Home

Version - User manual \(innehållsmässigt\)

Contents

1. 79 TS UMNO E T T T a TCCTEHHEDEEDEELCLTOLTCOEERTECTCCO ELCHE 85 4 85 1 Introduction The Pyrosequencing Assay Design Software is a tool for designing PCR and sequencing primers for Pyrosequencing assays Applications supported by the software are genotyping mutation analysis allele quantification AQ and sequence analysis SQA The software generates primer sets each primer set consists of a PCR primer pair and a sequencing primer that fulfill the specific requirements for Pyrosequencing analysis 1 1 The User Manual The User Manual contains user instructions for the software guidelines for PCR and sequencing primer design guidelines for PCR setup and optimization and hints amp tips for designing assays It also contains instructions for installing the software and computer requirements The appendix of the User Manual contains information on the Principle of Pyrosequencing assay types and general information on sample preparation Pyrosequencing systems and basic definitions 1 2 The Quick Guide The Quick Guide contains short concise instructions for performing a typical assay design The Quick Guide is available as a separate document on the installation CD When the installation CD is inserted into the CD drive an installation wizard will automatically start The wizard presents three different choices install Assay Design software view the User Manual or view the Quick Guide If the wiza
2. sl T p neo B Atn cp e NE E S gt box G nde ehenk Assay Bienes acti Eiseres es E ems BOSI i SNPs can be entered manually in the text file and should be denoted either with IUPAC codes or with slash notation e g C T Insertion Deletion polymorphisms should be typed in square brackets e g C 23 85 The sequence in GenBank format should be saved as a text txt file The text file should start with the LOCUS line The end of the sequence at the bottom of the text file should be a double slash An example of a GenBank text file is shown below Ele Edit Format Help OCUS NM 181587 1543 bp mRNA linear ROD 22 DEC 2003 DEFINITION Mus musculus RIKEN cDNA 6530404N21 gene 6530404N21Rik mRNA ACCESSION NM 181587 NM 181587 2 GI 31795586 Mus musculus house mouse ORGANISM Mus musculus Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Mammalia Eutheria Rodentia Sciurognathi Muridae Murinae Mus REFERENCE 1 bases 1 to 1543 AUTHORS Strausberg R L Feingold E A Grouse L H Derge J G Klausner R D Callins F S Wagner L shenmen C M Schuler G D Altschul s F Zeeberg B Buetow K H Schaefer C F Bhat N K Hopkins R F Jordan H Moore T Max S I Wang J Hsieh F Diatchenko L Marusina K Farmer A A Rubin G M Hong L Stapleton M Soares M B Bonaldo M a Scheetz T E Brownstein M J Usdin
3. e frameshifts if the InDel is not a multiple of three nucleotides e addition loss of one or several amino acids if the InDel is a multiple of three nucleotides 69 85 Frameshifts Frameshifts usually result in a non functioning or malfunctioning protein since the frameshift changes the reading frame three nucleotides give one amino acid and thereby alters the protein which may lose all activity Addition loss of one or several amino acids When the addition loss of one or several amino acids occurs the amino acid chain that builds up the protein changes This may alter the structure and or function of the protein Homozygous versus heterozygous InDels If the InDel is heterozygous see above there will still be one allele left expressing the correct protein and this can be sufficient in some cases If the InDel is homozygous see above there will not be any correct proteins expressed and the protein s function is lost or altered 10 3 4 Short tandem repeats STRs A short tandem repeat STR is a repeated DNA sequence in which the repeat elements are typically two to five base pairs long Different alleles have different numbers of the repeat element 10 3 5 Sequence database files Two of the major nucleic acid sequence databases are e GenBank http www ncbi nlm nih gov Entrez index html and e EMBL Nucleotide Sequence Database http www ebi ac uk embl GenBank is an annotated collection of
4. Because the Pyrosequencing reaction takes place at 28 C the Tm of the sequencing primer can be lower than for the PCR primers The default target Ta of the sequencing primer is 50 C The lowest possible Tm is not absolutely defined but primers with a calculated Tm of around 40 9C have been used with very good results However if using Single Strand Binding protein SSB in the Pyrosequencing reaction a slightly higher primer Tm is required and as low a Tm as 40 9C cannot be recommended for use with SSB The T is calculated using the nearest neighbor method Primer dimers duplexes and internal secondary structures As the sequencing reaction is run at 28 9C it is crucial to check the sequencing primer for self annealing especially at structures are detected by the the 3 end software and displayed in the e Avoid sequencing primer duplex formation Primer set details and in the report Possible dimer and hairpin Sequencing primers should be analyzed with regard to their ability to form duplexes Primers with four or more complementary nucleotides in the 3 end and 51 85 with 5 overhang should not be used Blunt ended duplexes will not give rise to background However if they have many stabilizing bonds they might self anneal to a high degree and lower Pyrosequencing signals Three complementary nucleotides in the 3 end are acceptable as long as other complementary nucleotides within the primer do not stabilize
5. References 1 Ronaghi M Karamohamed S Peterson B Uhl n M Nyr n P 1996 Real time DNA sequencing using detection of pyrophosphate release Analytical Biochemistry 242 84 89 2 Nordstr m T Nourizad K Ronaghi M Nyr n P 2000 Method enabling Pyrosequencing on double stranded DNA Analytical Biochemistry 282 186 193 3 Nordstr m T Alderborn A Nyr n P J 2002 Method for one step preparation of double stranded DNA template applicable for use with Pyrosequencing technology Biochem Biophys Methods 31 52 2 71 82 4 Clarke S C and Diggle M A 2003 A novel method for the preparation of single stranded DNA for Pyrosequencing Molecular Biotechnology 24 221 224 66 85 10 2 Pyrosequencing systems 10 2 1 Introduction Two different systems are available for Pyrosequencing analysis e PSQ 96MA System This system supports genotyping allele quantification AQ and sequence analysis SQA e PSQ HS 96A System This system is a highly sensitive system that requires less reagents than PSQ 96MA It supports genotyping and allele quantification AQ The two systems are used to perform the Pyrosequencing reaction on the PCR product Use Assay Design Software to design an assay suitable for Pyrosequencing analysis The assays can be saved as xml files which can be imported by PSQ 96MA Software version 2 1 and higher and PSQ HS 96A Software version 1 2 and higher 10 2 2 Genotyping and mutat
6. a On the Sequence tab mark where the primer anneals to the template sequence The Find function can be used to search for a primer sequence string in forward or complement reverse direction see section 4 2 4 1 b For each of the three primers right click and select one of the following e Set As PCR Primer Biotinylated if it is a PCR primer that should be biotinylated it will be set as forward or reverse depending on which side of the target region the marked sequence is situated e Set As PCR Primer Not biotinylated if it is a PCR primer that should not be biotinylated it will be set as forward or reverse depending on which side of the target region the marked sequence is situated e Set As Sequencing primer if it is a sequencing primer The primers appear on the Sequence tab and in their respective fields in the Primer set area forward reverse PCR primer field and sequencing primer field 5 All three primers are automatically locked as indicated by darkened buttons to the right of each primer in the Primer set area A score for the primer set will automatically be 29 85 generated as soon as you leave the primer entry fields Note By locking the primer s the software keeps this primer s constant and tries to find the best primer s complementing the locked primer s To toggle between locked unlocked primers click the blue button to the right of the primers Darkened buttons indicate locked primers and hig
7. Analysis of duplex formation between the forward and reverse PCR primers 43 85 Sequencing primer analysis Low sequencing primer Tm Primer with low complementarity Self annealing duplex detected Hairpin loop structure detected A homopolymer is detected adjacent to polymorphism Primer length shorter than min Position outside settings Melting temperature analysis Complementarity analysis Duplex formation analysis Hairpin loop analysis Homopolymer analysis is only performed for genotyping or allele quantification Only for manually entered primers Primer length analysis Only for manually entered primers Analysis of the distance between sequencing primer and target region Primer set analysis Duplex between sequencing primer and biotinylated PCR primer detected Mispriming site detected for sequencing primer Hairpin loop structure on biotinylated PCR primer detected Loop structure detected on template Analysis of duplex formation between sequencing primer and biotinylated PCR primer Mispriming analysis for sequencing primer within the PCR amplicon Biotinylated PCR primer hairpin analysis Template loop analysis 44 85 7 2 3 PCR primer analyses The following analyses are performed on the individual PCR primers GC content Complementarity Duplex formation Hairpin loops Melting temperature Mispriming Primer end stability Primer length Calcu
8. Ferriera S Wang G RA Zheng X te T J Sninsky J J Adams M D Cargill E A f javascript subgif document Forms 0 GwwwPageName DOownLoadOptPagetbqueryFromibdowntoadEntryGqueryNumbere i28 8B Internet 20 85 4 Change the view from EMBLSeqSimpleView to Complete entries and hit the Save button Ble Edt View Favorites Tools Help Heak gt O A A search Favorites Brstoy 4H QD Address hetp fsrs ebi c ukjsrsbinjcg binjwoetz Z eee tinks Google v sskpiwebben Gisokpisten P9 sio Jupp P Markera Saving Query EMBL AY399933 1entries C Browser Window HTML File text Number of entries to download 30 ASCII text table Save with view EMBLSeqSimpleView vj Column Separat x k SeqSimpleView FasteSeqs Generic XML forn SeaSimpview enere AML TTT MBLSedSimpleView Using tho ibad Feste2Se IS Specific XML format Using the loader SeqSimp 7 Using XML PrintMetaphors with id no valid metaphors found SRS Release 7 1 1 Copyright 1997 2003 LION bioscience AG All Rights Reserved Terms of Use Feedback 5 The Entry that opens is now in the correct format Select File Save As enter a file name of choice and change the file type to Text File txt Press the button Save osoft Internet Explorer Bile Edit View Favorites Tools Help Back gt amp A A Asearch Favorites Bristory D 4 GI 8
9. Melting temperature Melting temperature difference Menu bar Monitor Multiplex assay Name polymorphisms Operating system PCR Complementarity PCR duplexes PCR hairpin loops PCR Mispriming PCR primer analysis 11 11 17 33 39 33 41 72 20 18 19 71 72 27 29 46 23 18 61 73 74 46 47 42 80 60 12 33 34 17 17 36 80 49 33 18 18 61 12 32 81 81 17 81 34 30 81 81 48 47 82 32 48 19 41 10 32 32 46 47 11 83 26 46 47 46 46 46 85 85 PCR primer end stability PCR primer length PCR primer pair analysis PCR primer regions Performing an assay design Polymorphism Polymorphisms area Primer set analysis Print a report Printer Processor Quality RAM Report Report formats Result presentation Reuse primers Reverse assay Reverse PCR region Run automatic design Save a report Save an assay Score Scoring Search Seg primer complementarity Seq primer duplexes Seq primer Hairpin loops Seq primer length Seq primer melting temperature Sequence Editor tab Sequence tab Sequence to analyze Sequencing primer analysis Settings Buttons PCR Primer settings Primer set settings Sequencing primer settings Short tandem repeats Simplex assay Software setup Sort primers System requirements Target distance Target region Template loops 11 18 26 49 64 26 The Assay Design Software start screen Troubleshooting Type
10. Carninci P Prange C Raha S S Lo Abramson R D Mullahy s 3 Bosak s McKernan K J Malek 3 A Gunaratne worley K C Hale S Garcia A M Ga VATISTOR Boke Muzny D M Sodergren Fahey J Helton E Ketteman M Mad Sanchez A MEOS Mea Madan A You Bouffard G G Blakesley R w Touchm Dickson M C Rodriguez A C Grimwoo Butterfield Y S Krzywinski M I Sk Schnerch A Schein J E Jones S J TITLE Generation and initial analysis of mo human and mouse cDNA sequences PREDICTED REFSEQ The mRNA record is supported by experimental evidence however the coding sequence is predicted The reference sequence was derived from BC052355 1 On Jun 17 2003 this sequence version replaced gi 31712011 FEATURES Location Qualifiers source 1 1543 forganism Mus musculus mol_type mRNA db_xref taxon 10090 chr omosome 19 gene t agen 6530404 N21Rik JOURNAL Proc Natl Acad Sci U S A 99 26 eee ED taco ten PUBMED 12477932 db xref MG1 1915045 REFERENCE 2 bases 1 to 1543 cos 55 253 AUTHORS Kawai J Shinagawa A shibata K Y Arakawa T Hara A Fukunishi Y KO Aizawa K Izawa M Nishi K Kiyosa Saito T Okazaki Y Gojobori T Bo Kadota K Matsuda H Ashburner M Fleischmann w Gaasterland T Gissi Kuehl P Lewis S Matsuo Y Nikaid Quackenbush J Schrim L M Staubli wagner L washio T Sakai k okido Baldarelli R Barsh G Blake J Bo Carninci P de Bonaldo
11. InDel notation see Example 2 Example 2 Allele 1 ACGACGACG Allele 2 ACGACGACGACG Allele 3 ACGACGACGACGACGACG Assay Design Software will not position primers inside the selected target region In order for the sequencing primer to overlap the constant repeat region when possible two different notations and choices of target region should therefore be used for forward and reverse sequencing primers respectively Forward assay ACGACGACG ACG ACGACG The target region should be selected starting four nucleotides into the constant part of the repeat region In the example above choose CGACG ACG ACGACG as target region and generate only forward sequencing primers Tick the box Generate forward primers in the Current assay settings dialog By choosing this target area the forward sequencing primers that are generated will overlap between 0 and 4 nucleotides of the constant repeat region Reverse assay ACGACG ACG ACGACGACG The target region should be selected starting four nucleotides into the constant part of the repeat region In the example above choose ACGACG ACG ACGAC as target region and generate only reverse sequencing primers Tick the box Generate reverse primers in the Current assay settings dialog By choosing this target area the reverse sequencing primers that are generated will overlap between 0 and 4 nucleotides of the constant repeat region 76 85 11 1 3 Entering polymorphisms in Assay Design
12. Software The following rules apply when entering polymorphic positions in the software e Single nucleotide polymorphisms SNPs should be entered using e g A T or by using the corresponding IUPAC code Adenine Cytosine Guanine Thymine A C A G A T G C T C G T A C G A C T A G T C G T G A T C ZBNOUT lt K lt AKON SAZAGQAOND e Insertion deletion polymorphisms InDels should be entered using the square bracket notation e g AT Short Tandem Repeats STRs should be entered using the InDel notation See section 9 9 for further information e Polymorphisms involving a combination of SNPs and InDels should be entered using a combination of and see example 3 below e Nested polymorphisms are not allowed Example ATT C G The table below shows some examples of how to enter different kinds of polymorphisms in the software Example 3 Di allelic SNP A C G T A represents a tri allelic G C or S IUPAC represents either ACG or AG polymorphism where the Tri allelic SNP GC TT TT possible alleles are a T an A or A C G or V IUPAC represents either GCTTTT or neither deletion Tetra allelic SNP GCTT G A T C or N IUPAC 77 85 11 2 Sequence analysis SQA 11 2 1 Introduction Assay Design Software can be used to design assays PCR and sequencing primers for sequence analysis SQA in the PSQ 96MA System This part of the methodology provides information on how to ent
13. all publicly available DNA sequences and the genetic sequence database of the US National Institute of Health The EMBL Nucleotide Sequence Database is Europe s most comprehensive nucleotide sequence database Both databases use their own sequence file format consisting of a header which contains general information such as keywords author names source organism etc and the actual nucleic acid sequence Both GenBank and EMBL sequences can be saved in FASTA format see below GenBank sequence format The Gen Bank sequence format is identified by the entry ORIGIN at the beginning of the nucleic acid sequence and ends with See section 4 2 2 2 for detailed information EMBL sequence format The EMBL sequence format is identified by the entry SQ at the beginning of the nucleic acid sequence and ends with See section 4 2 2 2 for detailed information 70 85 FASTA sequence format The sequence file format FASTA is used by many sequence alignment and homology search programs A sequence in FASTA format begins with a single line description followed by lines of sequence data The description line is distinguished from the sequence data by a greater than symbol in the first column See section 4 2 2 2 for detailed information 10 3 6 Sequence to analyze genotyping and allele quantification The Sequence to analyze is a short part of a DNA sequence complementary to the sequence of the biotinylated strand that contains
14. an SNP Entry for the imported assay file Note 1 Import of assays is only supported by PSQ 96MA version 2 1 or higher and PSQ HS 96A version 1 2 or higher Note 2 Assays can only be imported into SNP Simplex Entries SQA Entries and SNP Multiplex Entries do not support import of assay files To import an assay file into PSQ 96MA or PSQ HS 96A Software 1 Open PSQ 96MA Software or PSQ HS 96A Software 2 Select SNP Simplex Entries to display the Simplex Entries tree view 3 Right click on the folder in which to save the assay file the Entry to import and select Import Entries from the pop up menu The Entry Import dialog opens 4 Click Browse locate and select the assay file xml to import and click Open Alternatively enter the file path to the file in the File to import field and click Enter on the keyboard The Entry in the selected file is shown in the Entries area of the dialog 5 Atimport a dispensation order will be generated for the assay By default if this dispensation order would generate warnings in the PSQ system software the import will be stopped and failed If you want to override this check the Import if dispensation order gives warnings box to import entries even if warnings are generated by the dispensation order algorithm 6 Click Import to import the assay file the Entry During import the software calculates a dispensation order and warnings and error messages may also be generated If there are
15. and sequencing primer design see Chapter 7 Guidelines for primer design For guidelines on PCR setup and optimization see Chapter 8 Guidelines for PCR setup and Optimization Non specific PCR Perform a homology search of the DNA sequence amplification Avoid placing PCR primers in highly homologous sequence regions For DNA analysis of genes in homologous sequence regions it may be wise to use a search engine e g BLAST Basic Local Alignment Search Tool to check possible homologous regions such as pseudogenes in the given genome Generated PCR primers can also be checked for sequence homology One such homology search engine can be found on the website of the National Center for Biotechnology Information NCBI at http www ncbi nlm nih gov BLAST If homologous sequences are found If highly homologous sequences were found during the homology search the sequences may be aligned using an alignment tool One such alignment tool can be found on the web site at the web site of Institut National de la Recherche Agronomique INRA http prodes toulouse inra fr multalin multalin html Template loop formation Add an extra nucleotide to the non biotinylated PCR primer self priming The 3 end of the template which can form a loop is defined by the 5 end of the non biotinylated PCR primer The 3 end complementarities of a template loop may be removed by modifying the non biotinylated PCR primer Enter a random extra nucleotide
16. area and associated with a warning message Use this information as a quick indication of which analysis results will need to be inspected more closely in the report In the primer set report warnings are displayed together with penalties for the individual analysis steps See section 7 2 2 Warning messages for a list of possible warnings and corresponding descriptions Differences in the analysis steps for the different assay types The analyses carried out by Assay Design Software when designing primers are e Individual PCR primer analyses e PCR primer pair analyses e Sequencing primer analyses e Primer set analyses Some analysis steps differ between the three assay types genotyping allele quantification AQ and sequence analysis SQA The following table gives an overview of the differences in the analyses PCR primer pair analysis Yes Yes Not for sequences Amplicon length containing unknown regions Primer set analysis Yes Yes No Generation of sequence to analyze Sequencing primer Yes with low Yes with high penalty No analysis penalty level level Homopolymers Sequencing primer No Yes No analysis A nucleotide in polymorphism 42 85 7 2 2 Warning messages A warning is issued if an analysis step receives a penalty larger than a certain threshold value 50 The warning serves as an indication that the triggering analysis step has detected a potentially serious problem The following tables giv
17. computer 2 Insert the Pyrosequencing Assay Design Software CD into the CD drive of the computer 6 85 4 Follow the Assay Design Setup wizard which automatically starts If the wizard does not start either open the CD drive in Windows Explorer and double click the autorun exe file or choose Run in the Windows Start menu specify the path to the CD drive and the file autorun exe e g D autorun exe xi Assay Design Software 1 0 0 Biotage Install Assay Design Software 1 0 o Install Acrobat Reader 6 0 N User Manual Quick Guide e Pyrosequencing Assay Design Software requires Microsoft NET Framework version 1 1 to be installed and running on the computer The installation wizard will automatically detect the presence of NET Framework or will if necessary install the program If the installation of NET Framework fails the installation will stop with an error message Confirm that you are logged in with administrator rights and restart the installation After installation of NET Framework installation continues with Pyrosequencing Assay Design Software Click Next to proceed with the installation fe PSQ Assay Design E 2151 x x Welcome to the PSQ Assay Design Setup Wizard e A The installer will quide you through the steps required to install PSQ Assay Design on your computer WARNING This computer program is protected by copyright law and international treaties Unauthorized duplication or dis
