User Manual - BGSU RNA group - Bowling Green State University
Contents
1. Color Codes no info one gap both gaps Isosteric NearIsosteric Heterosteric Forbidden so Sri CAO e e e KE hb thTh U AAGG A AAUG A AGGC G GGUC G GGUG C AGUG CCUG G GAUA U AUGA aj b Done E E i i A my Computer a Figure 31 The corrected alignment as seen again with the Alignment Viewer Obviously Figure 31 shows an improvement in the alignment new score 96 eventhough it created an insertion in Seq7 The manipulation also created an addition gap on the far right of Seq7 but this may not be a true gap because depending on the unseen nucleotides that are present beyond this motif remember for simplicity the alignment in this example is just an small extract from a bigger alignment The reason the alignment was enhanced to this degree is the fact that the cuc at the end of Seq7 fit better the isostericity rules of the tSH tWH tHS and cWW BPs they now occupy respectively as compared to the ccuc 40 References 1 Hall T A 1999 BioEdit a user friendly biological sequence alignment editor and analysis program for Windows 95 98 NT Nucl Acids Symp Ser 41 95 98 2 Leontis N B and Westhof E 2001 Geometric nomenclature and classification of RNA base pairs RNA 7 499 512 3 Jossinet F and Westhof E 2005 Sequence to Structure S2S display manipulate and interconnect RNA data from sequence to structure Bioinfo2. Done i H my computer y Figure 30 The HTML Alignment Viewer output showing areas where the alignment is not consistent with structure red and pink areas of Seq7 So clearly this alignment is not perfect and it needs some enhancement based on what is isosterically acceptable in 3D and what is not the score of this alignment based on the formula described in section 3 2 above is 87 The sequence that is the cause of the problem is also clearly identified Seq7 To fix the alignment we need to edit it in a sequence editor or text editor where nucleotides can be moved by hand an automatic aligner based on Stochastic Context Free Grammar Markov Random Fields or SCFG MRF is in preparation Mokdad A Sarver M Stombaugh J Zirbel C Leontis N B Ribostral also provides some insight on how the alignment should be fixed through its _Cov htm1 output files where a substitution table for each BP position is provided with isosterically inspired colored In this particular case and because of the specific BP interactions formed in this motif a small manipulation of Seq7 second part after the dots creates an insertion but fixes problems with forbidden and 39 heterosteric BPs After this manipulation the resulting alignment was viewed again with the HTML Alignment Viewer and Figure 31 shows the results ze Ribostral Alignment iewer Microsoft Internet Explorer File Edit View Favorites Tools Help ar
3. UAUGA This is an alignment of an internal loop motif similar to one seen in 16S rRNA internal loop 20 IL20 the four small dots in the middle are just to indicate strand dicontinuity Ribostral will consider points like this in an alignment as N s in other words they are assigned local numbers unlike the gap symbol It is hard to tell whether this is a good alignment or not by just looking at it Some purines and pyrimidines seem to be aligned well there also seems to be a couple of cWW interactions that are covarying nicely but it is difficult to tell anything specific Suppose that we know the 3D structure of the first sequence seen in this alignment the one labeled Structure and that it looks as in Figure 28 37 Figure 28 Structure representation of the first sequence in the alignment Althought the sequence alignment is modified to simplify the example this motif does occur several times in rRNA as in internal loop 20 IL20 of the 16S rRNA This structure can easily be encoded in Excel in the form that Ribostral can handle Figure 29 shows how this file looks like Ed Microsoft Excel tSH EWH EHS ais lolx File Edit View Insert Format Tools lt Data Window Help 8 X Hla nlg a dia E9 X f A REE Organism LB1 LB2 Interaction Structure 1 14 cVVV i Structure 2 13 tSH Structure 3 12 tVWVH Structure 4 11tHS Structure 5 10 cA Sheet2 lt re A Figure 29 The same
4. CUTU Figure 1 Ribostral default installation subdirectories Using folders marked with asterisk is optional on Windows platforms these are the locations where Ribostral starts browsing for the corresponding input files 1 2 Disclaimer No guarantee expressed or implied is made as to the suitability of this software for any purpose computer or person The author shall not be held responsible nor be liable for any damage occurring in any way to equipment or health while using this software Author contact information Ali Mokdad M D Ph D Department of Biological Sciences Bowling Green State University Bowling Green Ohio 43403 Email mali bgsu edu 2 Executing Ribostral Ribostral can run as a GUI or as a script under MATLAB by running the program ribostralNoGUI follow the header information to point to your files of interest The following discussion concerns mainly the default GUI version The startup window of Ribostralis a blank GUI with three menu options File Tools and Help Figure 2 The other GUIs of the program are activated from this window EE iol x Fie Tools Help Hint start from FILE Figure 2 Ribostral s main GUI To avoid ambiguity the GUI only displays options that are allowed at the specific stage of the analysis For example before doing any analysis the user needs to load a sequence alignment file on which the analysis will be performed So initially the GUI does not displ
