If on the other hand the new tree looks very different it means that the inferred tree is unreliable By re sampling a number of times it is possibly to put reliability weights on each internal branch of the inferred
418. the enzyme database for your installation see section CHAPTER 17 RESTRICTION SITE ANALYSES 211 If you wish to use all the enzymes in the list Click in the panel to the left press Ctrl A 38 A on Mac Add gt The enzymes can be sorted by clicking the column headings i e Name Overhang Methylation or Popularity This is particularly useful if you wish to use enzymes which produce e g a 3 overhang In this case you can sort the list by clicking the Overhang column heading and all the enzymes producing 3 overhangs will be listed together for easy selection When looking for a specific enzyme it is easier to use the Filter If you wish to find e g Hindlll sites simply type Hindlll into the filter and the list of enzymes will shrink automatically to only include the Hindlll enzyme This can also be used to only show enzymes producing e g a 3 overhang as shown in figure 17 20 Restriction Site Analysis 1 Select DNA RNA _ enzymes to be conside ahammar sequence s Enzyme list 2 Enzymes to be considered Are v Use existing enzyme list Popular enzymes v in calculation gency P Y Enzymes in Popular en Enzymes to be used Filter 3 Filter Name Overhang Methylat Popul Name Overhang Methyla Pop PstI Egca 5 N6 met Pee KpnI gtac S N met Peer Sacl agct 5 S meth SphI cata FAN Apal ggcc 5 S meth Ball Ann 5 N4 met
419. the graph below the sequence furthermore hydrophobic regions are colored on the sequence Red indicates regions with high hydrophobicity and blue indicates regions with low hydrophobicity The hydrophobicity is calculated by sliding a fixed size window of an odd number over the protein sequence At the central position of the window the average hydrophobicity of the entire window is plotted see figure 16 15 Hydrophobicity scales Several hydrophobicity scales have been published for various uses Many of the commonly used hydrophobicity scales are described below Kyte Doolittle scale The Kyte Doolittle scale is widely used for detecting hydrophobic regions in proteins Regions with a positive value are hydrophobic This scale can be used for identifying both surface exposed regions as well as transmembrane regions depending on the window size used Short window sizes of 5 generally work well for predicting putative surface exposed regions Large window sizes of 19 21 are well suited for finding transmembrane domains if the values calculated are above 1 6 Kyte and Doolittle 1982 These values should be used as a rule of thumb and deviations from the rule may occur Engelman scale The Engelman hydrophobicity scale also known as the GES scale is another scale which can be used for prediction of protein hydrophobicity Engelman et al 1986 As the Kyte Doolittle scale this scale is useful for predicting transmembrane regions in prot
420. the residues according to the polarity of amino acids Foreground color Sets the color of the letter Click the color box to change the color Background color Sets the background color of the residues Click the color box to change the color e Trace colors only DNA Colors the residues according to the color conventions of chromatogram traces A green C blue G black and T red Foreground color Sets the color of the letter Background color Sets the background color of the residues Nucleotide info These preferences only apply to nucleotide sequences e Translation Displays a translation into protein just below the nucleotide sequence Depending on the zoom level the amino acids are displayed with three letters or one letter Frame Determines where to start the translation x ORF CDS If the sequence is annotated the translation will follow the CDS or ORF annotations If annotations overlap only one translation will be shown If only one annotation is visible the Workbench will attempt to use this annotation to mark the start and stop for the translation In cases where this is not possible the first annotation will be used i e the one closest to the 5 end of the sequence CHAPTER 10 VIEWING AND EDITING SEQUENCES 126 Selection This option will only take effect when you make a selection on the sequence The translation will start from the first nucleotide selected Making a new selection w
421. the server you can borrow a license Borrowing a license means that you take one of the floating licenses available on the server and borrow it for a specified amount of time During this time period there will be one less floating license available on the server At the point where you wish to borrow a license you have to be connected to the license server The procedure for borrowing is this Click Help License Manager to display the dialog shown in figure 1 22 Use the checkboxes to select the license s that you wish to borrow Select how long time you wish to borrow the license and click Borrow Licenses You can now go offline and work with CLC Protein Workbench Oo BRB WN PB When the borrow time period has elapsed you have to connect to the license server again to use CLC Protein Workbench 6 When the borrow time period has elapsed the license server will make the floating license available for other users Note that the time period is not the period of time that you actually use the Workbench Note When your organization s license server is installed license borrowing can be turned off In that case you will not be able to borrow licenses CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 26 No license available If all the licenses on the server are in use you will see a dialog as shown in figure 1 20 when you start the Workbench No valid license found X XX CLC Network Licensing The Fo
422. tide 321 peptide 321 shared BLAST database 1 3 structure 154 UniProt 152 Db source 141 db xref references 159 Delete element 08 residues and gaps in alignment 292 workspace 80 Description 141 batch edit 69 DGE 312 Digital gene expression 312 DIP detection 311 Dipeptide distribution 214 Discovery studio file format 327 Distance pairwise comparison of sequences in alignments 298 DNA translation 228 DNAstrider file format 327 Dot plots 314 Bioinformatics explained 200 create 197 print 199 Double cutters 267 Double stranded DNA 123 342 Download and open search results GenBank 151 15 7 search results UniProt 154 Download and save search results GenBank 151 157 search results UniProt 154 Download of CLC Protein Workbench 12 Drag and drop Navigation Area 65 search results GenBank 151 157 search results UniProt 154 DS Gene file format 327 Edit alignments 292 313 annotations 137 139 312 enzymes 268 sequence 130 sequences 312 single bases 130 Element delete 68 rename 68 embl file format 329 Embl file format 327 Encapsulated PostScript export 111 End gap cost 284 End gap costs cheap end caps 284 free end gaps 284 Enzyme list 2 8 create 2 8 edit 280 view 280 eps format export 111 Error reports 28 Example data import 30 Excel export file format 329 Expand selection 129 Expect BLAST search 170 Export bioinformatic data 107