18. concentration of nucleic acid is not taken into account Tm 2 C x number of A and T bases 4 C x number of G and C bases 7 4 Guidelines for PCR primer design When using Assay Design Software to generate primer sets PCR and sequencing primers a number of parameters are taken into account The following section describes some of the underlying knowledge that has been incorporated in the analysis steps of Assay Design Software to select suitable PCR primer sets for PCR and Pyrosequencing analysis 49 85 7 4 1 General guidelines to apply when designing PCR primers Primer length The PCR primers should typically be between 18 and 24 bp in length GC content The PCR primers and the PCR product should have approximately the same GC content in 9o The typical GC content of PCR primers ranges from 40 to 60 For good specificity the primers should preferably be more GC rich in the 5 end and less in the 3 end Melting temperature The standard range for the melting temperature Tm is 60 70 C The default settings in Assay Design Software are 56 86 C for PCR primer T4 Forward and reverse primer should have similar melting temperatures By default the nearest neighbor method is used for calculation of Tm Amplicon length PCR product length Whenever the PCR amplicon size can be directed by primer design PCR products should be as short as possible preferably less than 250 bp The optimal range fo
19. design Once target regions position names and assay types have been selected for all open assays batch analysis can be started 1 Select Assay Setup Batch Assay Design This opens a dialog where the output directory for the resulting assay files and text report can be selected Select Assay Run Batch Assay Design to open the Batch Design Progress dialog Batch design is automatically initiated for all open assay windows Optional The analysis can be stopped at any time by clicking the Stop button After analysis all successful assays have automatically been saved and closed Only failed assays will remain open in Assay Design Software The output field of the Batch Design Progress dialog shows a short report with the best primer set generated per assay This report can also be saved to the output folder under the name PSQ Assay Design Log txt by clicking the Save button Note If a new batch design is started using the same output folder as for the first one the new primer set information will be added to the bottom of the PSQ Assay Design Log txt file Thus PSQ Assay Design Log txt will never be over written by consecutive analyses Close the Batch Design Progress dialog to regain access to Assay Design Software 39 85 6 Importing an assay file into PSQ system software The instructions below describe how to import assay files from within PSQ 96MA Software or PSQ HS 96A Software Import will automatically create
20. detected by the software and displayed in the Primer set details and in the report Mispriming To obtain specific amplification it is important to Potential alternate annealing sites select PCR primers that do not have alternate within the entered sequence are annealing sites on the template sequence detected by the software and displayed on the Sequence tab and in the report Primer specificity Primers with a stable 5 end high AG value and a The relative stability is calculated in relatively unstable 3 end low AG value typically the Primer end stability analysis step perform best because they are more stable and The AG graph on the Sequence tab specific and thereby less prone to mispriming in the Assay window visualizes AG values for the 5 and 3 ends of primers 7 5 Guidelines for sequencing primer design A number of parameters are taken into account when using Assay Design Software to generate primer sets PCR and sequencing primers The following section describes some of the underlying knowledge that has been incorporated in the analysis steps of Assay Design Software to select suitable sequencing primers for Pyrosequencing analysis 7 5 1 General guidelines for sequencing primer design Primer length The sequencing primer should typically be between 15 and The minimum and maximum 20 bp but longer sequencing primers can also be used sequencing primer lengths can be changed Melting temperature
21. e Use dedicated PCR use only pipettes micro centrifuges and disposable gloves e Use aerosol resistant pipette tips e Setup a PCR reaction under a laminar flow hood equipped with UV light e Use sterile techniques and always wear fresh gloves e Always use new and or sterilized glassware and plastics to prepare the PCR reagents and genomic DNA e Use PCR reagents and solutions only for PCR reactions and store these reagents in small aliquots e Always include a negative control all reaction components except DNA and a positive control e g a sample that has been successfully amplified in previous experiments 9 3 Tips for assay controls Controls in the PCR Always include a negative control that includes all reaction reaction components except DNA When setting up a new assay perform PCR optimization on a couple of control DNA samples that have been successfully amplified in previous experiments Controls in the Sequencing primer only Pyrosequencing reaction DNA template only without sequencing primer PCR negative control with sequencing primer 59 85 9 4 Tips for multiplex assay design The Assay Design Software does not support automatic multiplex assay design However it contains some useful support functions for multiplex design Duplex design Once a candidate primer set has been selected for one polymorphism check for mispriming of the primers in the template containing the second polymorphism an
22. file one assay window will be opened automatically and the respective sequence imported into the Sequence Editor tab The information in the header line preceding each sequence will be imported into the Notes field on the Final Primer Set tab and will also be the default name of the assay file created Assay Design APOE codon 112 APOE codon 112 Position1 AE file Edit Assay Windows Help l8j x em Assar Type Genoypng s e D is o0 Sequence Editor Sequence Final Primer Set Primer sequence Pos Bp GC Tm ID M EE M Sequence to analyze Unie seg mesas gt lo iiv 8l Created by PYROS as037se Created 2004 02 19 10 29 Modified 2004 02 19 10 29 Notes APOE codon 114 E C st do rcf CON EET He T c 3 o2 lem The FASTA file format is a common format for DNA sequence files Sequences in FASTA file format are preceded by a line starting with the symbol as the first character The rest of the line is the name and description of the sequence the header line The following lines contain the sequence data The sequences should not have any numbers e g line numbers and should contain a maximum of 10 000 characters each SNPs should be denoted either with IUPAC codes or with slash notation e g C T Insertion Deletion polymorphisms should be typed in square brackets e g C The sequences
23. generated and analyzed Thus if the number of shown primers is lowered fewer primer sets will also be generated possibly decreasing the chance of finding a high scoring primer set 3 3 4 Buttons Get Factory Click to load the factory settings Get Default In the Current assay settings dialog Click to load the latest default settings settings Set As Default Click to save the entered settings as default settings OK In the Current assay settings dialog Click to apply the settings to the current assay Cancel Click to cancel any changes made and close the dialog 3 4 Selected settings will affect primer set scoring Some settings will affect the primer set scores while others while noted will not impact the scoring Furthermore parameters that do affect scores will affect them differently Many parameters have an allowed range within which primers are generated but given a penalty i e a decreased score and an optimal range within which they are generated without penalty A detailed description of how different settings will affect primer set scores is shown below 3 4 1 PCR Primer score effects Min Primer Length 18 bp Primers within the set min and max bp lengths are generated This analysis Max Primer Length 24 bp 18 24 bp step can result in warning messages for bp short primers but will not affect primer set scores Optimal Amplicon 50 bp Amplicons within the set optimal range Length from bp are generated wit
24. gt 50 and a warning 7 2 5 Sequencing primer analyses The following analyses are performed on sequencing primers GC content in Complementarity Duplex formation Hairpin loops Melting temperature Primer length Target distance Homopolymers Calculates the GC content in percent Analyzes the level of complementarity between the sequencing primer and the annealing site Detects possible sequencing primer self annealing duplex formations which can cause background signals in the Pyrosequencing analysis Detects possible sequencing primer hairpin structures that can cause background signals in the Pyrosequencing analysis Calculates the deviation between the melting temperature of the sequencing primer and the optimal melting temperature Calculates the deviation of the actual primer length and the optimal primer length Calculates the distance between the sequencing primer and the target region Homopolymer analysis is only performed for genotyping medium score weighting and allele quantification high score weighting Detects if the polymorphisms in the target contain adjacent homopolymeric sequences 47 85 Automatically generated primers always have complete complementarity Manually added or edited primers will receive will receive a penalty and a warning for non complementary sequence motifs Non complementarity is penalized more for the 3 end of the primer
25. nucleotide A T C or G in the genome sequence is altered For example a SNP might change the DNA sequence AAGGCTAA to ATGGCTAA Both forms must occur with a frequency of 1 0 01 or greater in a large population to be classified as a SNP otherwise it is a random mutation SNPs are evolutionarily stable not changing much from generation to generation making them suitable to use as genetic markers in population studies Two out of three SNPs involve the replacement of cytosine C with thymine T SNPs occur on average every 100 to 300 bp along the 3 billion base human genome SNPs can occur in both coding and non coding regions of the genome Many SNPs have no effect on cell function whereas others could predispose people to disease or influence their response to a drug 10 3 3 Insertions and deletions InDels An insertion or deletion is a mutation where one or several nucleotides have been inserted deleted in a DNA sequence The size of the InDel can vary from single base to a part of a chromosome Homozygous single base deletion Homozygous single base insertion Allele 1 CA TCCGGA Allele 1 CAATCCGGA Allele 2 CA TCCGGA Allele 2 CAATCCGGA Heterozygous single base deletion Heterozygous single base insertion Allele 1 CA TCCGGA Allele 1 CA TCCGGA Allele 2 CAATCCGGA Allele 2 CAATCCGGA InDels in protein coding parts of genes have great impact on the protein s function activity and structure because they can cause
26. product if the 3 most base is paired with a target High T A consequence of a too high Ta value is that too little product will be amplified since the likelihood of primer annealing is reduced Another important consideration is that a pair of primers with very different T may never give appreciable yields of a unique product and may also result in inadvertent asymmetric or single strand amplification of the most efficiently primed product strand 8 2 PCR setup and optimization Specific for allele quantification Allele Optimization of PCR conditions quantification PCR conditions should be optimized to give a PCR product of high quality with a yield of at least a 50 and one specific band on an agarose gel It is very important not to use more primer than necessary in the PCR reaction lt 0 2 uM Excess biotinylated primer may result in decreased specific signals and may also give rise to background signal At least 10 ng genomic DNA should be added to the PCR reaction This will ensure that enough copies of both alleles are included in the amplification reaction to result in a correct representation of the allele frequency distribution 56 85 8 3 PCR protocol example As a standard use 10 ng genomic DNA in a 50 ul PCR reaction and 125 uM of each nucleotide Below is an example of what a typical PCR reaction mix can look like The example shows a PCR reaction mix 2 0 mM MgCl using AmpliTaq Gold for one and te
27. recommendations on organic and biochemical nomenclature symbols terminology etc L Locus The position on the chromosome at which a gene or a genetic marker is situated The locus may be occupied by any one of the alleles for the gene or the genetic marker M Melting temperature Melting temperature Tm of a primer is defined as the temperature at which 50 of the primer is annealed to the template and 50 is free in the solution The melting temperature depends on the primer sequence GC rich primers have higher melting temperatures primer length concentration and chemical properties of the buffer 81 85 solution There are different ways to calculate the melting temperature The method primarily used in the Assay Design Software is the Nearest neighbor method Mispriming Mispriming occurs when there are alternate annealing sites for the primer in the template sequence i e when the 3 end of a primer has significant homology with more than one site on the template sequence Multiplex assay Reaction with one or several different DNA templates and several sequencing primers in the same reaction one primer per polymorphism Mutation A change in the DNA sequence Mutations within a gene can alter the amino acid sequence of the encoded protein They can also alter the reading frame and thus the amino acid sequence of the encoded protein Mutations occuring more frequently than 196 in a population are SNPs Nearest Neighbo
28. region which is used for PCR primer design Note It is possible to manually redefine within which sequence region the forward and reverse PCR primers should be allowed to anneal This is useful for example when the sequence contains multiple polymorphisms that should be contained within the same PCR amplicon For further details see section 4 2 5 The assay setup is now complete Continue with the instructions in section 4 3 Search for a sequence motif in the entered sequence 1 4 5 Select Edit Find or use shortcut key Ctrl F The Find area is displayed at the bottom of the Sequence Sequence editor tab Enter the sequence motif to search for in the Find what field Note It is not possible to search for a polymorphism e g C T or a sequence string containing a polymorphism The search string should only contain the characters A C G or T There are four different search directions to choose from e Forward sequence search for a motif from left to right on the upper strand e Reverse sequence search for a motif from left to right on the lower strand e Forward complementary sequence search for a motif from right to left on the upper strand e Reverse complementary sequence search for a motif from right to left on the lower strand Click Next to find the next occurrence of the motif If found it is marked on the Sequence and Sequence Editor tabs Click Previous to search for the former occurrence of the m
29. than the 5 end Primarily extendable duplexes i e duplexes that are complementary in the 3 end are penalized If serious duplexes are detected the primer will receive a penalty 50 and a warning If serious hairpin loops are detected the primer will receive a penalty 50 and a warning If the T is lower than the optimal Tm the primer will receive a penalty Penalties gt 50 will trigger a warning If the primer is shorter than the optimal length it will receive a warning The primer score is not affected Analysis in PSQ software will be more difficult if homopolymeric regions are adjacent to polymorphisms Sequencing primers annealing over the homopolymeric region are favored because the homopolymeric sequence effect is reduced Polymorphism Polymorphism analysis is only performed for allele quantification AQ Checks for A nucleotide s in the target polymorphism If an A nucleotide s is are contained in the target polymorphism primers that result in the incorporation of T will be favored over primers resulting in incorporation of A 7 2 6 Primer set analyses Biotinylated PCR Primer Hairpins Duplex formation Mispriming Template loops Sequence to analyze Detects hairpin structures on the biotinylated PCR primer which may cause background signals in the Pyrosequencing analysis Detects sequencing primer and biotinylated PCR primer cross annealing duplex formations
30. the duplex e Avoid 3 end hairpin loops Sequencing primers should be analyzed with regard to their ability to form hairpin loops At 28 C as little as three complementary nucleotides in the 3 end may give rise to background If hairpin loops cannot be avoided then it may be possible to shorten the primer to give a blunt end hairpin that cannot generate background signal Positioning of the sequencing primer The sequencing primer should preferably be positioned with its 3 end as close to the target region as possible typically within about 5 bp However for multiplex design the sequencing primers may have to be moved back further from the target region For analysis of single base In Dels the selected primer should preferably be moved back from the target region to generate at least one reference peak before the variable position Mispriming Avoid 3 end mispriming Sequencing primers should be analyzed with regard to their ability to misprime within the PCR amplicon A primer that has six or more complementary nucleotides in the 3 end at an alternative priming site and be extra careful with GC rich 3 ends should not be used By default the sequencing primer is positioned between 0 and 3 nucleotides away from the target region This can be changed Potential alternative annealing sites within the amplicon are detected by the software and displayed on the Sequence tab and in the report 7 5 2 Seq
31. the file type to Text File txt Press the button Save TNEI Sequence Viewer Microsoft Internet Exclorer prr n 7 EJ Honk 9 QUE Al sess freres toy Sy GRE ao adress J http tjm nc nin nis gov entreziviener Icgirdbeudectidsdopte GenEankio 3 745686 sj ee ws Google v fensa TZ BeeekeBwcblen Siki sten PMR o up gt Brakes fE Serben 3S hi die 3Nucleotide Seah Nudecide for Gal amp ew Limits Previewindex History Clipboard Detalls Display detan Z stow 20 x Sendt Fe z GelSubsequence Festes T 1 NM 181587 Mus musculus RIKE 5 3 1795586 Links Locus NI 181587 1543 bp wHNi linear ROD 22 DEC 2003 DEFINITION Mus wusrslus DIKEN CDNA SS DADANZT mene LGENANINA IRIK INA ACCESSION NN 1815 EE VERSION NK 1815 z HE O Save i E Bicer mj eS ekEM SOURCE Mus sus ORGANISM Mus xus Eukaryo Marma i REFERENCE 1 bas AUTHORS Strauch Kiausne Altschu Hopkins Diazche stapler Scheetz Carnine Bhraneo MeKerna Worley Fie name BerBork Meuse SES Villalo Sees M Eu Fahey J Sanchez Encoding Westem Earcpean windows 7 Boutfar Dickson 5 LJ EGHWtz J Myers RI Butterfield Y 2a VU Smailus D E TITLE human and mouse cDNA sequences JOURNAL Proc Nati Read Sei U S A 39 26 16899 16903 2002 PUBMED 12477932 REFERENCE bases 1 to 1543 AUTHORS Nishi K Sojobori
32. ttctagtttt cttatttttc tgtacagcca tacttttctc cttccctcct tatcttatac 2520 tattttttct tcttagaagc aactgccttc tttggacctt ttcttgttct tgcatttgrt 2580 ttagtttttt gtcctctaac tggaagtcct cttctatgtc tgattcctct gtagcatcca 2640 atttcaacta atctctttat atttadagct acttctcttc ttad tcacc ttcgattttg 2700 aagttcttgt taatgtaatc tcttaatgca ttaacttctt cttcagttaa atccttaact 2760 ctagtttcag gattaacact tgtagctttt ataatttttc ttgaacttgt tagtcctata 2820 Ccatatatat aagttagacc tatttcaact cttttttctt ttggtaggtc aataccggct 2880 attctcgcca ttgacattta cacctcctgg ttttgtatat atttattatt tatattataa 2940 Jj agttttaggc aacaaagtgt vtaacctttt tattgaca 2978 19 85 Save a file on EMBL text format 1 Atthe web site http www ebi ac uk the Nucleotide sequences database can be searched either for a gene name or for a specific accession number Type in the search item and hit the Go button He Eit gew Favortes Toole Heb exta gt O A Al Geach ates Hor De 9 EX ED 2 acess E nien each sz es ints Google T Te BBs pAwedben Gisdknieten FERK E sidrfo or Piao European Bioinformatics Institute the path to knowledge ean 2ean Bioinformatics Training and Education atine EBL je TEC eet Ei 2 The search results in a number of hits Click the open up a sequence record ose Explorer E mE fie Edt Yiew Favorites Tools Help EN tex 5 OO ASh Fates iim Ehe Go res ET estia et aceboy dee e
33. which may cause background signals in the Pyrosequencing analysis Detects alternative annealing sites for each sequencing primer within the amplicon Detects possible loop structures in the biotinylated strand the template sequence for Pyrosequncing analysis which may cause background signals in the Pyrosequencing reaction An extra A is automatically added to the 3 end of the amplicon for an additional template loop check as Taq polymerase frequently adds an extra A to the 3 end of the amplicon during PCR A sequence to analyze is only generated for genotyping and allele quantification assays A sequence to analyze is generated for the import into PSQ 96MA or PSQ HS 96A SNP software 48 85 If serious hairpin structures are detected the primer set will receive a penalty 50 and a warning If serious duplex structures are detected the primer set will receive a penalty 50 and a warning Mispriming will only be detected on the biotinylated strand because the non biotinylated strand is removed during the sample preparation phase If serious alternative annealing sites are detected the primer set will receive a penalty gt 50 and a warning Template loop formation can cause self priming resulting in background signals in the Pyrosequencing reaction If serious extendable template loops are detected the primer set will receive a penalty 50 and a warning The sequence to analyze does no
34. 0 and a warning The PCR primer specificity increases if the 5 end is more stable than the 3 end If a manually entered primer is shorter than the minimum setting it will receive a warning The primer score is not affected 7 2 4 PCR primer pair analyses The following analyses are performed on PCR primer pairs Amplicon length Duplex formation GC content difference Melting temperature difference Calculates the deviation of the actual amplicon length from the optimal range Detects possible PCR primer cross annealing fwd rev duplexes Calculates the difference in GC content between forward and reverse PCR primer and between the PCR primers and the amplicon Calculates the deviation between the actual Tm difference between the forward and reverse PCR primers and the optimal Tm difference range 46 85 If the amplicon length deviates significantly from the optimal range the PCR primer pair will receive a penalty 50 and a warning If potentially serious duplexes are detected the PCR primer pair will receive a penalty 50 and a warning PCR primers with a GC differences within the optimal range 0 10 will be generated without penalty GC differences higher than 1096 but lower than 30 will be generated but given a penalty that increases linearly At penalty gt 50 the primer pair will receive a warning If the deviation is large the PCR primer pair will receive a penalty