5. BP Family In alignment gt gt Figure 19 Initial screenshot of the interactive analysis GUI Here both a FASTA file and an NT list file were loaded from Ribostral main GUI before starting this GUI If only a FASTA file was loaded some of the options or buttons seen here would not have been made visible The interactive analysis tool is a powerful tool providing yet a new array of functions not provided by the list analysis tools discussed in previous sections After specifying the right choices such as source organism name domain of interest etc the user can either analyze a specific family of basepairs or analyze a particular position 4 1 Interactive analysis of a family of basepairs The right hand side of the interactive analysis GUI contains the options and buttons capable of gathering statistics about a whole family of basepairs at once This option is only possible if the user has loaded a basepair list before opening this GUI An example of what can be done here is the analysis of all the occurrences of the tHS family in the archaeal part of the 5S rRNA alignment The result of such an analysis is shown in Figure 20 27 lt InteractiveAnalysis x Organism Halomari Counts z f rchaea 39 sequences Family tHS 6 hits in organisr 1Q 3 am SPE KI EE Pa 10 tHS 11 cSS 12 tSs 13 bifurcated Figure 20 Analysis of all occurrences of a basepair family All b
6. NT list analysis becomes available 2 4 Analyzing a list of nucleotides Upon activation of the button NT list analysis the program takes each row of the Excel NT list file and counts the number of each substitution of its nucleotides in corresponding columns of the alignment This process may take a few minutes for long lists of nucleotides or for large alignments The MATLAB command prompt or the DOS prompt if the compiled stand alone version of Ribostral is executed displays different messages and counters indicating the status and progress of the analysis Upon successful execution output files are created in the folder lt installation directory gt Output refer to Figure 1 with descriptive names indicating the NT list file and the alignment file they represent At the same time a new button providing a quick link to the main output file will also appear on the main GUI 3 Types of output files and their interpretations Depending on the type of input NT list several types of output files are possible Figure 11 The following sections discuss each type of these output files in detail 13 Without structure info With structure info BP types Figure 11 Output files produced depending on input files analyzed The first output file in each case is the main file accessed directly by clicking the button Display list results on the main GUI Outputs indicated by asterisks are produced optionally b
7. Nucleotides 1878 1864 414 2409 Archaea Number of sequences 24 cc 4 CG 58 GC 21 Gil 4 x For Help press F1 E A ab Figure 12 Percent output text file created upon sequence analysis of an NT list Sequence patterns are listed in alphabetical order A similar output file with counts instead of percents is also created This analysis is for the NT list shown in Figure 8 3 2 Output files for a basepair list In case the Excel input NT file gives a list of basepairs from an atomic resolution structure Ribostral generates output files that are better tuned and more informative for this kind of input These include colored HTML outputs in the form of tables with sequence substitution values The tables use different background colors to indicate for each basepair whether observed substitutions are isosteric near isosteric heterosteric or forbidden as compared to what is observed in the structure By default three HTML output files and a fourth text output in case the input Excel list is a basepair list with structure information are produced upon the analysis of a basepair list see Figure 11 The main output file accessed directly by the Display list results button on Ribostral is the percent covariation file whose name ends with _COV_Percents html A similar _COV_Counts html file shows the counts instead of percents Figure 13 displays a 15 snapshot of such an output A third file ending wi
8. always straight forward Using the 2D mask together with the other two masks makes it easier to at least find nested pairs 3 4 Other output formats Ribostral can also create other output files upon request by checking the option GU special in Preferences These files represent another way of classifying some similar isosteric groups together the way the G U wobble basepairs are analyzed in our previous work 6 Ribostral scripts can easily be modified to produce other similar outputs 4 Interactive analysis of nucleotides Besides analyzing lists of nucleotides or basepairs at once and producing dedicated output files that describe their results Ribostral is also capable of analyzing nucleotides of interest one by one and directly displaying their results on screen This can be done by activating the button Interactive analysis from the main GUI which starts a new GUI window Figure 19 This option becomes possible only after successful loading of a FASTA alignment file If a BP list file is also loaded before the interactive analysis is activated the new GUI will have more options and information extracted from that file In the following text we will discuss the options available in case a BP list is loaded before activating the interactive analysis GUI 26 lt InteractiveAnalysis a x Pick the desired options and Input NT numbers separated by or Organism Halomari Counts lt jArchaea 39 sequences