423. tion Prediction of signal peptides SignalP Transmembrane helix prediction TMHMM Secondary protein structure prediction PFAM domain search Viewer E Viewer y E Viewer Protein E Protein y EI Protein y DNA RNA Main DNA RNA Main DNA RNA Main 313 Genomics E Genomics E E Genomics E APPENDIX A COMPARISON OF WORKBENCHES Sequence alignment Multiple sequence alignments Two algo rithms Advanced re alignment and fix point align ment options Advanced alignment editing options Join multiple alignments into one Consensus sequence determination and management Conservation score along sequences Sequence logo graphs along alignments Gap fraction graphs Copy annotations between sequences in alignments Pairwise comparison RNA secondary structure Advanced prediction of RNA secondary struc ture Integrated use of base pairing constraints Graphical view and editing of secondary struc ture Info about energy contributions of structure elements Prediction of multiple sub optimal structures Evaluate structure hypothesis Structure scanning Partition function Dot plots Dot plot based analyses Phylogenetic trees Neighbor joining and UPGMA phylogenies Maximum likelihood phylogeny of nucleotides Pattern discovery Search for sequence match Motif search for basic patterns Motif search with regular expressions Motif search with ProSite patterns Pattern discovery Viewer E V
424. tions of one type right click an annotation of the type you want to remove Delete Delete Annota tions of Type type If you want to remove all annotations from a sequence CHAPTER 10 VIEWING AND EDITING SEQUENCES 141 right click an annotation Delete Delete All Annotations r The removal of annotations can be undone using Ctrl Z or Undo in the Toolbar If you have more sequences e g in a sequence list alignment or contig you have two additional options right click an annotation Delete Delete All Annotations from All Sequences right click an annotation Delete Delete Annotations of Type type from All Sequences 10 4 Element information The normal view of a sequence by double clicking shows the annotations as boxes along the sequence but often there is more information available about sequences This information is available through the Element info view To view the sequence information select a sequence in the Navigation Area Show in the Toolbar Element info 157 This will display a view similar to fig 10 13 Name Edit gt Description Edit Comments Edit KeyWords Edit Db Source Gb Division Length Modification Date Latin name Edit Common name Edit Taxonomy name Edit Figure 10 13 The initial display of sequence info for the HUMHBB DNA sequence from the Example data All the lines in the view are headings and the corresponding text can b
425. tire word is required before an extension is initiated so that you normally regulate the sensitivity and speed of the search by increasing or decreasing the wordsize For other BLAST searches non exact word matches are taken into account based upon the similarity between words The amount of similarity can be varied so that you normally uses just the wordsizes 2 and 3 for these searches e Matrix A key element in evaluating the quality of a pairwise sequence alignment is the substitution matrix which assigns a score for aligning any possible pair of residues The matrix used in a BLAST search can be changed depending on the type of Sequences you are searching with see the BLAST Frequently Asked Questions Only applicable for protein sequences or translated DNA sequences e Gap Cost The pull down menu shows the Gap Costs Penalty to open Gap and penalty to extend Gap Increasing the Gap Costs and Lambda ratio will result in alignments which decrease the number of Gaps introduced e Max number of hit sequences The maximum number of database sequences where BLAST found matches to your query sequence to be included in the BLAST report CHAPTER 12 BLAST SEARCH 165 The parameters you choose will affect how long BLAST takes to run A search of a small database requesting only hits that meet stringent criteria will generally be quite quick Searching large databases or allowing for very remote matches will of course take longer Click N
426. to copy Ctrl C 38 C on Mac select where to insert files Ctrl P 3 P on Mac or select the files to copy Edit in the Menu Bar Copy 755 select where to insert files Edit in the Menu Bar Paste 71 If there is already an element of that name the pasted element will be renamed by appending a number at the end of the name Elements can also be moved instead of copied This is done with the cut paste function select the files to cut right click one of the selected files Cut right click the location to insert files into Paste C gt or select the files to cut Ctrl X 38 X on Mac select where to insert files Ctrl V 38 Von Mac When you have cut the element it is greyed out until you activate the paste function If you change your mind you can revert the cut command by copying another element Note that if you move data between locations the original data is kept This means that you are essentially doing a copy instead of a move operation Move using drag and drop Using drag and drop in the Navigation Area as well as in general is a four step process click the element click on the element again and hold left mouse button drag the element to the desired location let go of mouse button This allows you to e Move elements between different folders in the Navigation Area e Drag from the Navigation Area to the View Area A new view is opened in an existing View Area i
427. to the hit sequences e Annotations BLAST can also be used to map annotations from one organism to another or look for common genes in two related species 12 5 2 Searching for homology Most research projects involving sequencing of either DNA or protein have a requirement for obtaining biological information of the newly sequenced and maybe unknown sequence If the researchers have no prior information of the sequence and biological content valuable information can often be obtained using BLAST The BLAST algorithm will search for homologous sequences in predefined and annotated databases of the users choice In an easy and fast way the researcher can gain knowledge of gene or protein function and find evolutionary relations between the newly sequenced DNA and well established data After the BLAST search the user will receive a report specifying found homologous sequences and their local alignments to the query sequence 12 5 3 How does BLAST work BLAST identifies homologous sequences using a heuristic method which initially finds short matches between two sequences thus the method does not take the entire sequence space into account After initial match BLAST attempts to start local alignments from these initial matches This also means that BLAST does not guarantee the optimal alignment thus some sequence hits may be missed In order to find optimal alignments the Smith Waterman algorithm Should be used see below In the followin
428. torial Align protein sequences 000 lt lt lt 1 54 2 8 1 The alignment dialog lt Bo eee Cee eee AR we SOS as 54 2 9 Tutorial Create and modify a phylogenetic tree 56 2 9 1 Me ast oe a ee ae AEDES Ad E 56 2 10 Tutorial Find restriction sites 1 ee 57 2 10 1 The Side Panel way of finding restriction sites 57 2 10 2 The Toolbox way of finding restriction sites 58 36 CHAPTER 2 TUTORIALS 37 This chapter contains tutorials representing some of the features of CLC Protein Workbench The first tutorials are meant as a short introduction to operating the program The last tutorials give examples of how to use some of the main features of CLC Protein Workbench Watch video tutorials at http www clcbio com tutorials 2 1 Tutorial Getting started This brief tutorial will take you through the most basic steps of working with CLC Protein Workbench The tutorial introduces the user interface shows how to create a folder and demonstrates how to import your own existing data into the program When you open CLC Protein Workbench for the first time the user interface looks like figure 2 1 CLC Protein Workbench 3 0 Current workspace Default SE File Edit Search View Toolbox Workspace Help DES Si DD EI E ok A Show New Import Expor Cut Copy Paste Delete Workspace Search Selection Zoom In Zoom Out y FED Y c ha CLC Data