35. 2 Address E hetpfjsrs ebiacunsrsbinfeatbinjwgetz SY ee Links Google s gos kp amp webben ys kp siten P39P Epsidinfo upp Markers AY399933 standard genomic DNA GSS 1618 BP AY399933 AY399933 1 13 DEC 2003 Rel 78 Created 17 DEC 2003 Rel 78 Last updated Version 2 Homo sapiens PKLR gene VIRTUAL TRANSCRIPT partial sequence genomic survey sequence GSS Homo sapiens human Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Mammalia Eutheria Primates Catarrhini Hominidae Homo 1 1 1618 Clark A G Glanowski S Nielson R Thomas P Kejariwal A Todd M A Tanenbaum D M Civello D R Lu F Murphy B Ferriera S Wang G Zheng X H White T J Sninsky J J Adams M D Cargill M Inference of extensive non neutral evolution from human chimp mouse orthologous gene trios Science 302 1960 1963 2003 2 1 1618 Clark A G Glanowski Nielson R Thomas P Kejariwal A Todd M A Tanenbaum D M Civello D R Lu F Murphy B Ferriera 3 Wang G Zheng X H White T J Sninsky J J Adams M D Cargill M Submitted 16 NOV 2003 to the EMBL GenBank DDBJ databases Celera Genomics 45 West Gude Drive Rockville MD 20850 USA oO dep Internet 21 85 GenBank Sequences in GenBank format derived from the NCBI GenBank database and saved in text format txt can be imported in Assay Design Software one at a time The
36. 3 9 RZ GTCTCCCAGCAAAGCAAAACACT 2 Check the Unique seq primers box to display unique sequencing primers Only the best primer set generated for each sequencing primer will be displayed in the list This mode of display may be useful when designing multiplex assays 31 85 4 4 4 View information on different primer sets By default the primer set with the highest score is selected as the final primer set by the software A dark gray box surrounding the primer set indicates that it is selected as final For a primer set with high quality i e labeled with color code blue none of the performed analysis steps have identified any problems of concern Thus a high score primer set can be used directly without any more checks or analyses For lower quality primer sets however a manual analysis may be required before use To view more information on primer sets follow the instructions below To view information on a primer set 1 Click on the desired primer set in the Primer set area The selected primer set is highlighted in light blue and displayed in the top fields of the Primer set area In the right click menu there is an option to Copy Primer Set A copied primer set can be pasted into the entry fields of a different assay window or into a text editor like Microsoft Word There is also a menu alternative for copying the whole list Copy AII Primer Sets to e g a Microsoft Word document On the Sequence tab the primers are d
37. 795586 KEYWORDS SOURCE Mus musculus house mouse From Par to 1543 11543 ORGANISH Mus musculus r Comple Eukaryota Netazca Chordata Craniata Teraa Mammalia Eutheria Rodentia Sciurogna REFERENCE 1 bases 1 to 1543 Set Range UnsstHsnge Dismiss AUTHORS Strausberg R L Feingold E Grouse Collins F S Wagner Zeeberg Diavehent EE Ciera d Scheetz T E Brownstein M J Usdin T B Toshiyuki S Carninci P Prange C Raba Loquellexo N R Petcrs 0 J Worley Villalon Fahey J Sanchez A Wiiting H Madan A Young A C Shevchenko Y Bourtard Dickson I Byexs R M Butterfie Smailus D E Sehaerch A m TITLE Generation and initial analysis of more than 15 000 full length human and mouse cDNA sequences JOURNAL Proc Natl Acad Sci U S A 99 26 15899 16903 2002 PUBMED 12477932 REFERENCE 2 bases 1 ta 1543 AUTHORS Kawai J Shinagewa A Shibata K Yoshinc H Itoh K Iehii Y Arakawa T Hara A Fukunishi aAdachi J Fukuda S gt Nishi k Kondo S Yamanaka I Kasukawa T Batalov S Casa W Gaagterland T Giasi C Kuehl Pse Lewis Matsuo Y Nikmido Quackenbush J Schrinl L R Staubli F r Tonita He a Erre Aan ca 6 ESL S LL pent ic aree Bon Assay o Brno jace i Beers Geenda Eder ome SOCHIOSLOMS uc 3 Select File Save As enter a file name of choice and change
38. 85 11 Appendix B Assay types 11 1 Genotyping and allele quantification AQ 11 1 1 Introduction Assay Design Software can be used to design assays PCR and sequencing primers for genotyping mutation analysis and allele quantification AQ in PSQ 96MA System or PSQ HS 96A System This part of the methodology provides information on e Polymorphisms for which assays can be designed in Assay Design Software e How to enter the polymorphism in the Assay Design Software 11 1 2 Polymorphisms for which assays can be designed Assay Design Software supports assay design for di tri and tetra allelic single nucleotide polymorphisms SNPs point mutations and insertions deletions InDels Di allelic SNPs A SNP position in a gene is said to be di allelic if two possible alleles nucleotides can occupy the SNP position for example C or T denoted C T or Y when entered in Assay Design software Tri allelic SNPs A SNP position in a gene is said to be tri allelic if three possible alleles nucleotides can occupy the SNP position for example G T or C denoted G T C or B when entered in Assay Design software A diploid individual can have any combination of two of these nucleotides Tetra allelic SNPs A SNP position in a gene is said to be tetra allelic if four possible alleles nucleotides can occupy the SNP position i e C T G and A denoted C T G A or N when entered in Assay Design software A diploid individual can have a
39. AG at 5 end 8 0 Maximum AG at 3 end 5 0 Minimum AG at 3 end 11 0 In addition the AG graph can be used to visualize the AG distribution in the different primers Here each data point represents the stability of a nucleotide pentamer The data points correspond to the Gibbs free energy of the nucleotide in that position and the four nucleotides immediately downstream in the sequence O AGGGCGCTGATGGACGAG Ha AGGAACT GAGGGCGCT GAT GGACGAGACCAT GAAGGA iTCOT TGACTCCCGCGACT ACCT GCTCTGGTACTTCCT The Delta G graph of the selected primer will include graph points from the 5 end of the primer to 5 nucleotides upstream from the 3 end boxed as each point represents the Delta G of a nucleotide pentamer 61 85 9 7 Tips for using the melting temperature T graph Each bar in the Tm graph represents the Tm for a primer of 20 bp by default with its 5 end in that position To view Tm for a selected primer of any length click on the wanted primer in the graphical representation on the Sequence tab This automatically adjusts the T graph to this primer length The Tm of this particular primer is represented by the bar at the 5 position The primer Tm can be adjusted by changing the primer length or the primer position This can be done either by adding removing nucleotides in the Primer set area or by dragging and dropping or resizing the primer on the Sequence tab 9 8 Tips for analyzing InDels in homopolym
40. G of 3 1 is a good starting point Optimization GC rich templates often need a higher annealing temperature and lower MgCl concentration to amplify well because high salt concentrations will stabilize secondary structures e Checking the PCR product On agarose Check an aliquot of the PCR product on a 1 5 agarose gel There should be a clear strong product band without excess primers primer dimers or other non specific products On PSQ 96MA System Use 15 25 ul of the PCR product and 16 pmol of sequencing primer 54 85 to give strong signals single peak heights of about 15 to 25 units On PSQ HS 96A System Use 5 10 ul of the PCR product and 3 6 pmol of sequencing primer to give strong signals single peak heights of 100 200 units The DNA material should be purified and of high molecular weight good integrity The recommended amount of genomic DNA for a standard 25 ul or 50 ul PCR reaction is a minimum of 10 ng of DNA With smaller amounts than 10 ng DNA in a PCR reaction there is a risk that there will be too few copies of the genome to give an accurate and robust representation of the genotype allele content in the sample resulting in false or skewed genotypes MgCl e General concentration Mg ions bind to both nucleotides and DNA and the concentration of free Mg ions therefore depends on the concentrations of compounds like nucleotides template DNA free pyrophosphate PPi and EDTA from certa
41. GRE 37 5 Performing batch assay design cccceeeeeeeeeeeeeeeeeeeeeeeeeeeeueueueeaeasaseseeeeeaueuauaeaeeeeenenss 38 E E eser Er 38 5 2 Run automatic batch assay design ccceee eect eee ee eee eee eee eee eee eaten teeta 39 6 Importing an assay file into PSQ system software ssssssssssunnunnnunnunnnnnnnnnnnnnnnnnn nn 40 7 Guidelines for PCR and sequencing primer design eene 41 7il AMCHOAUCE ONT RET EET III ey Peake oes E a A D u nents 41 7 2 Analysis steps performed by the software ccccceeeee nsec eee eee e eee e eens teens eee e mene 41 VOLUN REB o gore Vrorujo PEEL 41 72 2 Watning messa g8es EI t Me REA RATE RE ee MERE E EVE 43 7 2 3 gt PER primer analyses uris dt deed aa due ERE HIR De ERR RAE ERR tna EMERE dee SERRE AERA RR a beds 45 72 4 PCR primer pair analyses rena ae E Lh oe aeo the exes been Sok deg eae alsa dense venne alea age 46 7 2 5 Sequencing primer analyses 2 sce ece cence sehen hne hehehe snae sna esae EEE EEE 47 7 2 6 Primer set analySes ciii tr ea tta A y sree SE YR RR Rr e 48 7 3 Melting temperature cx forme aie Ree Et RR Fl n en RI rer EN E RI PEE 49 7 3 1 Methods for calculating the melting temperature Tm sesssssssse n 49 7 4 Guidelines for PCR primer design cceeeee cece eee eee eee eee eee ene eee eens 49 7 4 1 General guidelines to apply when designing PCR primers sessi 50 7 5 Guidelines for s
42. M F Brownst Fletcher C Fujita M Gariboldi M Hofmann M Hume D A Kamiya M Lee Marchionni L Mashima J Mazzarelli Ring B Ringwald M Rodriguez I S Sat K Schonbach C Seya T Shiba Toyo oka K Wang K H Weitz C whi wynshaw Boris A Yoshida K Hasegaw and Hayashizaki Y TITLE Functional annotation of a full lengt JOURNAL Nature 409 6821 685 690 2001 PUBMED 11217851 REFERENCE 3 bases 1 to 1543 AUTHORS sShibata K Itoh M Aizawa K Nagao gene 6530404N21Rik Codon start l produc RIKEN CDNA 6530404N21 protein_ NP_853618 1 db_xref GI 31712012 db xref GenerD 67795 db xref Locus1D 67795 db xref MGI 1915045 translations MSRVLVVGAGLTGSLCAALLRKEITAPLYLGL 1 ggggacggeg aggctccgcg tctctgtcag cggcctagcc gaagggcagc cgccatgtcc 61 cgggtactgg tcgtgggcgc tgggctaacc ggaagtctgt gtgccgcgct gctgaggaag 121 gaaataaccg cccccctgta cctcggcctg tgagacaadg gtggggatat agggggaaga 181 atgattactg ccagcagtcc tcataatccc cgatgcacag ctgacttggg agctcagtac 241 atcacctgct ctcctcatta tgtcaaagag caccaaaatt tttatgagga actgttagct 301 catggaattt tgaagcctct gacatccccc attgaaggaa tgaaagggaa ggaaggagat 361 tgcaactttg tggcacctca aggattttct tcagttatca agtactactt gaaaaagtca 421 ggtgcagaag tctccctcaa gcactgtgtg actcagatcc acctgaaaga taacaagtgg 481 gaagtctcca cagacactgg ctctgctgag cagtttgacc ttgtcatcct caccatgcca 541 gctcctcaga ttctggaact tcaaggtgac attgtgaact taattagtga acgccagagg 601 gagcaactga aatctgtgag ctactcctct
43. N automatic CeSIQN x eec ue ette eee dee dea eta eer aa eese Eneas 27 4 3 1 Generate NCW primer sets 2 uei er chinvcse sentences Be yrde uut ru aga e nan ERA eR AREE RR 27 4 3 2 Analyze previously designed primers ssssssssssssssssesee seinen ehh hne nenne nnn 28 Cr AMBITIO 30 4 4 1 View results OVOeFVIeW ii dti inta aa ee Sahara EAR heel Gad aenea EX eina ee be e RE hela Deeds 30 4 4 2 SCOFING ANG QUIY Pieniniai aaar he sao nube trina R UAR Rae etf RE ERR EEREN ENARA 30 4 4 3 S rt the primer Setlist i es ond OR E M nel ded Red gs Pd ee ale 31 4 4 4 View information on different primer sets essssssssssssssss sese nen 32 4 4 5 Optional Select a different primer set as final assesses nennen 33 4 5 Adj st an dsSay ied ch pices Sue teeta wen retenue EE wale a 58 venae enne xi Pa dae AE epe ter SR rd te 33 4 5 1 Change the assay design SCCCINGS 21csceccecec ener tenet eet e ne nnn EEA EAE AAT ED hh ehe nna nnn 33 4 5 2 Edit the PCR and sequencing primers 1cccceceecnc eee nt ene e ete sehen eher nnn 34 4 5 3 Select one or more primers from a primer set and re analyze the assay 35 CES E I ELCHE 35 4 7 MiGW an assay TepOrt zeolite tae recu E reus NE REI ETE 36 4 1l VleW a report i cei cei kde iae SX ARRA R e RE REFER ERE ARR ERAN RA TRA ARR A TERRE RR ER DER EE ieee 36 47 2 PRING VEDOT enr sete a a E e A aae dan teddies sistea ewan no dee a EAA Ra 37 47 3 SAVE G TOPOL
44. Primer set area and do one of the following e double click the primer set e choose menu alternative Assay View Report e click the button View assay report in the Assay window e right click on the primer set and select View Report e press the short cut key combination Ctrl R The Report window opens displaying detailed information on the primer set e g the different analyses performed for each primer and primer combinations There are four different report formats to choose from as listed in the left part of the Report window Report formats Complete A report in Html format The report contains detailed information Results about the assay and the selected primer set in particular It presents details of the analysis steps that have been performed and any penalties and or warnings that have been generated during the design Complete A report in text format The report contains detailed information Results about the assay and the selected primer set in particular It Text presents details of the analysis steps that have been performed and any penalties and or warnings that have been generated during the design Summary A short summary in Html format The summary contains information about primer sequences and biotinylation in a format that is suitable for ordering oligonucleotides Summary A short summary in text format The summary contains Text information about primer sequences and biotinylation in a format that i
45. Qraph cceecee eee ee ee eee ee eee ee memes 61 9 7 Tips for using the melting temperature Tm graph ccceceeeee eee ee ee ee eee estes neta eee eeeaeeeas 62 9 8 Tips for analyzing InDels in homopolymeric stretches c ceeeeeeeee eens eens ee eeeeeeaeeeas 62 9 9 Analyzing short tandem repeats STRS ccceceee cece eee e eee tees eee e teens eaten emen emen 63 9 10 Troubleshooting quide sooo e e e teen te kuse tuer ced ee a bee new nde 64 10 Appendix A Methodological backQround scscsesceeeceeeeeeeeeeeeeeeeeeeeeaeueeeueeeeeeenenenans 66 10 1 Sample preparation ie adie rhe rre iiiter tois dir Ee HSA ae atten dads 66 10 2 Pyrosequencing Systems 1 er eere eren hx ne x e a Ra ae RE ER er su ERES EAN 67 10 2 1 Introduction iis te e PUB eae 67 10 2 2 Genotyping and mutation analysis ssssssssssssessss essen hehehe nnne nnne nn 67 10 2 3 Allel quantification AQ eiae baud etna oo oix euer eden Lad foe be eed EAR ease 67 10 2 4 Sequence analysis SQA cessssssssssssssssssses esee hehehe nha esae nsa esa enn nnn 68 10 3 JDefinitlOnsiiue irre tree ne ideas EN II RU XR a E hee ap has Mana tae 68 10 3 1 Allele S 1 2 i Ede oe Eae e Re ARA RER RE AERE ER Ee ERR TE HEARDUR TEE De YEARS 68 10 3 2 Single nucleotide polymorphisms SNPS isses ehe hehehe nnne nn 69 10 3 3 Insertions and deletions INDEIS 1 ccccceccecee ence tenet een esee hehehe hne hne nnne nn 69 10 3 4 Short
46. User Manual TNZ ai Assay Design Software 1 0 User Manual Version 1 0 6 AA Legal Warranty and Liability Biotage AB warrants that the product supplied has been thoroughly tested to ensure that it meets its published specifications The warranty is only valid if the product has been installed and used according to the instructions provided by Biotage AB Biotage AB makes no warranties expressed or implied including without limitation the implied warranties of merchantability and fitness for a particular purpose regarding the product Biotage AB does not warrant guarantee or make any representations regarding the use or the results of the use of the product in terms of its correctness accuracy reliability currentness or otherwise The user assumes the entire risk as to the results and performance of the product Since the exclusion of implied warranties is not permitted by some jurisdictions the above exclusion may not necessarily apply Biotage AB shall in no event be liable for any direct indirect special or consequential damages including without limitation damages for loss of business income business profits business interruption loss of business information and the like arising out of the use or inability to use the product Since the exclusion of implied warranties is not permitted by some jurisdictions the above exclusion may not necessarily apply Trademarks and patents owned by Biotage AB Pyrosequenc
47. ach analysis results in a penalty based on secondary structures or other potential problems that may have a negative impact on the PCR or Pyrosequencing analysis The final score of the primer set is a cumulative weighted sum of the penalties from all analyses Within a given template sequence high scoring primer sets have a higher probability of success during PCR and Pyrosequencing analysis than lower scoring selections In a high quality primer set i e labeled with color code blue none of the performed analysis steps have identified any problems of concern Therefore a high score primer set can be used directly without any more checks or analyses Warnings and penalties Primers automatically generated by the software are always within the parameters defined in the Default assay settings However if the primers deviate too much from the optimal target settings penalties and sometimes warnings are issued Warnings and or penalties are also issued if the software detects non favorable conditions such as secondary structures in an analysis step A warning is only issued for potentially serious problems where the generated penalty is above a certain threshold value 50 41 85 Primers that are added manually or edited will receive warnings and or penalties when parameters outside the Default assay settings are detected in addition to the warnings and penalties described above Warnings are displayed graphically in the information
48. agtaact agatttctaa 360 i i i i gcatt ctct tctttggtca gtaggacgac Ctaatttacg ataacctgat gccatgccta 420 RA Lee H M Dubois J Qiu D Hitti J wolf ESTEE ttai SCENES SELCI ceavcctans GRE 480 R L Sabathe F tctgttcaac ttcttctaaa Qattttcttc caaggtttct aactttcatc atatcttcca 540 i tggatctttg agttaattct tgaacagtgt ttattcctgc tctctttaag caattataac 600 RA Doucette stamm L Soucaille P Daly M J i2Eisusi iigetcisgt tetcgeesg CCICCICRig saccteeece crereavere 660 Koonin E V Cttcttttfc taccattatc tccac tcat ctgcatgate tgtaagagtc ataaataatt 720 RA smith D R Taaaatgctc tataagtatc tttgctgcca aactaattge ttcttct gt cttatagtac 780 S SROG a catttcecca tacttetatg gtgagtttgt cgtaatcagt tatttgagea actcttQtat 840 RT Genome Sequence and comparative Analysis o tttcaacagt gaaattcact cttttgattg gagaatatat tgaatcaact gctattgttc 900 1 duci ctataggcat gtcatctctt ttattcttat tctgagtaac ataacctctg cctctgttaa 960 Solvent Producini ier Bae cttcaattte Catatatagc tttccatctt categagagt tgctatatgt aaatccttat 1020 RT Bacterium Clostridium acetobutylicum tgataacttc tacagatcca tcagttctta tatcagegce tgtaacctct ccaggtccat 1080 4 jcatctat atatattact ttagaatctt caccttccat tttgaggcat aaagctttaa 1140 RL J Bacteriol 183 16 4823 4838 2001 Egecasgeat caatectgea acgectrctt tesctecttt ascageegas aactestgsa 1200 box gtactccctc aattctcact gaattagcgg caactcctgg taaagatgaa agaagtattc 1260 itcttaatga attgcccagg Qtaattccat atcctctttc aagaggttct acaacaaacc 1320 RN 2 tgccataaga acc t
49. analyze AT CCGTCAAAGC will result in the Dispensation order GATCAGTCA The first nucleotide in the Dispensation order as well as the first nucleotide following a polymorphic position is usually a blank internal negative control and is not the same as the first nucleotide in the Sequence to analyze 71 85 10 3 8 Reference peaks and quality control window Reference peaks are used by PSQ 96MA Software and PSQ HS 96A Software to determine which peak height corresponds to the incorporation of a single nucleotide The single peak height level is then used to determine the multiplicity of all peaks generated in the Pyrogram and thereby the sequence and genotype of the sample All non variable peaks generated in the sequencing reaction with a multiplicity of three or lower are used as reference peaks by the algorithm To assess the quality of the sequence all dispensations within the quality control window are used including negative control dispensations Reference peaks included in the quality control window will affect the analysis The quality window will be positioned symmetrically around the polymorphic position The quality control window is static so if dispensations fewer than half the value for the quality control window size are available before the SNP the size of the actual quality window will be reduced In the case of multiple SNPs analyzed with one sequencing primer the quality control window for one SNP position will end at the
50. arget region should be selected starting four nucleotides into the constant part of the repeat region In the example above choose CGACG ACG ACGACG as target region and generate only forward sequencing primers Tick the box Generate forward primers in the Current assay settings dialog By choosing this target area the forward sequencing primers that are generated will overlap between 0 and 4 bases of the constant repeat region Reverse assay ACGACG ACG ACGACGACG The target region should be selected starting four nucleotides into the constant part of the repeat region In the example above choose ACGACG ACG ACGAC as target region and generate only reverse sequencing primers Tick the box Generate reverse primers in the Current assay settings dialog By choosing this target area the reverse sequencing primers that are generated will overlap between 0 and 4 nucleotides of the constant repeat region 63 85 9 10 Troubleshooting guide Generated primers are not shown in or disappear from the sequence graph of the Sequence tab Primers are positioned incorrectly in the sequence graph of the Sequence tab The application becomes slower and slower The same PCR primer mispriming or template loop is shown twice in the report No PCR primers can be generated Import in PSQ software fails because an Entry with the same name already exists in the database Inadvertent use of the buttons to show hide prime
51. bases Adenine forms base pairs with thymine and cytosine forms base pairs with guanine They are called bases because they are alkaline basic in the acidic DNA structure Base pair Represents two complementary bases bound together by hydrogen bonds In DNA adenine A is hydrogen bonded to thymine T and guanine G is hydrogen bonded to cytosine C Two strands of DNA bound together by base pairs form a double helix The number of base pairs is often used as a measure of the length of a DNA segment Batch assay design Simultaneous assay design on several DNA sequences Biotin A molecule that can bind very strongly to streptavidin Can be used for biotinylation of primers to bind a DNA strand to a streptavidin coated solid phase BLAST homology search BLAST Basic Local Alignment Search Tool is an algorithm that can be used to search sequence databases for homologous sequences It may be used to characterize unknown sequences or to find homologous or related sequences C Chromosome A physically distinct unit of the genome containing many genes The chromosomes are replicated duplicated during cell division Prokaryotic genomes often carry the entire 79 85 genome on one circular chromosome whereas eukaryotic genomes often have a number of chromosomes Cyclic dispensation order A repetitive dispensation order for nucleotide dispensation in the Pyrosequencing reaction Normally used in Pyrosequencing technology for sequenc