9. o are transformed into o and all other characters are capitalized When analyzing basepairs Ribostral only recognizes dashes which represent gaps or deletions o s and the four RNA nucleotide letters A C G and U as valid characters in sequences When analyzing longer motifs the program recognizes all characters 2 2 Dividing Sequences into Subgroups After choosing the alignment file of interest Ribostral looks in the same directory for an Excel file called KnownFASTAFilenames xls This is an optional user created Excel sheet that gives additional details about the FASTA file being read such as the names of different groups or domains of organisms it represents and the boundaries of these groups If this Excel file is not present in the same folder as the FASTA file being read or if the exact name of the FASTA file being read cannot be found in the Excel file this information will be ignored and all FASTA sequences in the alignment will be considered as one group The analysis of specific subgroups such as phylogenetic domains will not be possible later Figure 5 shows the format of the KnownFASTAFilenames xls file and the information it contains Ed Microsoft Excel KnownFastaFilenames xls 10 x File Edit wiew Insert Format Tools Data Window Help Type a question for help i of X DEHER nom Zio e ESHA 4 AB X f 55_ABE_2004_UNIQUE fasta A Se D 7 1 235_ABE_2004_UNIQUE fasta Archae
10. and the interaction they make is known the buttons will be colored according to their BP family isostericity matrix The family name also will be printed on the upper left corner If any of the buttons is clicked the names of sequences giving rise to the value in it are displayed in the lower part of the GUI Figure 22 Note it is possible to scroll between the basepairs from the input BP list one by one by clicking the green buttons labeled lt lt or gt gt that appear in the middle of the GUI InteractiveAnalysis Haienars fects s farchaea Figure 22 Basepair interactive analysis Notice that if the query is 22 26 instead of 26 22 the same result is obtained since this is a known BP present in the input BP list and it is always colored in the same way The BP identity present in the source organism also the crystal structure is CG printed in bold font If instead of a basepair position such as 22 26 the user enters only one or more that two numbers separated by commas or if the user enters two numbers separated by a dash or any logical combination of comma separated and dash separated numbers then a motif sequence analysis will be performed and results will be produced in a different format where the motif pattern and its number of occurrences will be printed in text instead of the colored table The 30 pattern present in the source sequence will also be indicated by the lt lt l
11. choose pairs of nucleotides one pair at a time and display substitution covariation patterns for them in BioEdit this is called Mutual Information Examination The resulting substitution table however provides no description of what the observed substitution patterns mean in terms of structure BioEdit does provide one kind of link between sequence alignments and structure if the sequence alignment contains a mask describing cis Watson Crick nested basepair BioEdit will color nucleotides in the alignment in terms of how well they conform to the basepair family A mask is a representation of the locations of basepairs occurring between nucleotides represented in the sequence alignment with paired characters such as and indicating the positions of basepairs in the alignment BioEdit however does not provide such information for any of the other eleven or so families of edge to edge interactions that are possible between basepairs comprising about one third of all interactions 2 BioEdit is designed to be primarily an alignment editor and viewer and not a tool for structural alignment of RNA the way Ribostral is Coseq a program that runs under UNIX platforms measures substitution patterns of basepairs without using any structural information Massire and Westhof unpublished The user then needs to analyze the structure manually to see if sequence substitution patterns agree with it Finally S2S is another more recent UNIX progra
12. installation directory gt Ribostra SCORES txt The formula used throughout this text is Individual BP score c SUM 3I 2NI H 2F 2G1 3G2 number of sequences Where c is the correction coefficient c 100 Highest positive weight in this case c 100 3 I NI H F G1 and G2 are the counts of sequences having substitutions that are isosteric nearly isosteric heterosteric forbidden gap on one side or gaps on both sides respectively The Highest positive weight is 3 in this case The correction coefficient c insures that the maximum score is 100 in this case the minimum score when all substitutions are gaps on both sides is 100 but that is not always the case depending on the formula used The formula used here is asymmetric unfavorable terms that contribute to its reduction are more numerous and weigh slightly more than favorable terms that contribute to its increase But for our purposes this is not a critical point What is important is to easily identify low scoring spots in the alignment to guide manual realignment efforts To better locate these trouble spots in the alignment the score is printed in red if it is worse than a certain threshold i e if it is below zero which is the score in case no structural data is available and in black otherwise The total score printed at the top of each HTML output file is an adjusted sum sum divided by number of basepairs studied of the individual BP scores