429. tree If the data was bootstrapped a 100 times a bootstrap score of 100 means that the corresponding branch occurs in all 100 trees made from re sampled alignments Thus a high bootstrap score is a sign of greater reliability Other useful resources The Tree of Life web project http tolweb org Joseph Felsensteins list of phylogeny software http evolution genetics washington edu phylip software html Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents Part IV Appendix Appendix A Comparison of workbenches Below we list a number of functionalities that differ between CLC Workbenches and the CLC Sequence Viewer e CLC Sequence Viewer m e CLC Protein Workbench m e CLC DNA Workbench m e CLC RNA Workbench m e CLC Main Workbench a e CLC Genomics Workbench m Data handling Viewer Protein Add multiple locations to Navigation Area E Share data on
430. ts 13 Open at UniProt ch Figure 11 3 The UniProt search view 11 2 1 UniProt search options Conducting a search in UniProt from CLC Protein Workbench corresponds to conducting the search on UniProt s website When conducting the search from CLC Protein Workbench the results are available and ready to work with straight away Above the search fields you can choose which database to search e Swiss Prot This is believed to be the most accurate and best quality protein database available All entries in the database has been currated manually and data are entered according to the original research paper e TrEMBL This database contain computer annotated protein sequences thus the quality of the annotations is not as good as the Swiss Prot database As default CLC Protein Workbench offers one text field where the search parameters can be entered Click Add search parameters to add more parameters to your search Note The search is a and search meaning that when adding search parameters to your search you search for both or all text strings rather than any of the text strings You can append a wildcard character by checking the checkbox at the bottom This means that you only have to enter the first part of the search text e g searching for genom will find both genomic and genome CHAPTER 11 ONLINE DATABASE SEARCH 153 The following parameters can be added to the search e All fields Te
431. tton 5 at the top of the Side Panel and click Save Settings see figure 2 8 Delete Settings Apply Saved Settings P Figure 2 8 Saving the settings of the Side Panel This will open the dialog shown in figure 2 9 y Save Settings Ez Please enter a name for these user settings my settings Always apply these settings Figure 2 9 Dialog for saving the settings of the Side Panel In this way you can save the current state of the settings in the Side Panel so that you can apply CHAPTER 2 TUTORIALS 43 them to alignments later on If you check Always apply these settings these settings will be applied every time you open a view of the alignment Type My settings in the dialog and click Save 2 3 2 Applying saved settings When you click the Save Restore Settings button i again and select Apply Saved Settings you will see My settings in the menu together with some pre defined settings that the CLC Protein Workbench has created for you see figure 2 10 za a Save Settings Delete Settings Apply Saved Settings H Black white Conservation color Mon compack Show annotations my settings CLC Standard Settings Figure 2 10 Menu for applying saved settings Whenever you open an alignment you will be able to apply these settings Each kind of view has its own list of settings that can be applied At the bottom of the list you will see the CLC Standard Settings which are the d
432. two options You can click Install Example Data c in the Help menu of the program This installs the data automatically You can also go to http www clcbio com download and download the example data from there lf you download the file from the website you need to import it into the program See chapter 7 1 for more about importing data 1 7 Plug ins When you install CLC Protein Workbench it has a standard set of features However you can upgrade and customize the program using a variety of plug ins As the range of plug ins is continuously updated and expanded they will not be listed here Instead we refer to http www clcbio com plug ins for a full list of plug ins with descriptions of their functionalities 1 7 1 Installing plug ins Plug ins are installed using the plug in manager Help in the Menu Bar Plug ins and Resources E or Plug ins in the Toolbar The plug in manager has four tabs at the top e Manage Plug ins This is an overview of plug ins that are installed e Download Plug ins This is an overview of available plug ins on CLC bio s server In order to install plug ins on Windows Vista the Workbench must be run in administrator mode Right click the program shortcut and choose Run as Administrator Then follow the procedure described below CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 31 e Manage Resources This is an overview of resources that are installed e Download Resources
433. u move the mouse pointer over the label the pointer will turn into a vertical arrow indicating that the sequence can be moved The sequences can also be sorted automatically to let you save time moving the sequences around To sort the sequences alphabetically Right click the name of a sequence Sort Sequences Alphabetically If you change the Sequence name in the Sequence Layout view preferences you will have to ask the program to sort the sequences again The sequences can also be sorted by similarity grouping similar sequences together Right click the name of a sequence Sort Sequences by Similarity 18 3 6 Delete rename and add sequences Sequences can be removed from the alignment by right clicking the label of a sequence right click label Delete Sequence This can be undone by clicking Undo in the Toolbar If you wish to delete several Sequences you can check all the sequences right click and choose CHAPTER 18 SEQUENCE ALIGNMENT 294 Delete Marked Sequences To show the checkboxes you first have to click the Show Selection Boxes in the Side Panel A sequence can also be renamed right click label Rename Sequence This will show a dialog letting you rename the sequence This will not affect the sequence that the alignment is based on Extra sequences can be added to the alignment by creating a new alignment where you select the current alignment and the extra sequences see section 18 1 The same pro
434. u want to show enzymes that cut the sequence only once This means that you should de select the Two restriction sites checkbox Click Next and select that you want to Add restriction sites as annotations on sequence and Create restriction map See figure 2 31 EB Restriction Site Analysis ES 1 Select DNA RNA Number of cut sites sequence s 2 Enzymes to be considered in calculation 3 Number of cut sites Display enzymes with No restriction site 0 One restriction site 1 E Two restriction sites 2 Three restriction sites 3 N restriction sites Any number of restriction sites gt 0 CSA Previous gt Next X cancel Figure 2 31 Selecting output for restriction map analysis Click Finish to start the restriction map analysis CHAPTER 2 TUTORIALS 59 View restriction site The restriction sites are shown in two views one view is in a tabular format and the other view displays the sites as annotations on the sequence The result is shown in figure 2 32 The restriction map at the bottom can also be shown as a act ATP8al mRNA E ATP8al MRNA GTGGGAGGCGCGGCCCCGCGGCAGCTGAGCCCTCTGCGG Filter All Pattern Overhang Number of c Cut position s agtacec 3 1 1208 cogcgg 3 1 119 Figure 2 32 The result of the restriction map analysis is displayed in a table at the bottom and as annotations on the sequence in the view at the top table of fragments produced by cuttin