52. ccupying a given locus position on a chromosome Allele frequency The estimation of the proportion of each allele at one gene locus for example the proportion of each allelic variant in a SNP in a population Allele quantification Allele quantification is used to estimate the allele frequencies for sample populations Amplicon The amplicon is the DNA sequence amplified in a PCR reaction and is defined by the PCR primers on either side of the target region forward and reverse primer Annealing The base pairing of a primer to a complementary single strand of DNA or RNA to form a double helix The bases are held together by hydrogen bonds formed during annealing Annealing over a SNP The user can allow the PCR and or sequencing primers to anneal over a SNP if necessary This is useful if a polymorphism needs to be genotyped where the surrounding sequence contains SNPs Assay An assay is defined as the information required to perform PCR amplification and Pyrosequencing analysis on a sample The unique feature of an assay is the target region which either contains the polymorphism s of interest in genotyping or AQ or the unknown SQA sequence An assay generated by Pyrosequencing Assay Design Software contains information on suitable PCR primers sequencing primers and corresponding sequences to analyze Base A base is a part of nucleotides which are the building blocks of DNA Adenine thymine cytosine and guanine are
53. ce Pyrosequencing Assay Design Software User Manual version 1 0 6 AA Copyright 2004 Biotage AB All rights reserved No part of this manual may be reproduced or transmitted in any form or by any means electronic or mechanical for any purpose without the expressed written permission of Biotage AB Biotage AB Kungsgatan 76 SE 753 18 Uppsala SWEDEN Phone 46 18 56 59 00 Fax 46 18 59 19 22 E mail info biotage com Web http www biotage com 2 85 Contents 1 In ppm 5 11 The Wser Manual tsscestessee LET 5 1 2 THe sQuiek Guide sie 5e sess eto em eee ss cae het ce nde eren oa Qe ek Roy ces Sena rud d eee ete de DRE NEL wee 5 2 EO LNEIUpDMM 6 2 1 System requiremients s cies cen erra ree Dx noe eon Cuca eaae de ahs xe evs R xe EU a ERN ERR DER pen 6 2 2 Installing the software icio reg tete Mer ur nied UE eR IPEA ree M deeds 6 2 3 Starting Pyrosequencing Assay Design Software csssssssssssssesen menn 9 2 4 The Assay Design Software start screen ssssssssssssseseeseenemeneme seems enn 10 3 Assay design settings ueeeeeeeieieieieiesesesn sese nu nune neat a tacui k ununi u aane n anna n uan 11 3 1 Change default assay design settings ccccee cece ee eee eee e eee eee ents eae ee tanta eaee 11 3 2 Change assay design settings for one ASSAY ceeeeee eee e eee e
54. cgctatgctc tgggcctctt ttatgaagta 661 ggcatgaaga ttggtgtccc ttggtcctgc cgctacctca gcagtcaccc ctgcatatgc 721 ttcatctcca ttgataataa gaagcgcaat atagagtcat Cagaatgtgg tccatccgtg 781 gtgatccaaa ccactgtcce atttggagtt caacacttgg aggccagtga ggcggatgtg 841 Cagaagttaa tgatccagca attggaaacc attctgccgg gtttgcctca gccagttgct 901 accatatgcc ataaatggac atattcacag gttacaagct Cagtttccga Cagacctggt 961 cagatgactc ttcatctcaa gcctttcctg gtgtgcggag gggatggatt tactcactcc 1021 aacttcaatg gctgcatctc ctctgccctg agtgtcatga aagttttaaa gcgttatatt 1081 tagtgtctgt gttcttgttc tctacattta ctttaggatt tttgttttca caatcctctg 1141 ttattaattg ttctattttc ctttctgtga agagaaatta ctttggaaaa tgtcttcact 1201 tactgttatt tttaatatga aatataaaaa caatgttata attttgatag ctaccaccca 1261 tagtaaagtg ataacttctt aggctatatt tcttcctcac ttttctgact ttattactaa 1321 tccttattgg agaaaaatgg tgcttctaag tcacaaataa cccaagccca gaattatact 1381 aaaccaaagt tctcattcat ggaaaatctt tattatcctt ttgaaaactt Cagttctaaa 1441 taaataacag ttactaccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1501 aaaaaaaaaa aataaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 24 85 4 2 3 Step 3 Name Polymorphisms and unknown sequences 4 2 3 1 Name polymorphisms and unknown sequences in the Polymorphisms area An unknown sequence is treated in the same way as a polymorphism in the Polymorphisms area 1 Display the desired polymorphism to give a Position name by using the arrows at the bottom of the Polymorphisms area M 4 203 gt 2 Enter a po
55. cleotde v forf Go Gea uns Previewindex History Cipbciai tals Display deut show 20 _Sencta Tea __GetSubsequence Features T E NM 181587 Mus musculus RIKE 3131795586 Ele nis Ted Locus p Oipcord near non 22 DEc 2005 DEFINITION iS RIKEN CINA 553040421 gene 65204ANZIRIK MENA accesso VERSION GT 31795586 KETOORIS mci ongANIsIE REFERENCE AUTHORS TITLE som U S A 99 26 16899 16003 2002 REFERENCE AUTHORS Kem ve Shibata E e Yo o Hu E CES F Rost 22 85 2 If the sequence is to long to be imported into the Assay Design Software i e more than 10000 characters press the button Get Subsequence and select the desired sequence range in the dialog that appears INCH Seu soft Internet Eerlorer zialki Fie cde vew s Tis eh ES wm gt gt QUIE Al semen ravos Grey gt e Ed S Adress ET tetp slower govfentreeinwer eg di udecliotpi Genaro ae 3L 79GS06 z ee Junie gt Google Gesehen Giskpdse PRK G up Bye lt a e Ag 3l Nucleotide t Searh Nudeoide for Go ceo Limits Previewindex History Clipboard Detalls Display defan show 20 _Sendto Tex s GetSubsequence Features F 1 NM 181587 Mus musculus RIKE gi31795586 Links Locus NW 181587 1543 bo RN DET ES 7 imi DEFINITION Mus wusculue RIKEN CDNA 6530404N21 gene ACCESSION NN 181587 Change Sequence Range VERSION NY 181587 2 GI 31
56. cttct gtagactcaa cacattctat tttaggtttt tctatttcca 1380 RP 1 2978 acatatctat aaccctcctt aaaaataaaa tggcaaccta tattgagggt aactgaatac 1440 agtcaaatta cttactatat aactcaacga tcaatgtctc tttaattgga gtatctatat 1500 RA childress D zeng Q smith D R cetctctatt tggctctgca actattttcg cttcaaaatt atcatagttt gcttcaatcc 1560 RT 1 atgctggtaa agtctttgga ttttctgcaa aagtcttgaa tttttcagtt cctctactct 1620 Limited h i tctcactaac agatactact tcattaactt tt atgt ta tgaaggtata tctactttct 2680 Sie ttccatttac aaggaaatgt ccgtgagtaa ctaattgtct tgctterttt cttgagctac 4 RL Submitted 24 3UL 2001 to the EMBL GenBan Catagcctaa tttgtaagcc acgttatcaa gtcttaattc caaaagctta agtaggtttt 1800 Cacctgatat tcctttaatt ctttcagctc ttttatagta tattctaaat tgtttttcaa 1860 gaactccata tattcttttt gctttttgtt tttctcttaa ttgtaatccg tagtttgaaa 1920 ftttctttct gctctgtcca tgtactcctg gtgcataacc tcttcttgta aatgcacatt 1980 tatctgtata gcatctatca cccttaagga ataatttcaa accttctctt ctgcataatc 2040 tgcatacage tccagtatat ctagccatta aatattcaca cctcctatat ttctattaaa 2100 ctcttcttct tttt gtggt ctgcagccat tgtgtggtat tggagtaaca tcttttatta 2160 atgtaacctc taatcctgca gcctgtaatg atcttattge agcttctctt cctgatcctg 2220 gtccttttac aaacacatca cactctta gtccgtgttc cattgctgtt ttagcagctg 2280 ittcagcagc catttgagca gcaaaaggag tgctttttct tgatccttta aatcctaatc 2340 cacct cact tgaccatgat aaagcatttc cattaacatc agttatagta actatagaat 2400 Tgttaaaagt tgatttaata tgtgcacaac cgtgctctat attttttitc tctttrcttc 2460
57. d Enter a longer sequence and make sure that the target polymorphism sequence is flanked by a sufficiently long sequence stretch 200 bp on either side of the target is recommended Open the assay file in Assay Design Software Choose File Save As and save the assay with a new name The new assay name will be stored inside the xml file Retry import in PSQ software Batch analysis fails for one or several assays A high scoring assay could not be run in PSQ 96MA or PSQ HS 96A If batch analysis fails with one of the three following messages the problem is caused by illegal characters within the sequence or lack of a target polymorphism Messages Skipped empty or un intelligible assay test 48 Test 48 Before running the analysis target area or polymorphism has to be defined Before running batch resolve the following errors Something went wrong when interpreting the sequence Example an AQ assay of three consecutive SNPs that share allelic bases e g MRS is not possible to analyze in PSQ because of confounded signals This is not flagged until the assay is imported entered in PSQ system software because Assay Design Software does not have the ability to generate dispensation orders 65 85 Confirm that all assays contain polymorphisms or SQA target regions Resolve any incorrect characters in the sequence and re start batch analysis Enter import assays in PSQ
58. d vice versa Do as follows o Open two assay windows and enter the two sequences of interest o Design primer sets for assay one o Select the desired primer set in the Primer set list right click and select Copy primer set from the right click menu o Switch over to the second assay window Select biotinylation of the same primer as for assay one This can for example be done by clicking the button Set sequencing primer direction in the Primer set list o Put the cursor in the top field of the Primer set list i e in the forward PCR primer field right click and select Paste from the right click menu The selected primer set from assay one will now be pasted into the fields of assay two o As soon as you leave the entry fields the primer set score is updated The score can be expected to be zero discard since the primers from assay one are not expected to be complementary to the sequence of assay two Look in the info field on the Sequence tab to see if the PCR primers from assay one form any serious misprimings in the sequence of assay two When designing the different primer sets in Assay Design Software increase the Maximum Distance from Target in the settings in order to obtain sequencing primers at varying 3 positions This will allow for a higher flexibility in design of the multiplex assay Another option is to widen the target region so that it includes a number of nucleotides preceding the polymorphism The program is the
59. djust the assay in a different way 4 6 Save an assay An assay can be saved in xml file format The stored assay file can be used either to re open the assay in Assay Design Software or to import an Entry in PSQ 96MA or PSQ HS 96A system software To save the assay 1 Select File Save or Ctrl S in the menu bar or click the Save e button in the Assay window The Save as dialog opens 2 Locate the folder in which to save the assay 3 Enter a name for the assay and click Save If changes are made to the assay and the assay is saved the assay file is overwritten with the new information To save the assay in a different folder and or with another name use the Save As command Note The xml format is not associated with the Assay Design Software application Therefore assay files must be opened from the Assay Design Software and not by double clicking on the file in for example Windows Explorer 35 85 To save the assay in a different folder and or with another name 1 Inthe menu bar select File Save As The Save as dialog opens 2 Locate the folder in which to save the assay 3 Enter a name for the assay and click Save 4 7 View an assay report There are several ways to display a report with detailed information for a selected primer set The report can be printed and or saved in either Html or text format 4 7 1 View a report To open the Report window select the primer set by clicking on it in the
60. e an overview of possible warnings and the analysis steps they are linked to Some of the warnings are only generated for manually entered primers never for automatically generated primers PCR primer analysis Deviation from optimal T Self annealing duplex detected Hairpin loop structure detected Mispriming site detected Primer with low complementarity Deviation from optimal 3 end stability Tm for PCR primer is outside settings Primer length shorter than min PCR primer pair analysis Amplicon length outside size limit Deviation from optimal amplicon size Deviation of GC in PCR primers and or amplicon Deviation of GC in PCR primers and or amplicon is outside settings Tm difference out of range Large T difference Cross annealing duplex detected Melting temperature analysis Duplex formation analysis Hairpin loop analysis Mispriming analysis within the entered sequence Primer template complementarity analysis PCR primer 3 end stability analysis Only for manually entered primers Melting temperature analysis Only for manually entered primers Primer length analysis Only for manually entered primers Amplicon length analysis Amplicon length analysis GC content difference analysis Only for manually entered primers GC content difference analysis Only for manually entered primers Melting temperature difference analysis Melting temperature difference analysis
61. e editor tab or from the Polymorphisms area How to set the target region in the Polymorphisms area and on the Sequence Tab respectively is described below Skip between polymorphisms and set target region in the Polymorphisms area 1 Ifthe entered sequence contains several polymorphisms skip between them in Sequence Editor by clicking the arrows in the Polymorphisms area 4 203 gt 2 Once the desired target polymorphism or unknown sequence region has been selected click the Set Target button e 25 85 Set the target region on the Sequence tab 1 Genotyping AQ On the Sequence tab mark the polymorphism s to analyze and the desired sequence length before and after the polymorphism s SQA On the Sequence tab mark the region to analyze If analyzing unknown regions include 2 3 known nucleotides flanking the unknown sequence in the target region Note It is possible to search for a sequence motif to set as target region see below Right click on the marked sequence and select Target Region Set Target Region Alternatively select Assay Target Region Set Target Region in the menu bar The target region for Pyrosequencing analysis is highlighted in blue On the Sequence Editor tab the nucleotides in the selected target region are highlighted in yellow Generated sequencing primers will be placed outside of this region The software automatically defines a PCR amplification region around the selected target
62. e frequencies manually The linear relationship between peak height and nucleotide incorporations in combination with high signal to noise ratios for the obtained peaks facilitate the estimation of allele frequencies even for low frequency SNP alleles For more information see the user documentation for PSQ 96 MA System or PSQ HS 96A System 67 85 10 2 4 Sequence analysis SQA PSQ 96MA Instrument is used together with dedicated software and kits for the analysis of DNA sequences typically 30 50 bp in length The PSQ 96MA SQA software automatically completes base calling and the sequence alignment function enables easy comparison to a master sequence Sequence data can also be exported to other systems for database comparisons Along with sequencing short strand DNA templates PSQ 96MA System SQA is capable of straightforward sequencing of cloned DNA material templates with strong secondary structures and complementary DNA This is achieved by the addition of single stranded DNA binding protein SSB after the primer annealing step To sequence DNA templates a suitable dispensation order must be entered in the Define SQA Entry dialog in PSQ 96MA SQA software For more information see the user documentation for PSQ 96MA System 10 3 Definitions 10 3 1 Alleles An allele is any one of a number of alternative forms of the same gene occupying a given locus position on a chromosome If an individual is homozygous for a certain
63. eaction producing a suitable PCR product for Pyrosequencing analysis follow the general guidelines below Optimize the PCR protocol and conditions to obtain good PCR results PCR primers e Primer concentrations In general a PCR primer concentration of 0 2 uM is recommended Lower concentrations may be exhausted before the reaction is completed resulting in lower yields of the PCR product However biotinylated PCR primer concentrations should be kept low to avoid interference in the Pyrosequencing analysis We strongly recommend that the biotinylated primer is purified by HPLC or an equivalent procedure to minimize the amount of free biotin e Storage of primers Biotinylated PCR primers are particularly sensitive to storage Keep a stock primer solution at 20 C For the working solution prepare small aliquots of diluted primers 10 uM and store at 20 C PCR product e PCR product and size If possible select PCR primers to form a PCR product x 250 bp The typical range for PCR amplicon size is 40 to 250 bp Smaller PCR products have several advantages e g the amplification is easier and the risk of background in the Pyrosequencing analysis is reduced However up to 600 bp long products have been tested with good results e GC rich PCR products Amplification of very GC rich regions gt 70 often benefits from adding 5 dimethylsulfoxide DMSO and or exchanging part of the deoxyguanosine with deoxyinosine A ratio dI d
64. ed in the PCR reaction Minimum melting temperature of PCR primers Maximum melting temperature of PCR primers Maximum allowed Tm difference between forward and reverse PCR primers Maximum allowed GC difference between the forward and reverse PCR primers and the amplicon 3 3 2 Sequencing primer settings Min Primer Length bp Max Primer Length bp Min Distance from target bp Max Distance from target bp Allow Primer Over SNP Generate Forward Primers Generate Reverse Primers Minimum sequencing primer length Maximum sequencing primer length Minimum distance between the sequencing primer and target region Maximum distance between the sequencing primer and target region Check the box to allow annealing over SNPs Note Primers will not be allowed to anneal over an SNP within the 7 last nucleotides of its 3 end Check the box to generate forward sequencing primers Check the box to generate reverse sequencing primers 12 85 18 bp 18 24b 24 bp P S0 ap 50 250 bp 250 bp 600 bp Unchecked Nearest s Neighbor 0 2 uM 56 0 C 68 74 C 86 0 C 10 0 C 0 2 C 30 0 10 15 bp 20 bp 15 20 bp 0 bp 3 bp Unchecked Checked Checked 3 3 3 Primer set settings Primer Set Z Number of primer sets to be listed in the Primer set area The number of primer sets listed in the Primer set area is tightly linked to the number of sets that will be
65. ee eee ee eee eee emen emen 11 3 3 Description of settings and buttons irissat orin anera eee eee eee eee eee eee oai 12 3 341 PCR Primer settings uoc iier perse dou need oxdadeengu tate ERE due bods stegseede bree bss 12 3 3 2 Sequencing primer settings io REA hn shee cate RR deen codecs EEA A 12 3 3 3 Primer s t settings oss sei eR e Era ERR RR ARR a AAE AEA Aaoi 13 3 3 4 IBULLOTIS iii n OH e Ue E edu Ue Xxx e eden a xugns ah acing esate gales Fees e na eke 13 3 4 Selected settings will affect primer set SCOFING cccceeeee eee e eee eee teens eee ee mme 13 3 4 1 PGR Primet score effects o ert sak lees eed eee RR MEHR RR E Vong E Lat EE line sara a 13 3 4 2 Sequencing primer score effects imersa dede oia dp dhe ad eru ase a e iaaea iaa aaa 14 4 Performing an assay design essseseseseseiesesessununuua usan a nane neue u iaa uiuunt 15 41 Introduction eR EID 15 4 2 Create an assdy setup ciceede tuer i rc eM ed EE p edna EM tate RE ERE 16 4 2 1 Step 1 Choose assay type and enter a description for the assay sess 16 4 2 2 Step 2 Enter the DNA SCQUCNCE uiii i eae ap aient a Ee ne eR EER eed eas Signet ketal s 16 4 2 3 Step 3 Name Polymorphisms and unknown sequences essen 25 4 2 4 Step 4 Optional Set the target region ssssssssssssssssesses essen enne rennen 25 4 2 5 Step 5 Optional Redefine PCR primer regions sssssssssssssses eene 27 4 3 R n A
66. egment 2 of 23 iu Display Options ae E FKLR L type pyruvate kinase evcr intron junctens human D Emassan sen TTE rin 2a view results using T Fma asaan FKLE L tyne pyruvate krase exon intron junctions human me e TOT D internet EMBL number of the record of choice to 3 Click the Save button in the left panel of the sequence record to save the entry ry Page Microsoft Internet Explorer Heak gt OA Reach Cairavortes Hristov Gh G Oi ID Address E http jsrs ebi ac uk srsbin cgi bin wgetz id 1fGfP1NaN2Y e EMBL AY399933 qnum 1 enum 1 Google z Gosckpiwebben GySoK pS sten PAA sano Eup sie Form Tools Results Databar Entry 1 of 38 from Query 1 3399933 standard genomic DNA GSS 1618 BP Entry a ation 1399933 41399933 1 13 DEC 2003 Rel 78 Created 17 DEC 2003 Rel 76 Last updated Version 2 Launch analysis tool pz Homo sapiens PELR gene VIRTUAL TRANSCRIPT partial sequence genomic Boen a DE survey sequence xx immo ku css xx Link to related CE Hono sapiens nunen OC Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Manmalia Link oc Eutheria Primates Catarrhini Hominidae Homo BEEN Save entry save RN 1 E BP 1 618 View RA Clark A G Glanowski S Nielson R Thomas P Kejariwal A Todd M A RA Tenenbaum D M Civello D R Lu F Murphy B
67. enhance the speed of a biochemical reaction without altering it EMBL Nucleotide Sequence Database The EMBL Nucleotide Sequence Database is Europe s most comprehensive nucleotide sequence database F FASTA format The FASTA file format is a common format for DNA sequence files A sequence in FASTA format begins with a single line description followed by lines of sequence data The description line is distinguished from the sequence data by a greater than gt symbol in the first column It is recommended that all lines of text are shorter than 80 characters in length Forward assay A forward assay is an assay where the sequencing primer is annealed to the complementary strand of the input DNA sequence Sequencing will be performed reading the polymorphism unknown region in forward direction i e generating the input sequence For a forward Pyrosequencing assay the reverse PCR primer should be biotin labeled Forward PCR region The area in the DNA sequence in which forward PCR primers will be placed when performing primer set generation 80 85 G GC content The percentage of G and C bases in a DNA molecule GenBank GenBank is an annotated collection of all publicly available DNA sequences and the genetic sequence database of the US National Institute of Health NIH Gene A specific DNA sequence from which a protein or RNA can be generated The human genome is estimated to contain 30 000 genes Genome The total genetic
68. entarity to the DNA template sequence This is a natural consequence of the addition of a long tail and does not mean that the primer is unsuitable for Pyrosequencing analysis The score and quality values should therefore be ignored in this case since they are based on partially irrelevant analyses Instead the important thing is to check if any serious new misprimings PCR duplexes or template loops have been formed and detected 9 6 Tips for using the Gibbs free energy AG graph The Gibbs free energy is used to determine a primer s priming specificity The AG values indicate how specific the primer is and how efficiently it will anneal to its intended target Primers with a stable 5 end i e low AG value and a relatively unstable 3 end i e high AG value typically perform best because they are both more stable and specific and thereby rarely misprime A primer with low stability at its 3 end will function well in a PCR because the base pairing close to the 3 end with non target sites are not sufficiently stable to initiate extension false priming Conversely primers with a stable 3 terminus need not anneal with the target along their entire length in order to prime efficiently This could result in non specific products false priming Assay Design Software assesses the relative stability of the PCR primers in the Primer end stability analysis For this the following software settings are used Optimal AG difference 2 0 Maximum