13. local numbers On top of each table that represents a basepair position the following information is provided 18 The names of the subgroups in the order they are analyzed the numbers of sequences in each subgroup the count of forbidden substitutions present in the table in red color with the letter F following the value the count of gaps present including gaps on both sides or just on one side in gray with the letter G following the value the count of isosteric substitutions in blue with the letter I following the value nearly isosteric substitutions in cyan with the letters NI following the value nom isosteric but not forbidden substitutions that we refer to as heterosteric substitutions in pink with the letter H following the value and finally individual scores of each subgroup at that basepair position The score is a value describing how structurally acceptable the sequence substitutions at analyzed basepair positions are This is based on the measure of how structurally compatible these substitutions are with the basepair in the reference organism which is typically the organism with known structure The individual basepair scores are currently calculated based on an ad hoc formula derived from experience with structures and knowledge of the patterns of allowed substitutions for each type of basepair This formula can be easily modified by the user by manually entering desired weight parameters in the file lt
14. than one of its edges at once e g in base triplets the number of bracket symbols of the opening and closing type may not be equal Therefore it is not possible from this mask alone to know which opening brackets correspond to which closing brackets Another limitation of both the 2D and 3D masks is that they do not tell anything about the type of basepairs formed The 13D mask is designed to solve this problem Here instead of just one or two pairs of symbols representing opening and closing of basepairs thirteen different pairs of symbols are used to represent all known families These codes are AB for the cWW family CD for tWW family and so on see Table 1 Since some families also are not symmetric C G cWS has a different meaning than C G cSW uppercase symbols and lowercase symbols are used to indicate the direction of the interaction The symbols must be read from uppercase to lowercase direction whenever the interaction is asymmetric with the uppercase symbol referring to the base edge of higher priority edge priority is assigned in the order W H S Thus if an interaction exists between columns universal numbers 38 42 and is represented by kL this means that nucleotides occupying these positions form a tSW interaction in the source organism or structure This is the same as saying that 42 38 is a tWS or Lk interaction The 13D mask has the same limitation as the 3D mask in that finding matching opening and closing symbols is not
15. the lower part of the GUI Sequence names are preceded by the corresponding nucleotide numbers from the source organism 4 2 Interactive analysis of individual basepairs or motifs In addition to analyzing a family of basepairs it is possible through this GUI to analyze a particular position by entering individual numbers separated by commas or dashes into the editable box in the middle of the GUI But first the source of local nucleotide numbers has to be specified If this GUI is activated after a BP or NT list is loaded then the sequence name of the first entry will be initially displayed in the white editable box and gray drop menu box that define the source sequence for the analysis upper left corner of the GUI Both of these boxes can be used to specify the source organism but in case the sequence names in these two boxes are different then the white editable box takes priority If universal numbers are desired the word universal should be typed in the white editable box and carriage return pressed If only two nucleotide numbers separated by are entered such as 22 26 the sequence substitutions for these two positions are displayed in the same output format seen for the basepair family analysis described in the previous section The value in the box that corresponds to the source oe 99 29 organism will be printed in bold If the nucleotide numbers entered are present in the input Excel BP list
16. the option Open NT list from the File menu A browser opens and on PC platforms search for Excel files starts in the local directory lt installation directory gt NT_lists Figure 7 aixi x File Tools Help v 55_ABE_2004_UNIQUE fasta Py acters A Pick a list of positions to study 6 Hm235_737 734 xls rrOo IK xls 4 rr0033_55_Class1 cwWw xls 34 rr0033_55_Class4 twH xls 94 rr0033_55_Class5 cw xls File name rr0033_55_AllClasses xls Files of type als x Cancel Figure 7 Browsing for an Excel nucleotide list The NT list Excel file must contain names of the source organisms and nucleotide numbers of interest The first row is a header row and anything in it will not be read Figures 13 and 14 show what such a file should look like Fd Microsoft Excel 23S 4 P_correction trial xis l X File Edit Yiew Insert Format Tools Data Window E Help f X En gt B A aa s Loo O e G a i Eaua maimon eas ott s Escherichia_coli 1878 1864 4 Haloarcula_marismorui 684 662 Ps Escherchia_col 649 639 e Haloarcula_marismonui 740 731 690 695 _ KEE Le Haloarcula _marismortui 2744 Ps Haloarcula marismortui 2758 1 Fe Haloarcula_marismortui 2375 Sheet3 4 H Ready Sum 4 Z Figure 8 Nucleotide list for analysis This Excel list from 23S rRNA allows the sequence analysis of four nucleotide