435. ucleotide sequence databases nr All GenBank EMBL DDBJ PDB sequences but no EST STS GSS or phase 0 1 or 2 HTGS sequences No longer non redundant due to computational cost refseq_rna MRNA sequences from NCBI Reference Sequence Project refseq_genomic Genomic sequences from NCBI Reference Sequence Project est Database of GenBank EMBL DDBJ sequences from EST division est human Human subset of est 321 APPENDIX D BLAST DATABASES 322 e est mouse Mouse subset of est e est others Subset of est other than human or mouse e gss Genome Survey Sequence includes single pass genomic data exon trapped se quences and Alu PCR sequences e htgs Unfinished High Throughput Genomic Sequences phases O 1 and 2 Finished phase 3 HTG sequences are in nr e pat Nucleotides from the Patent division of GenBank e pdb Sequences derived from the 3 dimensional structure records from Protein Data Bank They are NOT the coding sequences for the corresponding proteins found in the same PDB record e month All new or revised GenBank EMBL DDBJ PDB sequences released in the last 30 days e alu Select Alu repeats from REPBASE suitable for masking Alu repeats from query sequences See Alu alert by Claverie and Makalowski Nature 3 1 752 1994 e dbsts Database of Sequence Tag Site entries from the STS division of GenBank EMBL DDBJ e chromosome Complete genomes and complete chromosomes from the NCBI Referenc
436. ucleotide sequences and show the direction of the aligned strands Minus indicate a complementary strand e Query This is the sequence or part of the sequence which you have used for the BLAST search e Sbjct subject This is the sequence found in the database The numbers of the query and subject sequences refer to the sequence positions in the submitted and found sequences If the subject sequence has number 59 in front of the sequence this means that 58 residues are found upstream of this position but these are not included in the alignment By right clicking the sequence name in the Graphical BLAST output it is possible to download the full hits sequence from NCBI with accompanying annotations and information It is also possible to just open the actual hit sequence in a new view 12 2 4 BLAST table In addition to the graphical display of a BLAST result it is possible to view the BLAST results in a tabular view In the tabular view one can get a quick and fast overview of the results Here you can also select multiple Sequences and download or open all of these in one single step Moreover there is a link from each sequence to the sequence at NCBI These possibilities are either available through a right click with the mouse or by using the buttons below the table lf the BLAST table view was not selected in Step 4 of the BLAST search the table can be shown in the following way CHAPTER 12 BLAST SEARCH 1 1 Click the Show
437. ucture Le Ll ma i gt Next X Cancel Figure 12 2 Choose one or more sequences to conduct a BLAST search with Select one or more sequences of the same type either DNA or protein and click Next In this dialog you choose which type of BLAST search to conduct and which database to search against See figure 12 3 The databases at the NCBI listed in the dropdown box will correspond to the query sequence type you have DNA or protein and the type of blast search you have chosen to run A complete list of these databases can be found in Appendix D Here you can also read how to add additional databases available the NCBI to the list provided in the dropdown menu e BLAST at NCBI 1 Select sequences of same _Set parameters type 2 Choose program Choose program and database Program blastp Protein sequence and database lw Database Swiss Prot protein sequences swissprot M a afan Previous gt Next X Cancel Figure 12 3 Choose a BLAST Program and a database for the search BLAST programs for DNA query sequences CHAPTER 12 BLAST SEARCH 163 e BLASTn DNA sequence against a DNA database Used to look for DNA sequences with homologous regions to your nucleotide query sequence e BLASTx Translated DNA sequence against a Protein database Automatic translation of your DNA query sequence in six frames these translated sequences are then used to search a protein database e tBLAS
438. uence CHAPTER 11 ONLINE DATABASE SEARCH 150 e Gene Name Text The search parameters are the most recently used The All fields allows searches in all parameters in the NCBI database at the same time All fields also provide an opportu nity to restrict a search to parameters which are not listed in the dialog E g writing gene Feature key AND mouse in All fields generates hits in the GenBank database which contains one or more genes and where mouse appears somewhere in GenBank file You can also write e g CD9 NOT homo sapiens in All fields Note The Feature Key option is only available in GenBank when searching for nucleotide sequences For more information about how to use this syntax see http www ncbi nlm nih gov books NBK3837 When you are satisfied with the parameters you have entered click Start search Note When conducting a search no files are downloaded Instead the program produces a list of links to the files in the NCBI database This ensures a much faster search 11 1 2 Handling of GenBank search results The search result is presented as a list of links to the files in the NCBI database The View displays 50 hits at a time This can be changed in the Preferences see chapter 5 More hits can be displayed by clicking the More button at the bottom right of the View Each sequence hit is represented by text in three columns e Accession e Description Modification date e Length It i
439. uence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements Click Next to set parameters for the SignalP analysis 16 1 1 Signal peptide prediction parameter settings It is possible to set different options prior to running the analysis see figure 16 1 An organism type should be selected The default is eukaryote e Eukaryote default e Gram negative bacteria e Gram positive bacteria You can perform the analysis on several protein sequences at a time This will add annotations to all the sequences and open a view for each sequence if a signal peptide is found If no signal peptide is found in the sequence a dialog box will be shown The predictions obtained can either be shown as annotations on the sequence listed in a table or be shown as the detailed and full text output from the SignalP method This can be used to interpret borderline predictions e Add annotations to sequence CHAPTER 16 PROTEIN ANALYSES 235 E Signal Peptide Prediction BG 1 Select proteins Set paramel 2 Set parameters Organism group Eukaryotes Gram negative bacteria Gram positive bacteria X Previous gt Next Y ems Xana Figure 16 1 Setting the parameters for signal peptide prediction e Create table e Text Click Next if you wish to adjust how to handle the results see section 9 1 If not click
440. uences Nucleic Acids Res 18 20 6097 6100 Siepel and Haussler 2004 Siepel A and Haussler D 2004 Combining phylogenetic and hidden Markov models in biosequence analysis J Comput Biol 11 2 3 413 428 Smith and Waterman 1981 Smith T F and Waterman M S 1981 Identification of common molecular subsequences J Mol Biol 147 1 195 197 Sneath and Sokal 1973 Sneath P and Sokal R 1973 Numerical Taxonomy Freeman San Francisco Tobias et al 1991 Tobias J W Shrader T E Rocap G and Varshavsky A 1991 The N end rule in bacteria Science 254 5036 13 74 1377 von Heijne 1986 von Heijne G 1986 A new method for predicting signal sequence cleavage sites Nucl Acids Res 14 4683 4690 Welling et al 1985 Welling G W Weijer W J van der Zee R and Welling Wester S 1985 Prediction of sequential antigenic regions in proteins FEBS Lett 188 2 215 218 BIBLIOGRAPHY 338 Wootton and Federhen 1993 Wootton J C and Federhen S 1993 Statistics of local complexity in amino acid sequences and sequence databases Computers in Chemistry 17 149 163 Yang 1994a Yang Z 1994a Estimating the pattern of nucleotide substitution Journal of Molecular Evolution 39 1 105 111 Yang 1994b Yang Z 1994b Maximum likelihood phylogenetic estimation from DNA se quences with variable rates over sites Approximate methods Journal of Molecular Evolution 39 3 306 314