69. equencing primer design ssssssssssssse emen 51 7 5 1 General guidelines for sequencing primer design esses nnns 51 7 5 2 Sequencing primer design for genotyping and allele quantification susuusuu 52 7 5 3 Sequencing primer design for SQA essssssssssssssssess sehen hehehe hehehe nnne nsn nena 53 8 Guidelines for PCR setup and optimization eeeeseseseseeeen eene nnne nennen 54 8 1 Guidelines for PCR setup and optimization for Pyrosequencing analysis 54 8 2 PCR setup and optimization Specific for allele quantification cceeceeeeeee teen eee ee ees 56 8 3 PCR protocol example 2 55 eR recte ex E ea ie re eR Mer uds 57 8 3 1 Optimization of the PCR protocol and conditions esses 57 9 Hints amp po q 58 9 1 Tips for succeeding with difficult ASSAYS cc eceee eee e ee eee ee eee e eee neta emen enne 58 9 2 Tips for avoiding PCR cross contamination cccceeeee eee ee ee eee ee ee eee eee e eee eee ea teeta eenaeas 59 9 3 gt TIPS fOr assay controls idees a ee def pe EET eu cath Pewee PE sixe ui en RE eR Er RR 59 9 4 Tips for multiplex assay design dene enun naa en e aa a E aa aA 60 9 5 Tips for universal biotinylated PCR PriMerS cccceeee eee ee ee eee ee ee eee eee e neta eee ea nena eene 60 9 6 Tips for using the Gibbs free energy AG
70. er the sequences for sequence analysis in the Assay Design Software 11 2 2 Entering sequences for SQA into Assay Design software Sequence containing variable region with unknown variants Enter or import the sequence in Sequence Editor with a number of N nucleotides to denote the unknown sequence E g ACCAGTATTITAGGACCAGATTAGGNNNNNNNNNNNNNNNNNNNNNACCAGGATGACAGTAGACCC The default target region will then be the stretch of unknown nucleotides with three known non variable nucleotides flanking the unknown region on either side Default target region ACCAGTATTTAGGACCAGATTAGGNNNNNNNNNNNNNNNNNNNNNACCAGGATGACAGTAGACCC Sequence containing variable region with known variants 1 Enterthe sequence of the expected or most common variant Expected sequence variants ACGTGGCTGG CATGGCTGCT CG ACGTGGTG ACGTGCTG ACCCTCC CGTGCTGCAT GGCTGCGCTG CTG Enter ACGTGGCTGG CATGGCT GCI GCTGCAT GGCTGCGCTG CTG 2 Set the nucleotides highlighted in blue as target region i e the variable region flanked by three known non variable nucleotides on each side Confirming a known sequence Enter the expected sequence and mark the region of interest as the target region 78 85 12 Glossary A Adenine A A purine base that is a part of DNA and RNA molecules Adenine forms base pairs with thymine a pyrimidine base and uracil in the case of a RNA molecule Allele An allele is any one of a number of alternative forms of the same gene o
71. erate matching primers or enter a complete primer set and have it analyzed by the software View results and select a final primer set The results of the assay design are displayed as primer sets with different scores and quality It is possible to view detailed results for the different primer sets by double clicking on a set The primer set with the highest score is automatically selected as the final primer set can be changed if desired When importing an assay into PSQ system software the information for the final primer set will automatically be transferred to the created Entry Optional Adjust the assay It is possible to adjust the assay by changing the assay design settings Save the assay The assay can be saved as an xml format file with the file extension xml Note The xml format is not associated with the Assay Design Software application Therefore assay files must be opened from the Assay Design Software and not by double clicking on the file in for example Windows Explorer Assay files can be imported into PSQ system software automatically generating an SNP Entry for the assay in the PSQ database Note Once a primer set is imported into the PSQ software the created Entry cannot be edited If editing is required select menu choice Duplicate Entry in PSQ software to create an editable copy of the Entry View an assay report of the selected primer set A report of the assay containing detailed information on the
72. eric stretches If the polymorphic nucleotide s of an InDel can form a homopolymer with adjacent nucleotides i e three or more of the same nucleotide in a row then a sequencing primer should be selected that overlaps the homopolymeric nucleotides Enter two different notations for forward or reverse assays see Example 1 Example 1 Allele 1 TTTTT insertion Allele 2 TIT deletion Forward assay Enter sequence as TTT TT Select TT as target region Open Settings for the current assay and for the sequencing primer check only the Generate forward primers box Reverse assay Enter sequence as TT TT Select TT as target region Open Settings for the current assay and for the sequencing primer check only the Generate forward primers box 62 85 9 9 Analyzing short tandem repeats STRs To design an assay for STR analysis use the InDel notation see Example 2 Example 2 Allele 1 ACGACGACG Allele 2 ACGACGACGACG Allele 3 ACGACGACGACGACGACG Assay Design Software will not position primers inside the selected target region By default sequencing primers will be positioned 0 to 3 nucleotides from the border of the selected target area In order for the sequencing primer to overlap some part of the constant repeat region two different notations and choices of target region should be used for forward and reverse sequencing primer generation respectively Forward assay ACGACGACG ACG ACGACG The t
73. es The weighted sum of the penalties is used to calculate a total score for each primer set and the result is a list of primer set candidates with different scores for quality and suitability for PCR and Pyrosequencing analysis A number of parameters are taken into account when using Assay Design Software to generate primer sets PCR and sequencing primers Many parameters e g target distance can be changed in the software using the Default assay settings dialog The software always tries to achieve the set optimal conditions when designing the primer sets Primer sets where the conditions deviate significantly from the set optimal conditions will get a higher penalty and thereby a lower total score and quality The primer design part of the User Manual includes information on e The analysis steps carried out by the software i e which parameters and secondary structures are checked and detected by the software when designing PCR primers and sequencing primers e Guidelines for PCR primer design e Guidelines for sequencing primer design 7 2 Analysis steps performed by the software 7 2 1 Introduction When designing PCR and sequencing primers for Pyrosequencing analysis in Assay Design Software a number of analyses are carried out resulting in a list of primer set candidates with different scores 100 0 where 100 is the best score and quality Different analyses are used to assess the PCR primer pairs and sequencing primers E
74. folder e Type in a DNA sequence Type the sequence as a continuous text in 5 3 direction The allowed characters for sequence input are A C G and T representing the four possible nucleotides adenosine cytidine guanosine and thymidine as well as IUPAC codes for polymorphisms W R K Y S M B H D V N Enter up to 10 000 nucleotides Polymorphisms genotyping AQ and unknown regions SQA should be entered using the respective formats described in sections 12 1 and 12 2 Nested polymorphisms are not allowed e Copy and paste a DNA sequence Copy a sequence from a text editor e g Microsoft Word or Notepad or from an Internet browser and paste it into the empty area on the Sequence Editor tab If the sequence contains row numbers these will automatically be removed when pasting the sequence into Sequence Editor To copy a sequence from the Sequence Editor of one assay window to the Sequence Editor of a second assay window right click on the sequence and select menu alternative Copy Entered Sequence This is useful for example when designing assays for multiple SNPs within the same PCR amplicon 17 85 e Open a previously saved assay file xml Select File Open to open a previously saved assay file xml in the Assay window 4 2 2 2 Import DNA sequences FASTA file format Using FASTA format several sequences can be imported simultaneously into the Assay Design Software For each sequence in the FASTA text
75. g primer The ring on the reverse PCR primer arrow indicates that this primer is biotinylated Blue gt High quality Yellow Medium quality Orange Low quality Red Bad quality Discard the primer set 30 85 3 Primer set ID 58 Primer set score Fi Forward PCR primer ID R3 Reverse PCR primer ID S1 Sequencing primer ID For a primer set with high quality i e labeled with color code blue and with a score higher than 87 none of the analysis steps performed have identified any problems of concern The software algorithms are very stringent in their analyses and a high score primer set can therefore be used directly without any further manual checks or analyses For a primer set with quality medium or low i e labeled with color codes yellow or orange one or several of the analysis steps have identified problems that may be of concern The software algorithms are quite stringent and most medium score assays can be expected to work very well whereas low score assays only should be used after very careful consideration In general two primer sets with similar scores are of equal quality and either one of them can be selected for Pyrosequencing analysis For primer sets with equal scores it may sometimes be informative to see if the primer set score has been lowered because of a rather severe penalty in one analysis step or because of several individually lower penalties In this case the latter primer set with many
76. g the amplification reaction and therefore require more cycles than a protocol with ordinary DNA polymerases For best yield and consumption of all biotinylated PCR primers which is important for Pyrosequencing run 45 50 PCR cycles when using a hot start DNA polymerase compared to 35 50 cycles when using ordinary DNA polymerases e Typical PCR program A typical PCR program for PCR products of up to about 300 bp using 55 85 a hot start DNA polymerase 95 C 5min 45x 95 C 15s T C 30s 72 C 15s 72 C 5min 4 C The program takes about 1 hour and 45 minutes to run For PCR products longer than 300 bp the extension time at 72 C may need to be increased Annealing e Optimal T temperature T The T4 of PCR and sequencing primers is calculated by the Assay Design Software The optimal annealing temperature Ta for the PCR reaction is likely to be between 5 10 C below the lowest Tm of the pair of primers to be used Most primers will anneal efficiently in 30 sec or less unless the T is too close to the T4 or they are unusually long The typical T range is 54 62 C e T effect on annealing and amplification Low T One consequence of having a too low T is that one or both primers will anneal to sequences other than the true target since internal single base mismatches or partial annealing may be tolerated This can lead to non specific amplification and a consequent reduction in yield of the desired
77. hlighted buttons unlocked primers 6 Click the Save candidate button d to save the primer set to the primer set list An asterisk below the primer ID indicates that it is a manually entered primer set 4 4 View results 4 4 1 View results overview The results of assay design are displayed in the Primer set area in the form of a primer set list Every primer set has been assigned a score and quality which reflects its suitability for both PCR amplification and Pyrosequencing analysis By default the primer sets are sorted by primer set score 0 100 where 100 is the best score so that the best primer set ends up at the top of the list The primer set list can be sorted in other ways see section 4 4 3 One hundred primer sets are shown by default This value can be changed in the Default assay settings dialog if desired By default the top score primer set is e shown at the top of the Primer set area e displayed first in the primer set list and highlighted in light blue to indicate that it is selected e defined as the final primer set indicated by a dark gray box surrounding it the selection of the final primer set can be changed if desired see section 4 4 4 4 4 2 Scoring and quality Primer set description gt Fl ACAGGATAGACAGATAGGACA gt R3 TCTCTTGGTGTGTCTCTAATTC GGACAGATATAGACACCAGA The color in the left panel indicates the quality of the primer set The arrows indicate the direction of the correspondin
78. hout penalties Optimal Amplicon 250 bp Amplicons outside the optimal range Length to bp but shorter than the max length will be Max Amplicon 600 bp generated but given a penalty that Length bp increases linearly Allow Primer Over Unchecked If the box is checked a PCR primer is SNP allowed to overlap up to two SNPs located within the 5 end half of the PCR primer No penalty is associated 50 250 bp 13 85 Primer 0 2 uM Concentration HM Min Melting 56 0 C 68 74 C Temperature C Max Melting 86 0 C Temperature C Max Allowed Tm 10 0 C 0 2 C difference Max GC Difference 30 0 10 Primer concentration will affect the Tm calculations Primers with Tm in the middle of the set range 3 C will be generated without penalty i e with factory settings Tm between 68 74 C will not get penalized Primers outside the optimal Tm range but within the set min and max temperatures will be generated but given a penalty that increases linearly Tm differences within the optimal range 0 2 C will be generated without penalty Tm differences higher than 2 C but lower than the set max value will be generated but given a penalty that increases linearly with Tm difference GC differences within the optimal range 0 10 will be generated without penalty GC differences higher than 10 but lower than the set max value will be generated but given a penalty that increases l
79. in FASTA format should be saved as text txt files An example of a FASTA file containing multiple sequences is shown below ziBixi File Edit Format Help gt AGR T449C gatcttccttcctgtcccttcagtgccctaataccatgtatttaaggctggacacatcaccactcccaacctgcctcacccactgcgt c acttgtgatcactggcttctggcgactctcaccaaggtctctgtcatgccctgttataaC Tgactacaaaadcaagtcttacctatag gaaaataagaattataacccttttactggtcatgtgaaacttaccatttgcaatttgracagcataaacacagaacagcacatctttca atgcctgcatcctgaaggcattttgtttgtgtctttcaatctggctgrgctattgttggtgtttaacagtctccccadctacactggaa acttccagaaggcacttttcacttgcttgt tgttttccccagtgtcrattagaggcctttgcacagggtaggctctttggagcadgctg aaggtcacacatcccatgagcgggcagcagggt cagaagt ggcccccgtgtt cctaagcaagactctcccctgccctctaccctctgc acctccggcctgcatgtccct tggcctrcttgggggt acat ct cccggggcrgggt cagaaggccr gggt ggrtggcctr caggct gt ca cacacctagggagat ctcccgtttctgggaaccttggc APOE codon 112 ccgcagcggaggtgaaggacgtccttccccaggagccgactggccaatcacaggcaggaagatgaaggttctgtgggctgcgttgctggt cacattcctggcaggat gccaggccaaggt ggagcaagcggt ggagacagagccggagcccgagct gcgccagcagaccgagt qgcaga gcggccagcgctgggaactggcactgggt cgcttttgggattacct gcgctgggt gcagacact gt ctgagcaggt gcaggaggagct g ctcagctcccaggt cacccaggaact gagggcgct gat ggacgagaccat gaaggagtt gaaggcctacaaat Cggaact ggaggaaca actgaccccggtqgcggaggagacgcgggcacggct gt ccaaggagctgcaggcggcgcaggcccggct gggcgcggacat ggaggacg 18 85 EMBL Sequences in EMBL format derived from the EMBL Nucleotide Sequence Database and saved in text f
80. in a DNA sequence Universal biotinylated PCR primers Warning messages Warnings and penalties Work area 46 46 47 28 16 49 27 49 38 31 65 37 37 29 84 84 28 38 31 31 27 48 48 48 48 48 17 72 48 12 14 12 13 13 77 73 32 48 27 49 11 65 18 61 44 42 11
81. in buffers These compounds bind to the ions via their negative charges Therefore the concentration of Mg should always be higher than the concentration of these compounds e Mg Effect on stringency In general increased Mg concentrations will lead to an increased efficiency of the DNA polymerase and therefore to a higher incorporation rate but may also increase non specific amplification and reduce fidelity Excess Mg in the reaction can increase non specific primer binding and increase non specific incorporation It may also stabilize secondary structures in the DNA template This may decrease the amplification efficiency particularly for GC rich templates Lowered magnesium concentrations will generally make the amplification reaction more stringent but also less efficient leading to lower yields Too little Mg in the reaction can result in a lower yield of the desired product e Optimization of Mg concentration The optimal Mg concentration which may vary from 1 mM to 3 mM should be determined empirically while DNA and nucleotide concentrations are kept constant Perform a magnesium titration from 1 mM to 3 mM in 0 5 mM increments to determine the optimal magnesium concentration PCR cycling The PCR cycling program should be optimized conditions e Optimization of the PCR cycling program Hot start DNA polymerases such as AmpliTaq Gold Applied Biosystems and HotStar Taq Qiagen are activated gradually durin
82. in specific amplification during PCR it is important to select PCR primers that do not have alternative priming sites on the DNA template sequence Mispriming in the Pyrosequencing reaction occurs when the sequencing primer binds to the wrong part of the sequence which may result in an overlaid sequence in the pyrogram 10 3 11 Secondary structures Double or overlaid sequences broad peaks or signal compression in the pyrogram may indicate the occurrence of secondary structures in the DNA template Secondary structures can hinder the annealing of the sequencing primer which can result in low and or spurious peaks 73 85 e Secondary structures can be the result of self priming Self priming occurs when a part of the DNA sequence at the 3 end is complementary to another part of the DNA sequence and the sequence thus binds to itself and acts as a primer 5 This may result in an overlaid sequence in the pyrogram e Secondary structures in a DNA template can cause problems in the sequencing reaction due to interference with enzymes in the assay Secondary structures may interfere with the enzymes in the assay Secondary structures can hinder the DNA polymerase from binding to its priming site or obstruct the procession of the DNA polymerase which could lead to broad peaks and or incorrect sequencing context in the Pyrogram Example A secondary structure has been formed that inhibits the enzyme s activity 3 5 74
83. inearly with GC difference 3 4 2 Sequencing primer score effects Min Primer Length bp Max Primer 15 20 bp Length bp Min Distance 0 bp from target bp Max Distance 3 bp from target bp Allow Primer Unchecked Over SNP Generate Checked Forward Primers Generate Reverse Checked Primers 14 85 Primers within the set min and max lengths are generated This analysis step can result in warning messages for short primers but will not affect primer set Scores Primers within the set min and max distance from target are generated without penalties If the box is checked a sequencing primer is allowed to overlap up to two SNPs without penalty Note Primers will still not be allowed to anneal over an SNP within the 7 last nucleotides of its 3 end No penalty effect No penalty effect 4 Performing an assay design 4 1 Introduction Assay design can be performed for genotyping allele quantification AQ and sequence analysis SQA A proposed workflow for performing an assay design is shown below Work flow Create an assay setup Choose assay type enter or import a DNA sequence define the target region and PCR primer regions optional and assign position names to the polymorphisms to be analyzed Run an automatic design Run the automatic assay design to create a number of different primer set candidates It is also possible to re use one or two primers from a previous assay and gen
84. information in the top rows of the GenBank file will automatically be transferred into the Notes field on the Final Primer Set tab see example below Assay Design Assay5 Assay5 E Anl x Ele gdt Assay Windows Help IES Assay Type Genotyping Q eo O usus e 60 Sequence Editor Sequence Final Primer Sat ls Primer sequence Pos Bp GC Tm ID NS e S feo iz isi Sequence to analyze Cinese mesasa gt FO ak Created by PYROS as0375 Created 2004 02 19 11 42 Modified 2004 02 19 11 42 Notes LOCUS NM 181587 1543bp mRNA linear ROD 22 DEC 2003 zj DEFINITION Mus musculus RIKEN cDNA 6530404N21 gene 65 0404N2TRIK mRNA CCESSION NM 181 VERSION NM_181587 2 Gl 31795585 SOURCE Mus musculus house mouse Position Types Variation z alictes 0of0 eii Sequence files in GenBank format start with a number of information lines followed by the sequence The sequence will contain line numbers Only sequences up to 10 000 characters in length can be imported If the sequence in the database record is too long to import shorten the sequence using GenBank functionality See Save a file on GenBank text format Save a file on GenBank text format 1 Change the format to Text and press the button Send to EEE 7 ee us Search Nu
85. information of an organism Genomics The analysis and investigation of the genome Genotype The observed alleles at a genetic locus for an individual Gibb s free energy AG Gibbs free energy is a measurement of nucleic acid duplex stability A DNA duplex is more stable when its AG value is more negative The definition of free energy is AG AH TAS where H is the enthalpy S is the entropy and T is the temperature Guanine G A purine base that is a part of DNA and RNA molecules Guanine forms base pairs with cytosine a pyrimidine base H Hairpin See Hairpin loop Hairpin loop A self annealing nucleic acid strand forming stable hydrogen bonds AG zero with itself The hydrogen bonded region is referred to as a stem and the single stranded region is referred to as a loop Heterozygote An individual with different alleles at a given locus postion on the two corresponding chromosomes Homopolymer A stretch of identical bases in DNA In Pyrosequencing technology stretches of two or more identical bases are regarded as homopolymers Homozygote An individual with the same alleles at a given locus position on the two corresponding chromosomes I Insertion Addition of a base or a DNA segment into a chromosome Small insertions within a gene can alter the reading frame and thus the amino acid sequence of the encoded protein International Union of Pure and Applied Chemistry IUPAC An organization providing