17. 2701 2694 A G2701 o Coe y OO Az 202 oS U GD A 2702 2601 A OF A 2703 2601 A GD A 2703 2600 U lt H C 2704 2600 U GD Caro 2689 A U2z05 2689 A U 2705 5 3 5 3 3 Dimensional structure 2 Dimensional representations AUA EAN 13D Mask AsO aishe 7b kl _TbthpTb 3DMask 1 1 2DMask 1 Dimensional representations Figure 18 Schematic explanation of structural masks These are one dimensional representations of structure The example shown here is from Helix 95 containing the sarcin ricin motif in the large ribosomal subunit of H marismortui pdb file 1S72 Color codes correspond to basepairs Note that the 13D and 3D masks may have several symbols overwriting each other only one of the cyan RG and symbols displayed in this Figure on top of each other is displayed in the 13D and 3D masks produced by Ribostral 25 The 2D mask describes only nested basepairings irrespective of their geometric families most of these would be helical cWW basepairs Each nested basepair one which does not cross in 2D with other basepairs is represented by the two parentheses symbols for its opening or 5 nucleotide and for its closing or 3 nucleotide The 3D mask is the same as the 2D mask but in addition to representing nested basepairs it also represents other non nested basepairs using the bracket symbols and T When one nucleotide forms more than one basepair by using more
18. 64 69 99 103 h7IL1 128 131 231 233 h7IL2 133 136 227 229 The nucleotide positions are then extracted from the alignment the same way as if they were entered one by one in the GUI editable box An advantage here is that the user can assign specific names to the sub alignments containing these motifs instead of the otherwise generic names derived from nucleotide numbers extracted 6 4 Tool 4 Merge amp remove repeats from FASTA files This tool opens a new GUI from which the user can browse for a minimum of one and a maximum of three input FASTA files Each one of the files read will be checked for individual sequence lengths and repeats in sequence names If some sequence lengths are shorter than others gap characters are added to the ends of the short ones This prevents some errors when the alignments are analyzed If name repeats are found the name meant here is the first separate word after the gt sign in a FASTA header line only the sequence with most valid nucleotide letters A C G and U is kept it is possible to change this default criteria and choose sequences with shortest internal gaps this can be done by changing the value for the variable Criteria in the script file mFastaPrinter m The resulting unique alignment represents a more divergent set of phylogenetic taxa which may be more meaningful to analyze The unique FASTA files are saved in the same directory and under the same names w
19. Basepair list is analyzed a button labeled Plot scores also appears on the main GUI When activated this allows the user to quickly analyze the scores of each basepair and define places of misalignment or motif swaps Figure 16 Alignment file 5S_ABE 2004 UNIQUE mat BP list n0033_55 AllClasses_MANUAL xls Archaea scores average 62 Score Formula 50 2I NI 2F 2G61 3G2 num of seqs 100 ANTM AWA a IS a WN tira O O E n nnn O 5 10 15 20 25 30 35 40 45 50 55 Basepair in order of BP list Figure 16 Score plots for the archaeal 5S rRNA alignment The red curve describes the scores of corresponding basepairs and the blue line represents the average score or total score as defined in this text for the whole alignment based on the basepair list provided By modifying the ad hoc formula according to which scores are calculated this can be done by changing parameters in the SCORES txt file the user can plot any combination of one or more aspects of the alignment such as percent isosteric substitutions percent isosteric and near isosteric substitutions and so on 22 3 3 Ribostral Alignment Viewer There are additional output files not produced by default that can be obtained by changing the Preferences under the File menu options The reason for not producing those output files by default is not to overwhelm the casual user with too many output files and not to unnecessarily extend the execution time If t
20. E mat 4 235_ABE_20 55_Rfam_ABE_seed_Jesse_2_20_05 mat BacteriaFixe J gt File name Files of type R mat Saved alignment data faster Cancel fasta Row alignment data slower all files Figure 4 Browsing for an alignment file On PC machines the browser window initially starts looking for alignment files in the folder lt installation directory gt FASTA_alignments refer to Figure 1 Here there are two main options reading a raw alignment file in its text form with extensions such as fasta or txt or reading a MATLAB data file mat which is derived from the raw alignment file This last option is processed faster by Ribostral especially if the alignment of interest contains thousands of sequences like the 16S rRNA alignments A mat file is created after a FASTA alignment is read for the first time It is saved in the same directory and under the same name except for the extension Time can then be saved by reading the data file instead of the text file the next time the same alignment is used Note that a new data file is only created if no data file with the same name is already present in the directory Therefore if the raw FASTA alignment is deliberately modified in any way the old mat file referring to it needs to be deleted to allow for its re creation Notice that when a FASTA format file is read all characters indicating unknown nucleotides N n O and