441. ugins Note that you will need administrative privileges on your CHAPTER 2 TUTORIALS 56 system to install it 2 9 Tutorial Create and modify a phylogenetic tree You can make a phylogenetic tree from an existing alignment See how to create an alignment in the tutorial Align protein sequences We use the ATPase protein alignment located in Protein orthologs in the Example data To create a phylogenetic tree click the ATPase protein alignment in the Navigation Area Toolbox Alignments and Trees Create Tree 5 A dialog opens where you can confirm your selection of the alignment Click Next to move to the next step in the dialog where you can choose between the neighbor joining and the UPGMA algorithms for making trees You also have the option of including a bootstrap analysis of the result Leave the parameters at their default and click Finish to start the calculation which can be seen in the Toolbox under the Processes tab After a short while a tree appears in the View Area figure 2 28 Te protein align x P68053 Tree Settings P68046 ill E T r E Tree Layout Mode symbol Layout Standard P68345 Show internal node labels P68063 Label color Branch label color Mode color EE Line color Annotation Layout Branches Bootstrap k Text Format Figure 2 28 After choosing which algorithm should be used the tree appears in the View
442. up the dialog shown in figure 14 25 E q Manage motifs s 1 Please choose motifs AAA Select motif lists RE Example motifs Motif name Motif Description Type Motif name Motif Description Type N glycosyla N P ST N glycosyla Prosite SP6 G ITJATTTA SP6 promot Java Amidation site x G RK RK Amidation si Prosite Protein kina ST x RK Protein kina Prosite Bacterial his GSK F x 2 Bacterial his Prosite attB1 ACAAGTTT Gateway fo Simple attB2 AACCCAGC Gateway re Simple T TAATACGA T promote Java Cy CGCAAATG CMY promot Simple T3 GCAATTAA T3 promote Simple pGEX 5 GGGCTGGC pGEX 5 primer Simple T7 terminator GCTAGTTA T7 terminat Simple His tag CAT CACH Standard hi Java ETA Figure 14 25 Managing the motifs to be shown CHAPTER 14 GENERAL SEQUENCE ANALYSES 220 q L At the top select a motif list by clicking the Browse py button When the motif list is selected its motifs are listed in the panel in the left hand side of the dialog The right hand side panel contains the motifs that will be listed in the Side Panel when you click Finish 14 7 2 Motif search from the Toolbox The dynamic motifs described in section 14 7 1 provide a quick way of routinely scanning a sequence for commonly used motifs but in some cases a more systematic approach is needed The motif search in the Toolbox provides an
443. ur users interests in mind Therefore we welcome all requests and feedback from users and hope suggest new features or more general improvements to the program on support clcbio com 1 5 2 Report program errors CLC bio is doing everything possible to eliminate program errors Nevertheless some errors might have escaped our attention If you discover an error in the program you can use the Report a Program Error function in the Help menu of the program to report it In the Report a Program Error dialog you are asked to write your e mail address optional This is because we would like to be able to contact you for further information about the error or for helping you with the problem Note No personal information is sent via the error report Only the information which can be seen in the Program Error Submission Dialog is submitted You can also write an e mail to supportOclcbio com Remember to specify how the program error can be reproduced All errors will be treated seriously and with gratitude We appreciate your help CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 29 Start in safe mode lf the program becomes unstable on start up you can start it in Safe mode This is done by pressing and holding down the Shift button while the program starts When starting in safe mode the user settings e g the settings in the Side Panel are deleted and cannot be restored Your data stored in the Navigation Area is not deleted Wh
444. urposes You may not alter transform nor build upon this work CHAPTER 16 PROTEIN ANALYSES 249 aa aa Kyte Hopp Cornette Eisenberg Rose Janin Engelman Doolittle Woods GES A Alanine 1 80 0 50 0 20 0 62 0 74 0 30 1 60 C Cysteine 2 50 1 00 4 10 0 29 0 91 0 90 2 00 D Aspartic acid 3 50 3 00 3 10 0 90 0 62 0 60 9 20 E Glutamic acid 3 50 3 00 1 80 0 74 0 62 0 70 8 20 F Phenylalanine 2 80 2 50 4 40 1 19 0 88 0 50 3 70 G Glycine 0 40 0 00 0 00 0 48 0 72 0 30 1 00 H Histidine 3 20 0 50 0 50 0 40 0 78 0 10 3 00 Isoleucine 4 50 1 80 4 80 1 38 0 88 0 70 3 10 K Lysine 3 90 3 00 3 10 1 50 0 52 1 80 8 80 L Leucine 3 80 1 80 5 70 1 06 0 85 0 50 2 80 M Methionine 1 90 1 30 4 20 0 64 0 85 0 40 3 40 N Asparagine 3 50 0 20 0 50 0 78 0 63 0 50 4 80 P Proline 1 60 0 00 2 20 0 12 0 64 0 30 0 20 Q Glutamine 3 50 0 20 2 80 0 85 0 62 0 70 4 10 R Arginine 4 50 3 00 1 40 2 53 0 64 1 40 12 3 S Serine 0 80 0 30 0 50 0 18 0 66 0 10 0 60 T Threonine 0 70 0 40 1 90 0 05 0 70 0 20 1 20 V Valine 4 20 1 50 4 70 1 08 0 86 0 60 2 60 W Tryptophan 0 90 3 40 1 00 0 81 0 85 0 30 1 90 Y Tyrosine 1 30 2 30 3 20 0 26 0 76 0 40 0 70 Table 16 1 Hydrophobicity scales This table shows seven different hydrophobicity scales which are generally used for prediction of e g transmembrane regions and antigenicity SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use
445. using the Toolbox tools Usually you use the 1 reading frame which means that the translation starts from the first nucleotide Stop codons result in an asterisk being inserted in the protein sequence at the corresponding position It is possible to translate in any combination of the six reading frames in one analysis To translate select a nucleotide sequence Toolbox in the Menu Bar Nucleotide Analyses 4 Translate to Protein 7 or right click a nucleotide sequence Toolbox Nucleotide Analyses A Translate to Protein 5 CHAPTER 15 NUCLEOTIDE ANALYSES 229 This opens the dialog displayed in figure 15 5 F Translate to Protein EJ 1 Select nucleotide Jet eotia sequences Projects Selected Elements 1 o CLC Data oC ATPSal mRNA Example Data 2 ATP8al genomic sec xe ES Cloning Hd Primers 7 Protein analyses 5 Protein orthologs E RNA secondary strut ES Sequencing data Fl E ole 4 mw p Qy zenter search term gt A Figure 15 5 Choosing sequences for translation If a sequence was selected before choosing the Toolbox action the sequence is now listed in the Selected Elements window of the dialog Use the arrows to add or remove sequences or sequence lists from the selected elements Clicking Next generates the dialog seen in figure 15 6 Cc G Translate to Protein 1 Select nucleotide lez ameters sequences 2 Set parameters Translation