86. ing PSQ Pyrogram and are trademarks owned by Biotage AB Pyrosequencing technology is covered by patents including patents US4863849 US610891 US6258568 EPO932700 and EPO946752 and patent applications owned by Biotage AB In view of the risk of trademark degeneration authors intending to use the trademarked designations are respectfully requested to acknowledge the trademark status of the products at least once in each article Other patents and trademarks The PCR process is covered by several patents owned by Roche Molecular Systems and F Hoffman La Roche Ltd Intel and Pentium are registered trademarks of Intel Corporation or its subsidiaries in the United States and other countries Microsoft and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries Sepharose is a trademark of Amersham Biosciences Limited Adobe and Adobe Acrobat are either registered trademarks or trademarks of Adobe Systems Incorporated in the United States and or other countries All other trademarks are the property of their respective owners 1 85 Important user information The Pyrosequencing Assay Design Software and all associated products from Biotage AB are for research purposes only Not for use in diagnostics procedures for clinical purposes For in vitro use only Biotage AB reserves the right to make changes in the information contained herein without prior noti
87. ing unknown DNA sequences For example CTGA or TCGA can be used and repeated the desired number of times Cytosine C A pyrimidine base that is a part of DNA and RNA molecules Cytosine forms base pairs with guanine a purine base D Deletion Loss of a base or a segment of DNA from a chromosome Small deletions within a gene can alter the reading frame and thus the amino acid sequence of the encoded protein Deoxyribonucleic acid DNA The carrier of genetic information in most organisms Di allelic SNP A SNP where two possible bases can occupy the polymorphic position Diploid A diploid genome contains two copies of each chromosome Directed dispensation order Non cyclic nucleotide dispensation order that follows the known sequence To be used in Pyrosequencing technology when you know the sequence to be analyzed Example the sequence TCCAGAA should be dispensed TCAGA Direction The direction of an assay which can be either forward or reverse See Forward assay and Reverse assay respectively Dispensation order Defines the order in which nucleotides should be dispensed in a Pyrosequencing run Use a cyclic dispensation order if the sequence is unknown Use a directed dispensation order if analyzing a SNP or if the sequence is known DNA pool A mix of several DNA samples Duplex A double stranded nucleic acid formed e g by two primers that anneal to each other Enzyme A protein or RNA working as a catalyst to
88. inue with the instructions in section 4 3 4 3 Run an automatic design 4 3 1 Generate new primer sets Click the Run Assay Design button to perform the assay design The 1 progress of the analysis is shown in the form of a progress bar at the bottom of the work area While analyzing the button displays two rotating arrows 2 Q It is possible to stop an ongoing assay design by clicking the red Stop button or by using shortcut keys Ctrl Q 3 The resulting primer sets are displayed in the Primer Set area It is also possible to enter primer s and generate matching primers resulting in primer sets with scores for Pyrosequencing analysis Furthermore a complete primer set can be entered and analyzed to obtain a score and quality ranking See section 4 3 2 27 85 4 3 2 Analyze previously designed primers Re use one or two primers from a previous assay and generate matching primers 1 a On the Sequence tab mark where the primer anneals to the DNA sequence The Find function can be used to search for a primer sequence string in forward or complement reverse direction see section 4 2 4 1 b Right click and select one of the following e Set As PCR Primer Biotinylated if it is a PCR primer that should be biotinylated it will be set as forward or reverse depending on which side of the target region the marked sequence is situated e Set As PCR Primer Not biotinylated if it is a PCR primer that should not be biotiny
89. ion analysis The PSQ 96MA Instrument with PSQ 96MA SNP Software or PSQ HS 96A Instrument with PSQ HS 96A Software and SNP reagent kit perform highly accurate and reproducible Single nucleotide polymorphism SNP and mutation analysis Automatic multiplex genotyping of polymorphisms such as point mutations insertions deletions InDels and SNPs reduce the cost per accurate result and increase sample throughput Multiple SNPs from a short stretch of DNA and di tri and tetra allelic polymorphisms can be analyzed The internal control capabilities provide the sequence context around the SNP or mutation For more information see the user documentation for PSQ 96 MA System or PSQ HS 96A System 10 2 3 Allele quantification AQ Once polymorphisms are identified the frequency of alleles can be determined using the dedicated module AQ within the PSQ 96MA SNP Software or PSQ HS 96A Software Alleles from both pooled genomic and single mixed population samples can be quantified calculating allele frequencies in pooled sample populations can increase the efficiency and decrease the cost for large population studies because only a single PCR amplification reaction followed by one sequencing reaction is needed when analyzing the samples Allele quantification determination of multiple SNPs tri and tetra allelic SNPs and InDels can all be achieved In multiplex assays it is possible to obtain the relative peak heights and then calculate the allel
90. isk Cost Install PSO Assay Design for yourself or for anyone who uses this computer C Everyone Just me cesi esc 8 85 8 Review the installation settings and click Next to start the installation zx Confirm Installation e A The installer is ready to install PSO Assay Design on your computer Click Next to start the installation Cancel lt Back 9 The installation wizard shows the progress of the installation 10 Click Close to exit the wizard 2 3 Starting Pyrosequencing Assay Design Software 1 In the Windows Start menu choose Programs gt Biotage gt PSQ Assay Design Alternatively double click the PSQ Assay Design icon on the desktop 2 The first time the program is started a License dialog opens License Enter license code LO DT p U IE Biotage 3 The license key can be found on a slip of paper in the CD jacket Enter the license key information in the Enter license code field and click Add Assay Design Software v 1 0 will appear in the Installed products field Note Every user with an individual user account on the computer will need to enter the license key the first time they start the program Enter license code m a 4 Click OK The Assay Design Software start screen will open 9 85 2 4 The Assay Design Software start screen ITTDETETTTECTN Ele Edit Assay Windows Help Assay Type Genotyping De
91. isplayed together with the DNA sequence as well as the graphs of the melting temperature and Gibb s free energy The biotinylated PCR primer is marked with a ring at the 5 end Any warnings generated for the primers are indicated by a warning triangle next to the primer When a new primer set is selected in the Primer set area the graph on the Sequence tab is automatically updated The Assay overview area at the top right corner of the work area gives an overview of the entire sequence with symbols for polymorphisms target regions primers and mispriming sites Use this area to get an overview of the whole sequence of primer positions and mispriming sites Also use it to quickly navigate to a specific part of the sequence on the Sequence tab in the Assay window In the information area on the Sequence tab general information about the primers in the selected primer set including primer sequence length warnings sequence to analyze etc can be viewed If at least one analysis step has identified a problem of potential concern a warning triangle and an associated warning message will be displayed in the information area Use this as a quick indication of what analysis steps you need to check in the detailed report When a new primer set is selected in the Primer set area the information area is automatically updated To view even more detailed information on the highlighted primer set double click the primer set or choose menu alter
92. late The DNA template is commonly defined as the single stranded DNA that is used by the DNA polymerase to synthesize a complementary template copy In Pyrosequencing the template is specifically the single stranded DNA that is attached to the Sepharose beads by means of a biotin bridge in the well and which is used to perform sequencing by synthesis Tetra allelic SNP A SNP where any one of four bases can occupy the polymorphic position Thymine T A pyrimidine base that is a part of DNA molecules Thymine forms base pairs with adenine a purine base Tri allelic SNP A SNP where any one of three bases can occupy the polymorphic position 84 85 13 Index Amplicon length Analysis steps Annealing over a SNP Assay Assay controls Assay design settings for one assay Assay overview area Assay settings Assay setup Assay type Assay window Batch assay design Biotinylated PCR Primer Hairpins Computer Copy all primer sets Copy and paste a DNA sequence Copy entered sequence Copy primer set Default assay design settings Discard quality Dispensation order Edit primers EMBL Nucleotide Sequence Database Enter the DNA sequence FASTA format Final primer set Find sequence Forward assay Forward PCR region GC content GC content difference GenBank Hard drive High quality Homopolymers Import a DNA sequence Import into PSQ software InDels Installation License key Low quality Medium quality
93. lated it will be set as forward or reverse depending on which side of the target region the marked sequence is situated e Set As Sequencing primer if it is a sequencing primer The primer appears on the Sequence tab and at the top of the Primer set area in the correct field forward reverse PCR primer field or sequencing primer field Alternative a Atthe top of the Primer set area type or copy and paste the primer sequence in the appropriate field forward reverse PCR primer field or sequencing primer field Note Before a sequencing primer can be entered in the sequencing primer field its direction must be defined This is done by pressing the Set sequencing primer direction button I and selecting which PCR primer should be biotinylated b As soon as you leave the primer entry field the software automatically places the primer at its annealing site in the DNA sequence on the Sequence tab Check that this is the correct annealing site 2 Ifdesired repeat the above procedure for the second primer for example two PCR primers can be set and matching sequencing primers found by the software 3 The entered primers are automatically locked as soon as you leave the primer entry field This is indicated by a darkened blue button to the right of the respective primer in the Primer set area By locking the primer s the software keeps this primer s constant and tries to find the best primer s complementing the locked primer
94. lates the GC content in percent Analyzes the level of complementarity between the PCR primer and its annealing site Detects possible PCR primer self annealing fwd fwd and rev rev duplex formations Detects possible PCR primer hairpin structures Calculates the deviation between the melting temperature of the PCR primers and the optimal melting temperature By default the melting temperature algorithm used is the Nearest Neighbor method Detects alternate annealing sites for each PCR primer on the entered sequence and reverse complementary sequence Calculates the relative stability Gibbs free energy difference between the primer 5 and 3 ends Calculates the deviation between the actual primer length and the optimal primer length 45 85 Automatically generated primers are always completely complementary Manually added or edited primers will receive a penalty and a warning for non complementary sequence motifs Non complementarity is penalized more for the 3 end of the primer than the 5 end If potentially serious duplexes are detected the primer will receive a penalty 50 and a warning If potentially serious hairpin loops are detected the primer will receive a penalty 50 and a warning If the deviation is high the primer will receive a penalty gt 50 and a warning If potentially serious alternate annealing sites are detected the primer will receive a Penalty gt 5
95. many polymorphic positions in the sequence to analyze the dispensation order generation may take time 7 When import is complete a dialog appears saying that the import has finished It is possible to stop the import by clicking Stop Note It may take some time before the import is stopped because the dispensation order generation cannot be interrupted 8 The Entry in the Entries area is updated and shows the import status of the Entry Status column and if generated warnings Dispensation order warnings column and error messages Dispensation order errors column The status shows whether the Entry was imported or not and the reason if it was not imported An icon in front of the Entry also indicates the status Double click on an entry in the Entry ID column to display an overview of the information in a window that opens above the list Successfully imported entries can now be opened from the simplex entries tree view and used in run setups Note If import fails because an Entry with the same name already exists in the PSQ database refer to Section 9 10 Troubleshooting guide for help 40 85 7 Guidelines for PCR and sequencing primer design 7 1 Introduction Assay Design Software generates primers that fulfill the specific requirements of Pyrosequencing analysis Depending on the chosen assay type the software carries out a number of analysis steps Any potential problems detected in these analysis steps will generate penalti
96. n The polymorphisms can be positioned in the same DNA fragment or in different DNA fragments One sequencing primer per polymorphism is used in the sequencing reaction PSQ 96MA SNP Software and PSQ HS 96A Software supports duplex and triplex sequencing of single polymorphic positions di tri and tetra allelic SNPs and InDels 72 85 Example 1 Multiplex assay where the polymorphisms are positioned in the same DNA fragment 3 AACATTCGGCTTACAG ACAGCTGACCTAGCCTCGGATGAAC TACTTCGTCGAAC 5S TAAGCCGAATGT m S TGGATCGGAGCCT e One DNA fragment with two polymorphic positions that are sequenced at the same time The peaks come from two different sequencing reactions when the primers are extended Example 2 Multiplex assay where polymorphisms are positioned in two different DNA fragments 3 ACGTGGATTACATTCGGCTTACAG ACAGCTGAGTAAAGTTAGT 5 TAAGCCGAATGT e 3 IGITGACAGGACGTACGCTTACACOGTAAGGTGTGAAC TAGGCK I S ATGTGGCATTCCAC mmj Two different DNA fragments are sequenced at the same time The peaks come from two different sequence reactions when the primers are extended Note Assay Design Software does not support the automatic primer design for multiplex assays To use Assay Design Software for multiplex assay design design the primer sets separately and manually check the different primer combinations e g for duplex formation See Chapter 9 Hints amp Tips for more information 10 3 10 Mispriming To obta
97. n 50 ul reactions respectively The volumes are in microliters Use 45 pl of reaction mix and 5 ul 2 ng ul genomic DNA per tube well H O 10x PCR buffer II Applied Biosystems MgCl 25 mM 2 0 mM dNTP 2 5 mM 0 125 mM each Forward PCR primer 10 uM 10 pmol Reverse PCR primer 10 uM 10 pmol AmpliTaq Gold Applied 1U Biosystems Total Template DNA 2 ng ul 5 5 each 10 ng 8 3 1 Optimization of the PCR protocol and conditions For optimization start with the parameters annealing temperature Ta and MgCl concentration The annealing temperature typically falls in the range 54 C 64 C and the MgCl concentration is typically between 1 mM and 3 mM Select two DNA samples that can be used for all PCR optimizations A good starting point is to try three different temperatures e g 54 C 57 C and 60 C and three different MgCl concentrations 1 5 mM 2 0 mM and 2 5 mM while keeping all other parameters constant 57 85 9 Hints amp Tips 9 1 Tips for succeeding with difficult assays The software performs a number of analyses to differentiate between primer set candidates and thereby identify the best possible assay to use However for some assays it may be difficult to find an assay without flaws It is then often possible to work around or resolve the detected potential problem by using one of the tips below For information on which analyses the software performs as well as guidelines on PCR
98. n is shown below Create assay setups Enter or import DNA sequences into different assay windows choose assay type define the target region and PCR primer regions optional and assign position names to the polymorphisms to be analyzed See Chapter 4 Performing an assay design for further details Run automatic batch assay design Run batch assay design for simultaneous analysis and design of all open assays Start by defining the folder where the resulting assay files should be stored During batch analysis the program will be locked and cannot be accessed again until all open assays have been analyzed or until batch analysis has been stopped Save the results After batch assay design has finished the successfully analyzed assays will be automatically saved as xml format files with the file extension xml in a folder of choice together with a text report of the best primer set per assay Only failed assay files will remain open in Assay Design Software for evaluation and further analysis Optional Adjust the assays If an assay is to be adjusted open the relevant assay file and edit primers or change assay settings See Chapter 4 Performing an assay design for further details View an assay report of the selected primer set To view a detailed report of a designed assay open the relevant assay file and view the report See Chapter 4 Performing an assay design for further details 38 85 5 2 Run automatic batch assay
99. native Assay View Report or click the button View assay report in the Assay window or right click on the primer set and select View Report The Report window opens displaying detailed information on the primer set e g the different analyses performed for each primer and primer combinations 32 85 4 4 5 Optional Select a different primer set as final The final primer set is the set that will be imported into PSQ 96MA or PSQ HS 96A system software 1 Right click on the primer set of choice and choose Select as final Alternatively select the primer set and click the Select as final button Y A surrounding dark gray box marks the chosen primer set The final primer set is displayed on the Final Primer Set tab of the Assay window On the Final Primer Set tab it is possible to change the IDs of the primers edit the creator of the assay and add notes about the assay 4 5 Adjust an assay 4 5 1 Change the assay design settings If the software fails to generate acceptable primer sets using the default settings the results may be improved by changing the settings for PCR primer and sequencing primer design It is also possible to change the default settings to be applied on all new assays See Chapter 3 To change assay settings and re analyze the assay 1 e Click the Settings for this assay button in the Assay window The Current assay settings dialog opens Change the settings by entering the desired values checki
100. nd ad nbn Toe PR rM Sereno parco Bee ine Googte Bbsskodwebben isdiplsten FAEM sino Epp rai l Ea Quick Search Libra Query Form Tools Results jects Views Databanks est Query Dibs temioi ombleon NlTesPHLR found 36 entries next Apply Options to ACE say T Homo sapiens PLR gene VIRTUAL TRANSCRIPT partal sequence genomic survey sequence uncelacted ras eee Tr Pan troglodytas PKLR gene VIRTUAL TRANSCRIPT partial T sequence genomic survey Sequence sult Options U82191 Fels catus DNA Lyons LA Felis catus genomic similar to poruk Ont F Ema Fcueeisi ueia pyrware kinasa EKL DNA sequence eu eee T EMBL AYSSSSOS us musculus PKLR gene VIRTUAL TRANSCRIPT partial sequence isis genomic survey secuerice Ben ge mee Homo sapens pyruvate kinase iver and RAC PKLR aere ane F EMBLAYSIGSOllaysiosei PTT EE E 13322 Show tools relevant to these E UC TUA TT emaL aco2s737 acce omo sapiens pyruvate Kinase iver and RAG transcript variant L ue D RCORSTO mRNA cDNA clone MGC 34243 MAGE 5221607 complete cds gun RUBRICA n E EMBLHSEKL Human pyruvate kinase type L mRNA complete cds 2431 Jane IFRLR L tyze pyruvate kinase exon intron junctions human Save results save J 382180 cenomic 60 nt segment 1 of 25 s C paca FKLR L tyoe pyruvate kinase exon intron junctions human eA B59 99 Gonorie 90 nt s
101. nd or target sequences for SQA if unknown are automatically displayed in the Polymorphisms area It is possible to skip between polymorphisms and change target regions in the Polymorphisms area It is also possible to select which allele to display in the sequence on the Sequence Editor Sequence tabs The different polymorphisms can be assigned Position names which will then automatically be transferred to the Entry upon import into PSQ 96MA or PSQ HS 96A system software 4 2 2 1 DNA sequence entry Import a DNA sequence Select File Import Sequences or click on the button Import sequence to this assay window in the Assay window The Import Sequence dialog is displayed Locate the sequence to import txt and click Open Sequences in GENBANK EMBL and FASTA format can be imported FASTA files can contain multiple sequences for simultaneous import The different sequence formats are exemplified in section 4 2 2 2 Note To perform import of multiple sequences in FASTA format File Import Sequences should be selected The button Import sequence to this assay window only works for import of one sequence at a time By default the software only shows files with the ending txt If the text file containing the sequence to import is not shown in the list because it has a file ending other than txt e g if it originates from a Mac or UNIX system select All Files in the File format drop down list to display all files in the
102. next SNP To make the best use of the reference peaks in the Pyrosequencing analysis by PSQ 96MA Software and PSQ HS96A Software design the sequencing primer to obtain e reference peaks as close as possible to the polymorphism e single peaks as reference peaks e reference peaks both before and after the polymorphic position especially when analyzing insertions deletions or multiple SNPs 10 3 9 Simplex and multiplex assays With the PSQ 96MA and PSQ HS96A systems it is possible to create two different types of assays in SNP genotyping simplex and multiplex assays e Inthe simplex assay one sequencing primer is used which means that the polymorphic positions must be in the same sequence and that the first and last polymorphisms should preferentially be within 15 nucleotides otherwise consider running a multiplex assay One sequencing reaction is performed in the simplex assay e In multiplex assays one sequencing primer per polymorphic position is used in the sequence reaction The polymorphic positions do not have to be in the same sequence Several different sequencing reactions occur simultaneously Simplex assay In simplex assays one or several polymorphic positions are genotyped using one sequencing primer PSQ 96MA System and PSQ HS 96A System are optimized for genotyping within 15 nucleotides from the sequencing primer Multiplex assay In multiplex assays several polymorphic positions are analyzed in the same reactio