21. Ribostral An RNA 3D alignment analyzer and viewer based on basepair isostericities 1 Introduction Ribostral Ribonucleic Structural Aligner is a suite of programs designed to integrate known structural data with homologous sequence alignments with the purpose of evaluating the quality of the alignments and guiding efforts to improve them The main GUI Graphical User Interface of this program provides an expandable user friendly platform through which other related programs can be run The related programs gather and analyze atomic resolution structure data parse and automatically align sequences and perform other manipulations on sequence alignments including extracting sub alignments corresponding to individual motifs or domains removing repeated sequences from an alignment to build a unique alignment with higher phylogenetic diversity and creating a fasta alignment file from mat which is another alignment format used by Ribostral These tools are covered in detail at the end of this manual The main functions of the program namely analyzing evaluating and viewing RNA sequence alignments are discussed first RNA sequence analysis programs are common but none of them provides the valuable structural information provided by ribostral One of the most widely used editors for manual alignment of RNA sequences is the program BioEdit which runs under Windows platforms 1 BioEdit reads a sequence alignment file and allows the user to
22. a Bacteria Eukarya 0 24 208 345 2 165_ABE_2004_UNIQUE fasta Archaea Bacteria Eukarya 0 220 4695 9943 a 3 235_ABE_2003_UNIQUE fatsa Archaea Bacteria Eukarya 0 25 180 316 4 165_ABE 2003 UNIQUE fasta Archaea Bacteria Eukarya 0 229 4617 10004 5 5S ABE 2004 fasta Archaea Bacteria Eukarya 0 40500615 6 15S ABE 2004 UNIQUE fasta Archaea Bacteria Eukarya 0 39 390 667 tRNA ABE 2004 fasta Archaea Bacteria Eukarya 0 676 5056 6035 6 tRNA_ABE_2004_ UNIQUE fasta Archaea Bacteria Eukarya 0 217 2085 2226 9 itrial fasta ABCDE 2 12 29 30 35 45 10 BacteriaFixed_255 ABE 2004 UNIQUE fasta Archaea Bacteria Eukarya 0 24 208 345 M 4 gt Mi Sheeti Sheet f Sheet3 VE i ee BISA rom Ta Ready f Figure 5 A snapshot of the KnownFASTAFilenames xls file The highlighted entry number 6 is the FASTA file analyzed in this manual The Excel file is organized in the following way The first column contains the exact names of known FASTA alignments the second column contains the names of the subgroups the FASTA alignment contains separated by empty spaces and the third column defines the limits of these subgroups in the order of their names separated by empty spaces The highlighted file entry 6 5S_ABE_2004_UNIQUE fasta is the one used for the sample study in this manual It contains sequences from the phylogenetic subgroups archaea sequences 0 1 to 39 bacteria sequences 39 1 to 390 and eukarya sequences 390 1 to 667 To ignore the f
23. airs as one of the thirteen codes listed above and not the reverse form 11 cis Watson CrickHoogsteen cHW trans Watson Crick Hoogsteen pe c cov H WH cis Watson CrickSugar edge be eb WS Or t tr Wratson Crick S d ans atson ric ugar e ge sw cis Hoosgsteen Hoogsteen cHh trans Hoosgsteen Hoogsteen A tHH Op oP cis Hoosgsteen Sugar edge fd ee cis Sugar edge Sugar edge 5 wW Braam Table 1 Basepair codes used in Ribostral The last column shows the codes used for constructing the structural mask in Alignment Viewer discussed later in text If the type of basepairing is known for a pair of bases the output of the sequence analysis will be colored to indicate its isosteric subfamilies so the investigator can determine whether the aligned nucleotides for each sequence represent isosteric or near isosteric substitutions If no structural information is available the table output will only display sequence substitution data observed in the corresponding columns of the alignment After successfully reading a nucleotide list file Ribostral displays a new button allowing for the analysis of the whole list at once Figure 10 12 ribostral File Tools Help Jv 5S_ABE_2004_UNIQUE fasta j Vv rr0033_55_AllClasses xls Figure 10 The main GUI after successful loading of a nucleotide list file The status bar upper right hand corner displays NT list loaded and the new button
24. alyzed In such cases the user will be notified by the sound to carry on to the next step Enabling Consider Gaps means that the program will not ignore insertion characters seen in the alignment Disabling this option not only stops the program from showing gaps in the output it also ignores gaps from any other calculations such as score or percent substitution 33 calculations The consider Ns option does the same thing but with characters representing undetermined nucleotides usually symbolized by o O n and N which as stated earlier are all converted to o when the FASTA alignment file is originally read These two options do not affect analysis of nombasepairs such as base triples or longer motifs Several rounding options are available through a dropdown menu The choice here affects the presentation of all decimal calculations done by Ribostral Activating any of the Additional Output options results in the production of one or two more output formats that Ribostral does not produce by default discussed earlier Preferences are saved only if the SAVE button is activated Finally upon activating the button Modify Score Parameters the file containing score parameters lt installation directory gt Ribostra SCORES txt is opened for editing The parameters entered in this file affect all score calculations The resulting formula is shown on screen and is al