446. ve this type of annotations them from the sequence it will just hide them from the view Besides selecting which types of annotations that should be displayed the Annotation Types group is also used to change the color of the annotations on the sequence Click the colored square next to the relevant annotation type to change the color This will display a dialog with three tabs Swatches HSB and RGB They represent three different ways of specifying colors Apply your settings and click OK When you click OK the color settings cannot be reset The Reset function only works for changes made before pressing OK Furthermore the Annotation Types can be used to easily browse the annotations by clicking the small button next to the type This will display a list of the annotations of that type see figure 10 8 Clicking an annotation in the list will select this region on the sequence In this way you can quickly find a specific annotation on a long sequence CHAPTER 10 VIEWING AND EDITING SEQUENCES 136 Annotation types Y v cos E Y 4 Conflict E Exon E MN Y sen E1 19289 21080 EM 7 mana HEG2 34478 36069 HBG1 39414 40985 EA DJ td sedHeD 54740 58389 HBB 62137 63742 MM O Frece ee thalassemia lt 62187 62380 EA UU Repea MA Repeat unit Figure 10 8 Browsing the gene annotations on a sequence mM View Annotations in a table Annotations can also be viewed in a table select the sequen
447. w clcbio com download 1 2 1 Program download The program is available for download on http www clcbio com download Before you download the program you are asked to fill in the Download dialog In the dialog you must choose e Which operating system you use e Whether you would like to receive information about future releases Depending on your operating system and your Internet browser you are taken through some download options When the download of the installer an application which facilitates the installation of the program is complete follow the platform specific instructions below to complete the installation procedure t 1 2 2 Installation on Microsoft Windows Starting the installation process is done in one of the following ways 1 You must be connected to the Internet throughout the installation process CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 13 If you have downloaded an installer Locate the downloaded installer and double click the icon The default location for downloaded files is your desktop If you are installing from a CD Insert the CD into your CD ROM drive Choose the Install CLC Protein Workbench from the menu displayed Installing the program is done in the following steps e On the welcome screen click Next e Read and accept the License agreement and click Next e Choose where you would like to install the application and click Next e Choose a name for the Start Menu folder used t
448. w you want to save Save HD in the toolbar or Click the tab of the view you want to save Ctrl S 38 S on Mac If you close a view containing an element that has been changed since you opened it you are asked if you want to save When saving a new view that has not been opened from the Navigation Area e g when opening a sequence from a list of search hits a save dialog appears figure 3 11 In the dialog you select the folder in which you want to save the element After naming the element press OK 3 2 5 Undo Redo If you make a change in a view e g remove an annotation in a sequence or modify a tree you can undo the action In general Undo applies to all changes you can make when right clicking in a view Undo is done by Click undo in the Toolbar CHAPTER 3 USER INTERFACE 3 dl Save SEIECE name and TOC clon Tor nen OS Folder Update All CLC Data XX ATP8al mRNA fs ATP8al FEE alignment 1 4 ATP8al ortholog tree Fu P39524 Ss P57792 ifs Q29449 QONTI2 fee 09033 x i d Q lt enter search term gt Name GERZE La Xena Se Figure 3 11 Save dialog or Edit Undo or Ctrl Z If you want to undo several actions just repeat the steps above To reverse the undo action Click the redo icon in the Toolbar or Edit Redo or Ctrl Y Note Actions in the Navigation Area e g renaming and moving elements cannot be undone However you can re
449. which are prime candidates for holding functionally important sites e Comparative bioinformatical analysis can be performed to identify functionally important regions 18 6 2 Constructing multiple alignments Whereas the optimal solution to the pairwise alignment problem can be found in reasonable time the problem of constructing a multiple alignment is much harder The first major challenge in the multiple alignment procedure is how to rank different alignments i e which scoring function to use Since the sequences have a shared history they are correlated through their phylogeny and the scoring function should ideally take this into account Doing so is however not straightforward as it increases the number of model parameters considerably CHAPTER 18 SEQUENCE ALIGNMENT 300 20 40 ri 80 kvlgafsdglah l Q6WN27 muhltgeekaBvtalwokvnva ENGUEENGENNANGASARGANNANEcREGANAs 5 E ATT Q6WN20 muhltosekaavtalwokvnvxevagealoriEssaivvvopwtarffesfadisspdavmsnxkvkahgkkvlgafsdalah Q6WN29 myhltodekaguta HER HEHE EH OOOO t HA tt afisdglah Q6WN25 muhItgeekaavtalwgkvnvdevggea logros Ivvypwtarffosftadistodavmsnpkvkahokkvigafsdglan Q6WN22 MWh tgeeksavttIwokvnvdevogea lor iS 1vvypwtarffestodisspdavmonpkikahokkvigafsdalan P68225 MWh EpeBknawkt EHEHE EEE TREE H h P68053 yhltgegkaavtalwokvunvdevagealori EsSivvypwtarffosfodisspdavmonpkvkahokkvInsfseglkn P68046 ENhiEEAa DO RO EO ee O RO N sfsdglkn P68231 muhISgdeknavhalwskvkva AA ETT HM P68228 m
450. will undoubtedly result in a noisy background of the plot You can imagine that there are many successes in the comparison if you only have four possible residues like in nucleotide sequences Therefore you can set a window size which is smoothing the dot plot Instead of comparing single residues it compares subsequences of length set as window size The score is now calculated with respect to aligning the subsequences e Threshold The dot plot shows the calculated scores with colored threshold Hence you can better recognize the most important similarities Examples and interpretations of dot plots Contrary to simple sequence alignments dot plots can be a very useful tool for spotting various evolutionary events which may have happened to the sequences of interest CHAPTER 14 GENERAL SEQUENCE ANALYSES 201 Below is shown some examples of dot plots where sequence insertions low complexity regions inverted repeats etc can be identified visually Similar sequences The most simple example of a dot plot is obtained by plotting two homologous sequences of interest If very similar or identical sequences are plotted against each other a diagonal line will occur The dot plot in figure 14 7 shows two related sequences of the Influenza A virus nucleoproteins infecting ducks and chickens Accession numbers from the two sequences are DQ232610 and DQ023146 Both sequences can be retrieved directly from http www ncbi nlm nih gov gquery gquer
451. with a full sequence 12 2 Output from BLAST searches The output of a BLAST search is similar whether you have chosen to run your search locally or at the NCBI If a single query sequence was used then the results will show the hits found in that database with that single sequence If more than one sequence was used to query a database the default view of the results is a summary table showing the description of the top database hit against each query sequence and the number of hits found 12 2 1 Graphical overview for each query sequence Double clicking on a given row of a tabular blast table opens a graphical overview of the blast results for a particular query sequence as shown in figure figure 12 8 In cases where only one sequence was entered into a BLAST search such a graphical overview is the default output Figure 12 8 shows an example of a BLAST result for an individual query sequence in the CLC Protein Workbench Detailed descriptions of the overview BLAST table and the graphical BLAST results view are described below 12 2 2 Overview BLAST table In the overview BLAST table for a multi sequence blast search as shown in figure 12 9 there is one row for each query sequence Each row represents the BLAST result for this query sequence Double clicking a row will open the BLAST result for this query sequence allowing more detailed investigation of the result You can also select one or more rows and click the Open BLAST Out