103. ng part of a DNA sequence Short tandem repeat STR An STR is a polymorphism where two or more bases are repeated after each other with different limited multiplicities in different alleles Simplex assay Sequencing reaction with one sequencing primer from which one or several polymorphisms are analyzed in a reaction Compare with Multiplex assays Single nucleotide polymorphism SNP SNPs involve the change of one DNA base to another SNPs and point mutations are structurally identical differing only in their frequency Variations that occur in 1 or less of a population are considered point mutations while those occurring in more than 1 are SNPs SNPs can occur in coding regions of the genome cSNPs in regulatory regions rSNPs or most commonly in junk DNA regions in which case they are referred to as anonymous SNPs SNPs can be di tri or tetra allelic polymorphisms However in humans tri allelic and tetra allelic SNPs are rare STR See Short tandem repeat Streptavidin A molecule that binds very strongly to biotin 83 85 T Target region The target region defines the region to be analyzed by Pyrosequencing In genotyping and AQ the target region will contain the polymorphism s that will be genotyped In SQA the target region will contain the known or unknown sequence to be sequenced Primers generated by Pyrosequencing Assay Design Software will always be placed outside the defined target region Temp
104. ng unchecking the desired boxes and clicking OK The selected settings will only be used for the assay in which the Settings for this assay button was clicked To view a description of the parameters that can be changed see section 3 3 Click the Run Assay Design button to re analyze the assay and generate primer sets based on the new settings The results are displayed in the primer set list in the Primer set area View the results and select a final primer set see sections 4 4 3 and 4 4 4 or try to adjust the assay settings in a different way 33 85 Buttons in the Current assay settings dialog Get Factory Click to get the factory settings Get Default Click to get the default settings Set as Click to set the current settings as default settings and use the settings in Default the assay The settings will also be applied in the Default Assay Settings dialog OK Click to apply the selected settings for the assay in which the Settings button was clicked Cancel Click to cancel any changes made and close the dialog 4 5 2 Edit the PCR and sequencing primers It is possible to edit PCR and sequencing primers both on the Sequence tab and in the Primer set area The edited primers will automatically be reanalyzed to generate a new primer set score Editing primers can be useful e g when generating an assay with a universal PCR primer tail or when adding nucleotides to the 5 end of a PCR primer to circumvent a template loo
105. ny combination of two of these nucleotides Mutations A point mutation is a DNA sequence variation that occurs when a single nucleotide A T C or G in the genome sequence is altered mutated For example a mutation might change the DNA sequence AAGGCTAA to ATGGCTAA The only difference between a point mutation and a SNP is that for a mutation to be classified as a SNP all alternative forms must each occur with a frequency of 1 0 01 or greater ina population Therefore a point mutation is regarded as a di allelic SNP when designing assays using Assay Design software Insertion deletion polymorphisms InDels An InDel is a mutation where one or several nucleotides have been inserted deleted in a DNA sequence Use square brackets when entering InDels in Assay Design Software It does not matter if the InDel is an insertion or deletion when entering it in the software see Example 1 If the InDel is located in a homopolymeric stretch see Hints amp Tips for more information 75 85 Example 1 A C G represents the two alleles ACG and AG GC TT TT represents the alleles GCTTTT and GCTT AT AT AT can be used to represent a short tandem repeat with alleles AT ATAT and ATATAT Select AT AT as target region Short tandem repeats STRs A short tandem repeat STR is a repeated DNA sequence in which the repeat elements are typically two to five base pairs long To design an assay for STR analysis use the
106. o currently open assays as well as to all future new assays To view a description of the parameters that can be changed see section 3 3 3 2 Change assay design settings for one assay 1 Inthe Assay window click the button Settings for this assay The Current Assay Settings dialog opens where the settings for the active assay are set 2 Change the settings as desired by entering new values checking unchecking the desired boxes and clicking OK The settings are changed for this assay only as long as the button Set As Default has not been clicked To view a description of the parameters that can be changed see section 3 3 11 85 3 3 Description of settings and buttons 3 3 1 PCR Primer settings Min Primer Length bp Max Primer Length bp Optimal Amplicon Length from bp Optimal Amplicon Length to bp Max Amplicon Length bp Allow Primer Over SNP Melting Temperature Algorithm Primer Concentration HM Min Melting Temperature C Max Melting Temperature C Max Allowed Tm difference Max GC Difference 9o Minimum length of the PCR primers to be generated Maximum length of the PCR primers to be generated Lower limit of the optimal amplicon range Upper limit of the optimal amplicon range Maximum allowed length of the amplicon Check the box to allow annealing over SNPs in the DNA sequence Choose algorithm for calculation of PCR primer Tm Primer concentration us
107. on the cursor at the end of the sequence and click Enter to parse it i e to get the sequence numbered and the nucleotides in the sequence divided up in blocks of ten If the entered sequence contains characters that are not allowed the first one will be highlighted in red These characters must be corrected or removed before assay design can be performed On the Sequence Editor tab the entered sequence is arranged in blocks of ten nucleotides The polymorphisms are highlighted in bold and the position number for the first nucleotide of a row is shown in the column to the left of the sequence The first polymorphism is automatically selected as target region the target region can be changed if desired The sequence can be edited As soon as the sequence is edited the numbering and nucleotide grouping will disappear Therefore when editing has been finished position the cursor at the end of the sequence and click Enter to parse the sequence again On the Sequence tab the entered sequence is displayed together with its complementary strand In addition to the sequence a melting temperature Tm graph and Gibbs free energy AG graph of the template sequence are displayed Default 16 85 target region and PCR regions can be viewed The Assay overview area gives an overview of the entire sequence with symbols for polymorphisms and target regions It can be used to quickly navigate in the sequence on the Sequence tab Polymorphisms a
108. on when editing primers A new primer set has been created The primers are automatically re analyzed and the score updated When satisfied with the results click the Save candidate icon jk in the Primer set area to save the primer set to the list The primer set will receive a unique primer set ID and be placed at the bottom of the list A star will indicate that it has been manually 34 85 added to the list View the result and select a final primer set see sections 4 4 3 and 4 4 4 or try to adjust the assay in a different way 4 5 3 Select one or more primers from a primer set and re analyze the assay It is possible to lock one or two of the primers in a primer set and re run the analysis to generate different candidates complementing the one or two locked primers Example 1 Select a certain sequencing primer and then generate a list of primer sets with PCR primers matching the sequencing primer Example 2 Select the forward PCR primers and generate a list of primer sets with a matching reverse PCR primer and sequencing primer To lock a primer and generate matching pairs 1 Click on the blue buttons to the right of the desired primers to lock them the blue buttons will darken 2 Click the Run Assay Design button in the Assay window to re analyze the assay 3 Theresults are displayed in the Primer set area View the results and select a final primer set optional see sections 4 4 2 and 4 4 3 or try to a
109. one or several polymorphisms to be analyzed using PSQ 96MA System or PSQ HS 96A System The Sequence to analyze always starts with the first nucleotide after the sequencing primer i e where the sequencing reaction starts Example of a Sequence to analyze AT CCGTGT T CCCA T C denotes a di allelic SNP T denotes an insertion deletion polymorphism This is the sequence to analyze for the following DNA sequence with annealed sequencing primer GGCACGAATCGACTT CCGTGCTTAGCTGAATA GGCACA A GGGT Biotin Assay Design Software automatically generates a Sequence to analyze for genotyping and allele quantification assays When importing the assay into PSQ 96MA System or PSQ HS 96A System a dispensation order is generated based on the Sequence to analyze and an SNP Entry is thereafter automatically created containing the Sequence to analyze and dispensation order 10 3 7 Dispensation order The dispensation order determines in which order the nucleotides will be added dispensed to the Pyrosequencing reaction by the PSQ 96MA Instrument or PSQ HS 96A Instrument The Sequence to analyze which has automatically been generated by Assay Design Software for genotyping and allele quantification assays is used to generate a Dispensation order when the assay is imported into PSQ 96MA Software or PSQ HS 96A Software Note The Dispensation order for SQA assays must be entered into the PSQ 96MA Software by the user Example Sequence to
110. ormat txt can be imported into the Assay Design Software one at a time The information in the top rows of the EMBL file will automatically be transferred into the Notes field on the Final Primer Set tab see example below z eo S usus SSS 60 Sequence Editor Sequence Final Primer Set Primer sequence Pos Bp GC Tm ID Sequence to analyze Cjuewese marsase enfe cv i Created by PYROS as037se Created 2004 02 19 11 02 Modified 2004 02 19 11 02 Notes ID AEQO7807 standard genomic DNA PRO 2378 BP a JAC AE007807 AE001 437 SV AEQU7807 1 DE Clostridium acetobutylicum ATCCB24 section 295 of 356 of the complete DE genome JOS Clostridium acetobutylicum a Position tme vaio oo SSSCS S Alieles H I oto leui Sequence files in EMBL format start with a number of information lines followed by the sequence The sequence will contain line numbers Only sequence files up to 10 000 characters in length can be imported SNPs can be entered manually in the text file and should be denoted either with IUPAC codes or with slash notation e g C T Insertion Deletion polymorphisms should be typed in square brackets e g C The sequence in EMBL format should be saved as a text txt file The text file should start with the ID line The end of the sequence at the bottom of the text file should be a double slash For further information ab
111. otif Click the cross button x to close the Find area 26 85 4 2 5 Step 5 Optional Redefine PCR primer regions The software automatically defines PCR priming regions around the target which are used for the PCR primer design It is possible to manually redefine within which sequence region the forward and reverse PCR primers should be allowed to anneal Note If there are two or more polymorphisms that should be contained within the same PCR amplicon the PCR primer regions need to be manually defined Select the PCR primer regions so that the forward PCR primer is only allowed to anneal upstream of the first SNP and the reverse PCR primer is only allowed to anneal downstream of the last SNP Define forward and reverse PCR primer regions 1 On the Sequence tab or Sequence Editor tab mark the part of the sequence within which the forward PCR primer should be generated 2 Right click on the marked sequence and select PCR Regions Set Forward PCR Primer Region 3 Mark the part of the sequence within which the reverse PCR primer should be generated 4 Right click on the marked sequence and select PCR Regions Set Reverse PCR Primer Region 5 Bars placed over the sequence on the Sequence tab indicate the selected regions On the Sequence Editor tab the selected regions turn blue To hide the bars on the Sequence tab right click on the sequence and deselect View PCR regions 6 The assay setup is now complete Cont
112. out how to save an EMBL sequence record on the correct text format see Save a file on EMBL text format An example of an EMBL text file is shown below Eime clostridium noterad Tal Ele Edit Format Help AEOO7807 standard genomic DNA PRO 2978 BP AE007807 AE001437 AE007807 1 DT 31 JUL 2001 Rel 68 Created DT O02 sEP 2002 Rel 72 Last updated version 2 DO DE Clostridium acetobutylicum ATCC824 section 295 of 356 of the complete DE genome DO KW emo Clostridium notepad es E px Ele Edt Format Help os clostridium acetobutylicum Er se ES OC Bacteria Firmicutes Clostridia Clostridi ae Clostridiaceae FT 3107 Pri FT pi bosomal protein s13 oc clostridium FT Yprotein_id Aak81047 1 box FT translation MARIAGIOLPKEKRVEIGLTYIYGIGLTSSRKIIKATSVNPETRV FT KDLTEEEVNALRDYINKNFKIEGDLRREVALNIKRLVEIGCYRGIRHRRGLPVRGQKTK RN 1 FT TNARTRKGPKKAVASKKKK RP 1 2978 EQ sequence 2978 BP 774 A 629 ci 364 G 1211 T 0 other RX MEDLINE 21359325 ctacttgcaa agcagtttat ttatttatta aaat gggac taagctagct taatcccttt E 1 aaattagtaa ctacactaat tctagtataa caatttctgc tcc tcgcct cttcttggtc 2 RX PUBMED 11466286 EUG GEO Laccesceae tecccecsee Gtacceeane geascerent 186 RA Nolling J Breton G Omelchenko m v Mar Caaataattt tttaacaact gtttcttctg ttacaaagct aagaacttgt cttctagcat 240 ib ghaaatctcc tctctttgca agagtgatca ttttttctgc taaacttcta gtttcttttg 300 Q Gibson R tcttgttac agtagtctct attttaccat gctttaaaaa act
113. p For more hints amp tips when editing primers see Chapter 9 Hints amp Tips To edit PCR and sequencing primers 1 If the primers to edit are locked indicated by darkened blue buttons click on the buttons to unlock them unlocked buttons are highlighted D a Drag the primer along the sequence or change its length On the Sequence tab click on the primer to edit and drag the primer along the template sequence to change its position If the mouse pointer is instead pointed at the end of the primer the pointer icon will change to a double arrow symbol Dragging will then change the primer length rather than moving the primer around The sequence of the primer displayed in the Primer set area is automatically adjusted to the template sequence to which it anneals A new re calculated score and quality color are displayed at the top right corner of the Primer set area b Add remove nucleotides in the primer sequence In the Primer set area click in the desired primer field and enter delete the desired nucleotides in the primer sequence Only A C G or T can be added to the primer sequence A new re calculated score and quality color are displayed at the top right corner of the Primer set area as soon as you leave the primer field Note PCR primers with universal tails or primers with non specific tails to avoid template loop formation can be created in this way See Chapter 9 Hints amp Tips for more informati
114. polymorphic position the allele sequences in that position are identical i e the same nucleotide occupies the position If an individual is heterozygous for a certain polymorphic position the allele sequences differ at that position Example 3 polymorphisms in one gene reading forward Be E EN GdlaccrAa TAlicc 3 Chromosome 1 317 CACCTCTA CGATCGATT ATGGC 5 Biotinylated DUPIN E lm GCHAGCTAA TAIICG 3 Chromosome 2 3M CATCTCTA CGATCGATT ATAGC 5 Biotinylated Heterozygous Homozygous Heterozygous Genotype G A Genotype T T Genotype C T DNA is obtained from both alleles when amplifying the samples with PCR and will be analyzed simultaneously in the Pyrosequencing reaction 68 85 If there are three possible polymorphic positions reading forward for example G A T C and C T 27 different theoretical outcomes are possible G G T T C C A A T T C C G A T T C C G G T T T T A A T T T T G A T T T T G G T T C T A A T T C T G A T T C T G G C C C C A A C C C C G A C C C C G G C C T T A A C C T T G A C C T T G G C C C T A A C C C T G A C C C T G G C T C C A A C T C C G A C T C C G G C T T T A A C T T T G A C T T T G G C T C T A A C T C T G A C T C T 10 3 2 Single nucleotide polymorphisms SNPs Single nucleotide polymorphisms SNPs are DNA sequence variations that occur when a single
115. r PCR amplicon size is 50 to 250 bp Nevertheless up to 600 bp long fragments have been tested with good results and even longer amplicons work for some assays In general shorter PCR products have several advantages compared to longer ones e g the amplification efficiency increases and the risk of mispriming or secondary structure formation is reduced Primer dimers duplexes and internal secondary structures The Assay Design Software checks the PCR primers for dimers and hairpins Excess biotinylated primer in the PCR reaction can disturb the subsequent Pyrosequencing reaction There it can cause background if it can form a hairpin loop with a 5 overhang or a duplex with the sequencing primer See Chapter 8 Guidelines for PCR setup and PCR optimization for how to avoid formation of primer dimers duplexes and internal secondary structures 50 85 The minimum and maximum PCR primer lengths can be changed However remember that the shorter the PCR primer the greater is the risk that it matches more than one region in the genome thereby increasing the risk of non specific amplification The allowed difference in GC content can be changed Method for Tm calculation can be changed Furthermore the minimum and maximum T and the allowed Tm difference between primers can also be changed The optimal amplicon range and the maximum amplicon length allowed can be changed Possible dimer and hairpin structures are
116. r algorithm A common method for calculating the melting temperature Tm of primers Nucleoside A compound consisting of a purine or pyrimidine base covalently linked to a pentose Nucleotide A nucleoside phosphorylated on one or more of the hydroxyl groups of the sugar A nucleotide is the monomer unit of nucleic acids P PCR See Polymerase chain reaction PCR primer A primer used for amplifying a part of a DNA sequence by Polymerase chain reaction PCR PCR primer pair In order to conduct PCR two primers are necessary one forward and one reverse Hence PCR primers always come in pairs PCR product See Amplicon PCR region The forward PCR region is the region in the DNA sequence where the forward PCR primer will be positioned and the reverse PCR region is the region in the DNA sequence where the reverse PCR primer will be positioned Point mutation A variation in a single nucleotide position The most common type of genetic variation Point mutations that occur in more than 1 of the members of a population are called SNPs Polymerase chain reaction PCR A technique for amplifying a specific segment of DNA more than 1 million times Multiple cycles of denaturation annealing with primer and extension using a thermostable DNA polymerase produce an analyzable amount of DNA Polymerases Enzymes that catalyze the synthesis of nucleic acids assembling DNA or RNA from deoxy ribonucleotides Most polymerases need one st
117. rand of pre existing nucleic acid as template and a double stranded end to begin from Polymorphism Genetic variations broadly encompassing any of the many types of variations in DNA sequence that are found within a given population Specific subtypes of polymorphisms include mutations point mutations SNPs and insertions deletions Primer A short DNA oligonucleotide that anneals to a template DNA strand The primer provides a free 3 OH end from which the DNA polymerase can start synthesizing a complementary DNA strand Primers can be labeled with various molecules e g biotin Primer annealing site The primer annealing site is the location on the template DNA strand where the primer can anneal In general the primer has been designed to be 100 complementary to the template DNA strand which allows specific primer annealing 82 85 Primer set A primer set for Pyrosequencing consists of a PCR primer pair and a sequencing primer Pyrogram The resulting graph from a sequencing reaction performed using Pyrosequencing technology Each incorporated nucleotide is shown as a peak in the pyrogram R Reference peak Reference peaks are used as internal controls in sequencing reactions in Pyrosequencing technology Repeat See Short tandem repeat Reverse assay A reverse assay is an assay where the sequencing primer is annealed to the input DNA sequence strand Sequencing will be performed reading the polymorphism unknown region in
118. rd does not start automatically the CD drive can be opened in Windows Explorer and the wizard started by double clicking on the autorun exe file x Assay Design Software 1 0 6 Biotage Install Assay Design Software 1 0 Install Acrobat Reader 6 0 User Manual Quick Guide o e Exit Assay Design o 5 85 2 Software setup These installation guidelines describe how to install Pyrosequencing Assay Design Software on a computer fulfilling the system requirements listed below The software usage is restricted by a license key The license key information which can be found on a slip in the installation CD folder is needed when the software is started for the first time 2 1 System requirements Computer The computer used to run Assay Design Software should be a PC with the following preferred specifications Processor 2 6 GHz RAM 1 GB Hard drive 100 MB free space Monitor 1024 x 768 resolution Medium color quality 16bit Operating system Microsoft Windows 2000 or Microsoft Windows XP English versions only Printer All printers supported by Windows 2000 or XP are suitable Backup of data Good data management requires that data backups be made on a regular basis Note Biotage AB is not responsible for the User s backup routines 2 2 Installing the software 1 Before starting the installation of Pyrosequencing Assay Design Software confirm that you have administrator s rights on the
119. reby forced to generate sequencing primers further away from the SNP For more information about multiplex assay design visit http techsupport pyrosequencing com 9 5 Tips for universal biotinylated PCR primers It is possible to design and analyze PCR primers with a universal tail for use with a universal biotinylated PCR primer This allows the use of the same biotinylated primer in different PCR reactions assays To design PCR primers with a universal primer tail Generate primer sets or enter a previously designed primer set according to the description in Chapter 4 Select a primer set by clicking in the Primer set list Position the cursor in the biotinylated primer field at the top of the Primer set area Edit the biotinylated PCR primer to include a certain number of additional nucleotides at the 5 end which make up the universal tail This can be done either by Copy Paste or by typing in the appropriate sequence 60 85 e As soon as you leave the primer entry field the score and quality will automatically be updated The new score and quality are displayed at the top right corner of the Primer set area The Sequence tab of the Assay window has been updated to display the primer with its associated tail Note A primer set with a universal tail can be expected to receive low score and quality because the tailed primer will receive a substantial penalty for having a high T value as well as for having low complem