25. anism in the NT list Excel file if such a spelling cannot be found in the FASTA file an error message will appear and the program will stop If universal numbers for the alignment are desired the word universal can be used instead of an organism name similar to entry 8 in Figure 9 Structural information i e basepair type is allowed only for basepair lists These are lists comprising two columns of nucleotide numbers like the one shown in Figure 9 Basepair types are named by reference to the interacting edges of the nucleotides These are the Watson Crick edge W Hoogsteen edge H and sugar edge S Edge to edge interactions in addition may be cis c or trans t with respect to the glycosidic bond This gives rise to twelve main families of basepairs 2 4 and some intermediate families 5 only one of which with currently characterized isostericity matrix the bifurcated cWW tWH family These thirteen basepair types are coded as follows in order for Ribostral to understand them case insensitive for all except tSs because it is a directional interaction with asymmetric isostericity matrix cWW tWW cWH tWH cWS tWS cHH tHH cHS tHS cSS tSs and bif Asymmetric codes such as cWS can be reversed so in the table if nucleotides 22 and 26 in this order are coded as tSW this is the same as 26 and 22 being tWS see Table 1 for a definition of all the basepair codes used by Ribostral Ribostral always presents data about basep
26. asepairs of family 10 tHS are analyzed here in the archaeal 5S rRNA alignment The GUI shown in Figure 20 states on the top right the number of sequences in the chosen domain in this case the domain archeae has 39 sequences Below that the number of basepairs forming this particular interaction in the source organism is shown In this case six such tHS basepairs are found The isosterically colored buttons that appeared in the middle of the GUI display the substitutions in the sequence positions corresponding to all these six positions at once Notice how most of them are clumped in the yellow isosteric subfamily the colors are the same as defined previously for the HTML output files Upon clicking on any of these colored buttons the names of sequences giving rise to them preceded by the corresponding nucleotide numbers in the source organism will be displayed in the lower part of the GUI Figure 21 This allows for easy identification of organisms with potential mistakes in their alignments so that the investigator can realign them by hand 28 InteractiveAnalysis ee es ee F Ade i m g 105 76 Eu Methferv 105 76 Eu_Methform 105 76__Eu_Thercele 105 76_ Eu_Therm_ac 93 90 Eu_Methrumi 93 90_ Eu_Methvacu Figure 21 Getting sequence names with specific substitution patterns Upon clicking any button in the substitution matrix button GU is clicked here the names of sequences giving rise to the substitutions become displayed in
27. ay the buttons that start the analysis These buttons will appear only after the sequence alignment file is loaded into the program To provide additional help for the user the upper right hand corner of the main GUI is reserved for messages describing the status of the program any errors in execution or hints on how to proceed further 2 1 Loading an alignment file To start the analysis the user needs to follow the hint that appears in the upper right hand corner of the main GUI Hint start from FILE By clicking on File from the menu bar the user sees the options Open FASTA alignment Open NT list and Preferences Figure 3 CE 01 xi File Tools Help Open FASTA alignment Open NT list Preferences Hint start from FILE Ctrl P Figure 3 File menu options The first thing to do is to open an alignment file In its current version Ribostral reads only alignments in FASTA format the simplest and most common sequence alignment format available In this format each sequence is represented by a header line preceded by the symbol gt and one or more sequence lines Upon choosing Open FASTA alignment from the File menu the user can browse the local drive for alignment files Figure 4 Look in S FASTA_alignments fe c EJ OLD FastaFixing programs 55_RFAM_Se OLD mat alignments e 165_ABE_20 RFAM 165_elected 55_ABE_2004 mat 235_ABE_20 55_ABE_2004_UNIQU
28. e major areas for local alignment mistakes or motif swaps are located Note that if base triplets are present 1 e two basepairs with one nucleotide in common the basepair listed earlier in the Excel BP list file takes priority in coloring If the user prefers to give priority to cWW interactions for example then these should be listed first in the BP list SS de Alignment Viewer Microsoft Internet Explorer Fie Edt Wen Faventes Tods Hap re 5 a WAS a sI s Or 3 x 2 7S po sewn Greats G 3 1 SO BE i Addes E ciare Ha lbworkiN ATLAN programe aigner Anshis Out pyro 5 A Clssses is 55 ADE Z0 UNIQUE mat A gemer hiri Uns a5 ARE 09 UNIQ BP iist 2073 SS _AlCkewes 2b BP family symbols tsed in ED mash noo syraeetnic BPs nest be wed flom UPPERCASE to biases to reflect edass m the onder WHS CAWS Aed CWH a WH gh zel Color Codes mo WG GG 0 rake I po DOCUCAUUUOGAAC DOUOGUVICSASOCDI SCATUCCGAACICGY CGUGAAAA O6 UG AAAUGC C2040 col GAGARA 0 SARE me Figure 17 Ribostral HTML alignment viewer The visible part of the 5S alignment describes how well sequences agree with structure represented here by Eu_Halomari or sequence number 11 The color code is printed at the top Note that the whole content including colors can be copy pasted into Excel or other editors for manipulation Ribostral Alignment Viewer starts with several title lines containing the names of the analyzed files as well a