452. wn At the bottom of the pane you can click Next E to see the next 50 hits see figure 4 3 lf a search gives no hits you will be asked if you wish to search for matches that start with your search term If you accept this an asterisk will be appended to the search term Pressing the Alt key while you click a search result will high light the search hit in its folder in the Navigation Area CHAPTER 4 SEARCHING YOUR DATA 85 Fe laa Lal td CLC_Data se Example Data 6 Fa 2 Fo Nucleotide a Protein E README ee Recycle bin 14 Figure 4 3 Page two of the search results In the preferences see 5 you can specify the number of hits to be shown 4 2 2 Special search expressions When you write a search term in the search field you can get help to write a more advanced search expression by pressing Shift F1 This will reveal a list of guides as shown in figure 4 4 Wildcard search Search related words e Include both terms AMD Include either term OR Any field search contents Mame search name Length search length START TO ENC J Organism search organism Figure 4 4 Guides to help create advanced search expressions You can select any of the guides using mouse or keyboard arrows and start typing If you e g wish to search for sequences named BRCA1 select Name search name and type BRCA1 Your search expression will now look like this name BRCA1 The gu
453. xt searches in all parameters in the UniProt database at the same time e Organism Text e Description Text e Created Since Between 30 days and 10 years e Feature Text The search parameters listed in the dialog are the most recently used The All fields allows searches in all parameters in the UniProt database at the same time When you are satisfied with the parameters you have entered click Start search Note When conducting a search no files are downloaded Instead the program produces a list of links to the files in the UniProt database This ensures a much faster search 11 2 2 Handling of UniProt search results The search result is presented as a list of links to the files in the UniProt database The View displays 50 hits at a time can be changed in the Preferences see chapter 5 More hits can be displayed by clicking the More button at the bottom right of the View More hits can be displayed by clicking the More button at the bottom left of the View Each sequence hit is represented by text in three columns e Accession e Name e Description e Organism Length It is possible to exclude one or more of these columns by adjust the View preferences for the database search view Furthermore your changes in the View preferences can be saved See section 5 5 Several sequences can be selected and by clicking the buttons in the bottom of the search view you can do the following e Download and open
454. y In figure 14 11 you can see a dot plot window length is 3 with an inversion Low complexity regions Low complexity regions in sequences can be found as regions around the diagonal all obtaining a high score Low complexity regions are calculated from the redundancy of amino acids within a limited region Wootton and Federhen 1993 These are most often seen as short regions of only a few different amino acids In the middle of figure 14 12 is a square shows the low complexity region of this sequence Creative Commons License All CLC bio s scientific articles are licensed under a Creative Commons Attribution NonCommercial NoDerivs 2 5 License You are free to copy distribute display and use the work for educational purposes under the following conditions You must attribute the work in its original form and CLC bio has to be clearly labeled as author and provider of the work You may not use this work for commercial purposes You may not alter transform nor build upon this work CHAPTER 14 GENERAL SEQUENCE ANALYSES 204 Figure 14 11 The dot plot showing a inversion in a sequence See also figure 14 6 SOME RIGHTS RESERVED See http creativecommons org licenses by nc nd 2 5 for more information on how to use the contents 14 2 4 Bioinformatics explained Scoring matrices Biological sequences have evolved throughout time and evolution has shown that not all changes to a biological sequence is equally likely to happen
455. y fcgi Figure 14 7 Dot plot of DO232610 vs DQ023146 Influenza A virus nucleoproteins showing and overall similarity Repeated regions Sequence repeats can also be identified using dot plots A repeat region will typically show up as lines parallel to the diagonal line If the dot plot shows more than one diagonal in the same region of a sequence the regions depending to the other sequence are repeated In figure 14 9 you can see a sequence with repeats CHAPTER 14 GENERAL SEQUENCE ANALYSES 202 Direct repeats gt PDD TO ACDEFGHIACDEFGHIACDEFGHIACDEFGHI Inverted repeats dt l ACDEFGHIIHGFEDCAACDEFGHIIHGFEDCA Figure 14 8 Direct and inverted repeats shown on an amino acid sequence generated for demonstration purposes Figure 14 9 The dot plot of a sequence showing repeated elements See also figure 14 8 Frame shifts Frame shifts in a nucleotide sequence can occur due to insertions deletions or mutations Such frame shifts can be visualized in a dot plot as seen in figure 14 10 In this figure three frame shifts for the sequence on the y axis are found 1 Deletion of nucleotides 2 Insertion of nucleotides 3 Mutation out of frame Sequence inversions CHAPTER 14 GENERAL SEQUENCE ANALYSES 203 E Figure 14 10 This dot plot show various frame shifts in the sequence See text for details In dot plots you can see an inversion of sequence as contrary diagonal to the diagonal showing similarit
456. y showing two additional columns Positive and Query start These should simply be checked in the Side Panel Now sort the BLAST table view by clicking the column header Positive Then press and hold the Ctrl button 38 on Mac and click the header Query start Now you have sorted the table first on Positive hits and then the start position of the query sequence Now you see that you actually have three regions with a 100 positive hit but at different locations on the chromosome sequence see figure 2 16 Why did we find on the protein level three identical regions between our query protein sequence and nucleotide database The beta globin gene is known to have three exons and this is exactly what we find in the BLAST search Each translated exon will hit the corresponding sequence on the chromosome If you place the mouse cursor on the sequence hits in the graphical view you can see the reading frame which is 1 2 and 3 for the three hits respectively CHAPTER 2 TUTORIALS AAA16334 niBL ORD IDJ0 ni BL ORD IDJ0 IMIBL_ORD_IDJO IMIBL_ORD_IDJO IMBL_ORD_IDJO IN BL_ORD IDJ0 BL ORD IDIO MT el DR Eb AAA15334 BLAST 49 HBB Rows 17 Summary of hits From query 44416334 E value Hit start 2 35E 54 5204729 2 55E 54 5204350 1 06E 16 5203407 3 06E 50 5211794 9 456 16 5210773 3 06E 50 5212141 1 05E 39 5232095 2 o8E 31 5247257 EXE 1111 he a ME T gt ail Bla