120. rs in the sequence graph The 3 end of an edited primer is positioned at the same site as the 3 end of the original primer even if nucleotides have been added or changed at its 3 end If the contents of an entry field in the Primer set area are changed by deleting the primer sequence and then typing pasting a new sequence in the field the software considers this as an edited primer Thus it does not reposition the primer on the Sequence tab The application has been running for a long time without restart which may lead to consumed memory resources If the sequence contains multiple polymorphisms each haplotype is analyzed separately I e if a PCR primer misprimes over an SNP the mispriming to both alleles will be shown in the report thereby seemingly showing the same mispriming twice The sequence entered on the Sequence Editor tab may be too short to allow positioning of suitable PCR primers Changing the name of the xml file in Windows Explorer before import in PSQ software will not solve the problem The Entry name is stored inside the xml file 64 85 Click the appropriate button to the left of the sequence graph to show the primer s that is are hidden To enter new primers in the Primer Set area press the New Primer Set button e to blank the primer entry fields before the new sequence string is entered pasted Restart the application to relieve memory No action is neede
121. s To toggle between locked unlocked primers click the blue button to the right of the primers Darkened buttons indicate locked primers and highlighted buttons unlocked primers 28 85 4 Click the Run Assay Design button to start the assay design 5 The results primer sets containing the locked primers in combination with matching primers are listed in the list in the Primer set area Enter a complete primer set and obtain a score for Pyrosequencing analysis 1 If a new primer set is to be added to a list of generated primers click the New primer set button The fields at the top of the Primer set area are emptied to allow entry of new primer sequences t Click the Set sequencing primer direction button in the Primer set area The following primer set options appear zx aa x 1 Biotinylation not defined elele 2 Biotinylation of forward PCR primer reverse assay ex 3 Biotinylation of reverse PCR primer forward assay 3 Select the desired primer set combination The fields in the Primer set area are cleared and new primers can be entered 4 a Atthe top of the Primer set area type or copy and paste the three primers in the appropriate fields forward reverse PCR primer field and sequencing primer field b The software automatically places the primers at their respective annealing sites on the DNA sequence on the Sequence tab Check that these are the correct annealing sites Alternative
122. s read 7 5 3 Sequencing primer design for SQA Positioning of sequencing primer The positioning of the sequencing primer should be as close as possible to the sequence to be read This will maximize read lengths Nevertheless it is recommended to start the sequencing with 2 3 known bases preferably single bases I e include 2 3 known bases flanking the unknown sequence in the target region These bases as well as known sequence motifs anywhere along the sequence can be utilized by the PSQ software algorithm when calling the unknown sequence Primers for sequence analysis of cloned material Position the sequencing primer in the multiple cloning site Select the target region so that it includes 2 6 bases before the start of the insert In this way the first bases of the sequence are known Directed dispensations of these bases may improve the sequence quality 53 85 The software will penalize homopolymers harder when assay type AQ has been selected than when Genotyping has been selected Avoidance of A peaks is only considered when assay type AQ has been selected Consider sequencing both strands In some cases it may be useful to perform Pyrosequencing reactions in both directions and gather complementary sequence information in order to confirm the sequence 8 Guidelines for PCR setup and optimization 8 1 Guidelines for PCR setup and optimization for Pyrosequencing analysis To set up a PCR r
123. s suitable for ordering oligonucleotides 36 85 4 7 2 Print a report 1 Choose which report format to use by clicking on the desired report format in the column to the left If Complete Results Text or Summary Text are selected a report in text format can be printed Otherwise the report will be in HTML format Click the Print report button in the Report window P d Click the Print preview button in the Report window to preview the printout Click Close to close the preview The standard Print dialog opens Select the printer on which to print the report and click Print 4 7 3 Save a report A report can be saved in text or html format 1 Choose which report format to use by clicking on the desired report format in the column to the left If Complete Results Text or Summary Text are selected a report in text format can be saved Otherwise the report will be saved in HTML format In the Report window click the Save report button e The Save as dialog opens Locate the folder in which to save the report Enter a name for the report and click Save 37 85 5 Performing batch assay design 5 1 Introduction Automatic batch assay design on several sequences can be performed for genotyping allele quantification and sequence analysis When running batch analysis all open assay windows will be analyzed with their respective settings A proposed workflow for performing a batch assay desig
124. scription Tol a is is n s Editor sequence Final Primer set iB xi laix e he div Menu bar Work area Assay window Assay overview area Primer set area Polymor phisms area The menu bar contains 5 different drop down menus File create open and close assays import sequences or change default settings Edit cut and paste sequence information for analysis or search for a sequence string Assay analyze assays individually or in batch view and change analysis settings Zoom in out on the sequence Windows hide show the results panel and arrange windows and Help The work area displays the Assay window A and the results panel at start The results panel is divided into three areas the Assay Overview area B the Primer set area C and the Polymorphisms area D The Assay window and the results panel are used together to perform an assay design For more information see Chapter 4 Performing an assay design This window can be used to choose assay type enter or import the DNA sequence set target and PCR primer regions optional run the assay design view results adjust the assay primers optional save the assay and view an assay report For more information see Chapter 4 This area gives an overview of the entire sequence with symbols for polymorphisms target regions primers and mispriming sites This area can be used to ge
125. selected primer set can be viewed saved and printed Two different report formats are available Complete Results and Summary 15 85 4 2 Create an assay setup 4 2 1 Step 1 Choose assay type and enter a description for the assay 1 After launching Assay Design Software a new assay file is automatically displayed in the Assay window Choose assay type by selecting the desired assay genotyping allele quantification AQ or sequence analysis SQA from the Assay Type drop down menu The analysis steps and primer scoring are automatically adjusted to the chosen application Assay Type Genotyping Genotyping Allele quantification Sequence analysis SOA Note A new Assay window will by default be the same Assay Type as the last assay that was analyzed If desired enter a description for the assay in the Description field 4 2 2 Step 2 Enter the DNA sequence 1 Enter a DNA sequence on the Sequence Editor tab There are four ways of entering a sequence see sections 4 2 2 1 and 4 2 2 2 for details e Import a DNA sequence in GenBank EMBL or FASTA format e Type in a DNA sequence e Copy and paste a DNA sequence e Open a previously saved assay file xml Note The entered sequence may not be longer than 10 000 characters The entered sequence is displayed on the Sequence Editor tab and on the Sequence tab of the Assay window If the sequence was typed or pasted into the Sequence Editor positi
126. sition name in the Name field This is the position name that will be imported into the PSQ 96MA or PSQ HS 96A Entry 4 2 4 Step 4 Optional Set the target region Genotyping allele quantification Assay Design Software automatically sets the first polymorphism in the entered sequence as the target region for Pyrosequencing analysis This is indicated by a light blue highlight of the polymorphism on the Sequence tab and is shown in the title bar of the Assay window Sequence analysis The last unknown region including three known nucleotides flanking either side of the unknown sequence area is automatically set as the target region for Pyrosequencing analysis by Assay Design Software This is indicated by a light blue highlight of the nucleotides on the Sequence tab Note1 It is possible to select a target region that covers more than one polymorphism If there are two polymorphisms in close proximity that are to be analyzed in the same Pyrosequencing reaction the target region needs to cover both positions Otherwise the Sequence to analyze generated by the program will be cut to exclude the second polymorphism Sequencing primers generated by the program will never overlap any part of the selected target region Note2 For selection of target region of repeat polymorphisms STRs see Section 9 9 in Hints amp Tips for special guidelines 4 2 4 1 Change the target region The target region is set either on the Sequence tab on the Sequenc
127. small penalties rather than the first one with a single severe structure will probably be the better choice A potentially severe structure in an analysis step is indicated by a Penalty 50 which will generate a warning visible in the information field on the Sequence tab The higher the penalty the larger is the risk of problems If an individual analysis step gets Penalty 100 the problem is considered serious enough to set the whole primer set score to 0 and thereby make it Discard quality Penalties lower than 50 are in general nothing to be concerned about To view detailed information on different primer sets see section 4 4 3 for further instructions 4 4 3 Sort the primer set list In addition to sorting the results by primer set score it is possible to sort the results by PCR Score Seq Score and Seq Position and ID This can be useful if the sorting of the primer set list by primer set score gives a low variability among the primer sets For example if you do not find enough different forward PCR primers to be displayed in the list The option to display Unique sequencing primers may be useful when performing multiplex assay designs To sort the primer set list 1 Select the desired sorting option from the drop down list in the Primer set area Unique seq Primer set score io 4 v primers Frimer set score F1 PCR score CGTGATC Seq score CCCATCAA Seq position 2 Fz TTGGCCAGGTTGGTCTTG n
128. system software generate dispensation orders and confirm that assays are analyzable before primers are ordered 10 Appendix A Methodological background 10 1 Sample preparation The starting material for a Pyrosequencing reaction is a PCR amplified single stranded DNA template with a sequencing primer hybridized to it Several methods to generate templates for Pyrosequencing analysis have been described e g Ronaghi et al 1996 Nordstr m et al 2000 Nordstr m et al 2002 Diggle and Clarke 2003 The principle for PCR and sample preparation prior to Pyrosequencing analysis is outlined below When using streptavidin coated DNA denaturation and magnetic or Sepharose beads for PCR primer annealing sample preparation Rhonagi et al 1996 one of the PCR primers should be biotin labeled B for immobilization to the beads The other PCR primer should be unlabeled PCR iF 8 As free biotin will compete with the biotinylated PCR product for binding to streptavidin thereby lowering the signal level we strongly recommend purifying the biotinylated PCR primer by HPLC or equivalent procedure to minimize the amount of free biotin and maximize the proportion of biotinylated primer B After immobilization using NaOH denaturation and annealing of the sequencing primer the immobilized strand can be analyzed using the Pyrosequencing technology Strand separation iF Sequencing primer V annealing
129. t influence the primer set scoring 7 3 Melting temperature The melting temperature of a primer depends among other things on salt concentration strand concentration sequence and length For PCR primers one of two algorithms can be selected for the calculation of Tma Nearest Neighbor default or 2 x AT 4 x GC For sequencing primers only the Nearest Neighbor algorithm is used 7 3 1 Methods for calculating the melting temperature Tm e Nearest Neighbor The melting temperature T4 of an oligonucleotide duplex is calculated using the nearest neighbor thermodynamics approach Rychlik et al 1990 SantaLucia 1998 and the following equation Tm Primer i 16 6 log M 237 15 AH Enthalpy for helix formation AS Entropy for helix formation R Molar gas constant 1 987 cal C X mol C Concentration of the probe M Molar concentration of monovalent cations Values of AH and AS used Breslauer et al 1986 were obtained in 1 M NaCl The values used for molar concentration of monovalent cations M and primer concentration C are 150 mM and 0 2 uM respectively for PCR primers and 50 mM and 0 33 uM respectively for sequencing primers The default PCR primer concentration of 0 2 uM can be changed in the Default assay settings dialog e 2xAT 4xGC This equation adds 2 C for each A and T and 4 C for each G and C nucleotide It is a simple but less accurate method for primer Tm calculation In this approach the
130. t an overview of the whole sequence including primer positions and mispriming sites It can also be used for quick navigation to a specific part of the sequence on the Sequence tab in the Assay window For more information see Chapter 4 This area can be used to view a list of primer set candidates and to select the final primer set to be imported into PSQ system software It is possible to edit and modify primers Assay Design Software can also analyze previously designed primers by pasting or typing the sequences in to this area For more information see Chapter 4 The polymorphisms or unknown sequences entered into the Sequence editor in the Assay window are automatically displayed in the Polymorphisms area This area can be used to name the polymorphic positions Position names step between polymorphisms and to define the target region for the next analysis For more information see Chapter 4 10 85 3 Assay design settings 3 1 Change default assay design settings 1 Select File Default Settings in the menu bar The Default Assay Settings dialog opens 2 Change the settings as desired by entering new values checking unchecking the desired boxes and clicking Set As Default 3 A message box with the following question appears Do you want to apply your new settings to all open assays If you choose No the new settings will only be applied to new assays Click the button Yes to make the new default settings apply t
131. tandem repeats STRs sssssssssssssssssssses eese haee reser eres enn 70 10 3 5 SSequence database files 1 3 idera p oe FER dunes AER RR och RRRR AN RR EAEREARR LERE Ar TAR 70 10 3 6 Sequence to analyze genotyping and allele quantification esses 71 10 3 7 Dispensation OFder s neede run gant ge ru itae na e e Mn om FRRERUAE AR RES MERE RR a HE AERA RARE R ATA EAR 71 10 3 8 Reference peaks and quality control windOw sessi nnne 72 10 3 9 Simplex and multiplex assays sssssssssssssesees esee hehe hh rehenes resa enn nnn 72 10 3 10 MISDENTNOG LR 73 10 3 11 Secondary str ct les s dde itio aeree ea A uR e RR EE a are Hen EE ce iter she tiara LAE RER HE eves 73 11 Appendix B Assay types eesesesesseesesesenenenenehn anas ki unu u ananira asas u uuu un unn 75 11 1 Genotyping and allele quantification AQ cceceeeee eee ee ee eee ee eset neta eens mmm 75 Ti d 1 Introduction di E EE d ELENA tog eee als anaes 75 11 1 2 Polymorphisms for which assays can be designed sse 75 11 1 3 Entering polymorphisms in Assay Design Software sessssssssssse nennen 77 11 2 Sequence analysis SQA eiecit eee cs ted sus re horn Er Tey daa Ene Eine deep TERRAS 78 11 2 1 Introduction oi EL EE aed eps R E urba urea Haedui di Va 78 11 2 2 Entering sequences for SQA into Assay Design software esessss en 78 PEMICLLIII IMM
132. the reverse direction i e generating the complementary strand to the input DNA sequence The forward PCR primer should be biotin labeled for a reverse Pyrosequencing assay Reverse PCR region The area in the DNA sequence in which reverse PCR primers will be placed when generating primer sets S Secondary structures Structures formed by single stranded DNA such as hairpins and loops Often seen in GC rich or repetitive stretches of the DNA Secondary structures may be difficult for polymerases to read through Self priming Self priming occurs when a part of the DNA sequence at the 3 end is complementary to another part of the DNA sequence and the strand binds to itself and acts as a primer In the Pyrosequencing reaction self priming may result in an overlaid sequence making the pyrogram difficult to interpret Sepharose beads Streptavidin coated sepharose beads that can be used for preparation of biotinylated PCR products Sequence format The way in which a DNA sequence is recorded in a computer file Different programs for database searches use different formats Examples of formats are FASTA GenBank and EMBL formats Sequence to analyze A short part of a DNA sequence starting directly after the sequencing primer which contains one or several polymorphisms to be analyzed using PSQ 96MA or PSQ HS96A systems See also The Sequence to Analyze in the methodology part of the manual Sequencing primer A primer used for sequenci
133. to the 5 end of the non biotinylated PCR primer in the Primer set area The score and quality of the primer set will automatically be updated as soon as you leave the field the new score is shown in the upper right corner of the Primer Set area Continue to try different nucleotide additions until the loop has disappeared and the score has improved Hairpin loops 3 end Check if generated background signal would affect the duplexes or misprimings polymorphic position that cannot be avoided If hairpin loops 3 end duplexes or sequencing primer misprimings cannot be avoided check what nucleotides will get incorporated to determine if the generated 58 85 background signal would affect the target region to be analyzed Also check if the dispensation order could be modified to avoid the background signal from showing up in the variable positions Hairpin loops or 3 end Modify the sample preparation procedure duplexes that cannot be If the sequencing primer forms hairpins or duplexes add avoided an extra wash step to the sample preparation to remove background signal After annealing transfer the beads to a new PSQ 96 MA or PSQ HS 964 Plate containing 40 ul or 10 ul respectively of fresh 1x Annealing buffer per well leaving excess sequencing primer behind 9 2 Tips for avoiding PCR cross contamination e Set up physically separated working places for template preparation PCR set up and post PCR analysis
134. tribution of this program or any portion of it may result in severe civil or criminal penalties and will be prosecuted to the maximum extent possible under the law Cancel 7 85 6 Click I Agree and Next to accept the terms in the license agreement i PSQ Assay Design License Agreement Please take a moment to read the license agreement now If you accept the terms below click Agree then Next Otherwise click Cancel Software License Agreement and Warranty Statement IMPORTANT PLEASE READ THIS LICENSE AGREEMENT CAREFULLY BIOTAGE AB LICENSES THIS SOFTWARE TO YOU LICENSEE ONLY UPON THE ACCEPTANCE OF ALL OF THE TERMS IN THIS LICENSE AGREEMENT BY USING THIS SOFTWARE PACKAGE LICENSEE CONSENTS TA DC DOALIBIN D AMD IO DCAM A MANT TA TUIC ACNOCCKACKIT C Do Not Agree Agree te ce Ta 7 Choose the destination folder for the installation files The default location is C Program Files Biotage PSQ Assay Design Click Next to proceed Note If the Just me box is checked the program will only be visible to the user who was logged in when the installation was performed ji PSQ Assay Design Select Installation Folder The installer will install PSQ Assay Design to the following folder To install in this folder click Next To install to a different folder enter it below or click Browse Eolder C Program Files Biotage PSQ Assay Design Browse D
135. uencing primer design for genotyping and allele quantification Positioning of sequencing primer The positioning of the sequencing primer is flexible within about 0 15 nucleotides from the polymorphism Genotyping analysis in PSQ 96MA Software and PSQ HS 96A Software is in some instances improved by including a reference peak before the variable position This is specifically the case when analyzing single base insertion deletion polymorphisms Avoid homopolymers at the polymorphic position 52 85 The default setting in Assay Design Software is a primer distance from target of 0 3 bp The SNP can be sequenced on either strand If the polymorphism is located in a homopolymer the software will select a primer with a 3 end that overlaps the homopolymer region Avoiding homopolymers is especially critical for AQ analysis For AQ Avoid A peaks Reason for avoiding A peaks The use of dATPaS in the Pyrosequencing reaction results in A peaks that are 10 20 higher than for the other three nucleotides This must be corrected for by measurement on a heterozygote sample when doing allele frequency measurements on polymorphisms containing A To avoid A peak height corrections the software chooses the opposite strand for sequencing primer design whenever possible However it is better to have a high quality sequencing primer from which A is read than a poor primer that generates nonspecific background but from which T i

Download Pdf Manuals

Related Search

Related Contents

Manuales de instrucciones  CHM-S630  Severin TO 2028  Installation instructions - Columbia Pipe & Supply Co.  guide_Keypad（pdf形式）  MANUAL DE USUARIO - Franklin Electric    Notas de la versión de Sun Java System Web Server 6.1 2006Q2  Rangemaster 110 (Gas) Kitchen Range - C:\Documents and Settings\cwuk  Packard Bell iMedia S2870  

Copyright © All rights reserved. Failed to retrieve file