457. ynisgdeknavholwskvkvdevagealori EsSivvypwtrrffesfodistadavmnnpkvkahoskvInsfgdglsh NP 058652 myhltdasksavscimakynpdevogealoriEnasivvypwtaryfosfodissasaimonpkvkahgkkvitafneglknl NP 032246 HEBEL BEET EERE EEE HES eH h Q6H1U7 muhitaceknaitsIwgkvaieatogea lor if FR 1ivypwtsrifohtadisnakavmsnpkviahgakvlvafodaikn P68945 EvhwtaeekaTitolwokvnvadcgacalar ic cc 1ivypwtarffssfanissptailonpmvrahokkvitsfogdavkn P68063 EvhWwtae8ka Fita iag HH EEE EEE HA n NP 032247 mynftaeektlinglwskunveevagealori ESSivvypwthrffosfonissasaimonprukahokkvltafgesiknl CAA32220 myhftacekaaitsiwdkvdlekvogetloriEssanivypwtarffokfontssagaimonprikahokkvEtsglavkni CAA24102 HEBER FER ESE BRSEEEE CRRCEREEEERNNNEEEEEEEEEEA ERRERA E E HEE AA ni P04443 GUNFLoeekoBitsiWGkWAI ckVGGeRNGFISEEENiWYOWLGREToKTonissocainonprikah kkvitsTglavkni Q6WN28 mvhItgeeksavtalwokvnvdevogealoriERS21vvypwtarffesfodistodavmnnpkikahokkvigafsdglth Q6WN21 MWh EgeBksavtt AHHH HH EAH et A P67821 muhitaceksavttIiwgkvnvdevggea Gr IESESivvyoWtaGrhfoSfgdist pdavmnApkWKERGKKV gafsdgith CAA26204 MWh tpeeksavtalwokvnvdevogea lor ivsrituvypwtarffesfodistodavmonpkvkahokkvigafsdalan P68873 MuhIEpesksavtalwokvnvdevgusa lor ic 22 luvypwtartfesfadistpodavmonpkvkahgkkvIgafsdglan Figure 18 16 The tabular format of a multiple alignment of 24 Hemoglobin protein sequences Sequence names appear at the beginning of each row and the residue position is indicated by the numbers at the top of the alignment columns The level of sequence
458. you need more time for evaluating another two weeks of demo can be requested We use the concept of quid quo pro The last two weeks of free demo time given to you is therefore accompanied by a short form questionnaire where you have the opportunity to give us feedback about the program The 30 days demo is offered for each major release of CLC Protein Workbench You will therefore have the opportunity to try the next major version when it is released If you purchase CLC Protein Workbench the first year of updates is included When you select to request an evaluation license you will see the dialog shown in figure 1 2 In this dialog there are two options e Direct download The workbench will attempt to contact the online CLC Licenses Service and download the license directly This method requires internet access from the workbench CHAPTER 1 INTRODUCTION TO CLC PROTEIN WORKBENCH 1 License Wizard ES BD CLC Protein Workbench Request an evaluation license Please choose how you would like to request an evaulation license Direct Download The workbench will attempt to contact the CLC Licenses Service and download the license directly This method requires internet access from the workbench Go to License Download web page The workbench will open a Web Browser with the License Download web page From there you will be able to download your license as a file and import in the next step Ifyou experience a
459. you through to Step 2 which is displayed in figure 16 9 The Window size is the width of the window where the antigenicity is calculated The wider the window the less volatile the graph You can chose from a number of antigenicity scales Click Next if you wish to adjust how to handle the results see section 9 1 If not click Finish The result can be seen in figure 16 10 See section B in the appendix for information about the graph view The level of antigenicity is calculated on the basis of the different scales The different scales add different values to each type of amino acid The antigenicity score is then calculated as the CHAPTER 16 PROTEIN ANALYSES 244 EB Create Antigenicity Plot X 1 Select protein sequences set parameters 2 Set parameters Antigenicity scale v Welling Kolaskar Tongaonkar Window size Number of residues must be odd 11 2 A Previous l gt Next X Cancel Figure 16 9 Step two in the Antigenicity Plot allows you to choose different antigenicity scales and the window size Antigenicity plot of ATP8a1 0 15 0 10 0 05 0 00 Antigenicity 0 05 Welling O 100 200 300 400 500 600 700 800 300 10001100 Position Figure 16 10 The result of the antigenicity plot calculation and the associated Side Panel sum of the values in a window which is a particular range of the sequence The window length can be set from 5 to 25
460. ys apply these settings Y save MX Cancel Figure 5 11 The save settings dialog The settings are specific to the type of view Hence when you save settings of a circular view they will not be available if you open the sequence in a linear view If you wish to export the settings that you have saved this can be done in the Preferences dialog under the View tab see section 5 2 2 The remaining icons of figure 5 10 are used to Expand all groups Collapse all groups and Dock Undock Side Panel Dock Undock Side Panel is to make the Side Panel floating see below CHAPTER 5 USER PREFERENCES AND SETTINGS 97 Save Settings k Sequence layout Delete Settings Annotation layout Apply Saved Settings P Compact k Annotation types Mon compact no wrap Non compact with translations k Restriction sites P Rasmol colors k Residue coloring Show translation Nucleotide info CLC Standard Settings k Find k Text Format Figure 5 12 Applying saved settings 5 5 1 Floating Side Panel The Side Panel of the views can be placed in the right side of a view or it can be floating see figure 5 13 Number of rows 5 Accession Definition Modificati Length mM15292 Pymaniculat j 7 4PR 1993 110 Figure 5 13 The floating Side Panel can be moved out of the way e g to allow for a wider view of a table By clicking the Dock icon 3 the floating Side Panel reappear in the right sid
461. ysis and it is almost identical for all the analyses so we explain it in this section in general E Convert DNA to RNA Do 1 Select DNA sequences as 2 Result handling Result handling Open Save A Previous Next wf Einish X Cancel Figure 9 1 The last step of the analyses exemplified by Translate DNA to RNA In this step shown in figure 9 1 you have two options 118 CHAPTER 9 BATCHING AND RESULT HANDLING 119 e Open This will open the result of the analysis in a view This is the default setting e Save This means that the result will not be opened but saved to a folder in the Navigation Area If you select this option click Next and you will see one more step where you can specify where to save the results see figure 9 2 In this step you also have the option of creating a new folder or adding a location by clicking the buttons 15 15 at the top of f BB Convert DNA to RNA EJ 1 Select DNA sequences Savenbdr EEE 2 Result handlin g o 3 Save in folder Folder Update All CLC Data Example Data 2 ATP8al genomic sequence xx Sw ATP8al Cloning Primers Protein analyses Protein orthologs RNA secondary structure Sequencing data Qy lt enter search term gt A Figure 9 2 Specify a folder for the results of the analysis 9 1 1 Table outputs Some analyses also generate a table with results and for these analy
462. z NCBIE Linear Basic NCBI Entrez NCBI E Baws m m m Figure 5 Select the relevant files and export them as an archive through the File menu This will produce a file with a ma4 pa4 or oa4 extension Back in the CLC Workbench click Import E and select the file Importing single files In Vector NTI you can save a sequence in a file instead of in the database see figure 6 This will give you file with a gb extension This file can be easily imported into the CLC Workbench Import select the file Select You don t have to import one file at a time You can simply select a bunch of files or an entire folder and the CLC Workbench will take care of the rest Even if the files are in different formats You can also simply drag and drop the files into the Navigation Area of the CLC Workbench The Vector NTI import is a plug in which is pre installed in the Workbench It can be uninstalled and updated using the plug in manager see section 1 7 CHAPTER 7 IMPORT EXPORT OF DATA AND GRAPHICS 107 Save s ES save As File Save in DNA ANAs Database As Remote Sources Save jn mM Desktop E Al oR pace E File name Adenoz gb Files format DNA RNA Documents gb OF Cancel Figure 7 6 Saving a sequence as a file in Vector NTI 1 1 3 Export of bioinformatics data CLC Protein Workbench can export bioinformatic data in most of the formats that can be imported There are a few
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