29. es in its table will be colored in neutral beige like the second BP listed in Figure 13 Figure 15 Basepair families and their isosteric subfamilies Gray colors indicate forbidden combinations of nucleotides i e combinations that cannot form basepairs due to steric clashes or incompatible distribution of H bond donor or acceptor atoms In each family isosteric subfamilies have the same color and nearly isosteric families have similar colors the five similar color groups are shown on the bottom right corner Letters correspond to the original reference 4 Asterisk corrected from the original reference 20 21 One more output file is produced by default in case a basepair input list with structure information is analyzed This is the _Statistics out file which is best viewed with Microsoft Excel or WordPad This output summarizes some of the results by stating the percentage of basepairs analyzed that have mostly allowed substitutions containing lt 10 forbidden or gaps the percent of basepairs that have gt 10 forbidden substitutions and the percent of basepairs having gt 10 of sequences with gaps at their positions It also shows the percent of basepairs among the ones mostly allowed that have only isosteric substitutions Instead of the total score which is just one value that describes the quality of the alignment studied this output file gives a more quantitative measure of the alignment quality When a
30. he AlignViewer option is selected in Preferences an HTML format alignment viewer is created where the sequence alignment is shown in colors indicating substitutions that are compatible with the 3D structure and those that are not This tool is the first of its kind taking into consideration all thirteen families of basepairs instead of just the classical cis Watson Crick family The sequences in the Alignment Viewer are colored in a way to describe how well each column more precisely each pair of columns in the alignment agrees with structure Figure 17 Basepairs from each sequence are colored individually based on their isosteric agreement with the homologous basepair from the reference sequence which is usually the sequence with known 3D structure If the substitution pattern is isosteric to the one in the reference sequence it is colored in green if it is nearly isosteric to it it is colored blue heterosteric is pink forbidden is red gap on one side is dark gray and gap on both sides is light gray If no basepair information is available the nucleotides are printed in black these color assignments can be modified by changing the MATLAB script file mColorCode m For easy interpretation the color scale is printed at the top of the alignment viewer HTML file Thanks to this tool it is now possible to look at the sequence alignment and directly get a good idea about whether the alignment is structurally valid or not and where th
31. in the editable box All positions homologous to these numbers will be extracted from the original alignment and saved as a new sub alignment in FASTA format If there are one or more commas separating two or more groups of numbers the sub alignment will represent these breaks in 35 continuity by four consecutive dots This continuity separation code is understood by other Ribostral tools such as the alignment parser tool discussed in the previous section Naturally nucleotide numbers refer to the source or reference organism specified in the GUI Similarly to the interactive analysis GUI in case there is disagreement between the sequence name in the white editable box and that in the gray drop menu box the white editable box takes precedence The user can choose to ignore up to five organisms from the top of the sequence alignment for instance if these are structure masks or sequences that do not belong to the same phylogenetic group and can choose to extract one domain or all domains at once The extracted sub alignment is saved in the same directory as the original source alignment file If there is more than one motif to be extracted the user can create a tab delimited input text file containing the names of all of them and their nucleotide numbers for batch processing The nucleotide numbers should correspond to the Reference Organism Name selected in the GUI The input text file would look like this h5IL 53 56 356 358 h6IL
32. in the study so it is actually an average of the individual scores This means that its maximum is also 100 and its minimum in case the formula presented here is used is 100 Note that another valid formula used can be something like Individual BP score c SUM 2I NI 3F number of sequences Both formulas are ad hoc and the difference between their weights is not essential The ease of reading and understanding normalized values between 100 and 100 or 150 is the reason for using a correction coefficient 19 The most obvious and important element of the HTML output files discussed thus far is the background color patterns that appear in their tables These colors reflect the isostericity matrices of the thirteen characterized families of basepairs Each family has its own pattern of isosteric nearly isosteric heterosteric and forbidden substitutions Boxes with the same colors are isosteric Boxes that are nearly isosteric with each other have similar colors There are five groups of these similar colors pink red orange red orange yellow so substitutions with pink and yellow backgrounds are not nearly isosteric to each other blue cyan dark green light green and brown Figure 15 Forbidden boxes are colored in dark gray and gaps in light gray Boxes with nom similar colors indicate that they are heterosteric to each other If the basepair family for a certain basepair is not specified all nucleotide box
33. information in Figure 28 BPs observed in 3D structure written into Excel in the form required by Ribostral Given this structural information we can objectively measure the quality of the alignment All that needs to be done is to load both the FASTA file and the Excel file into Ribostral and to analyze the output files produced We will first look at the HTML Alignment Viewer file which displays the alignment with nucleotides colored in a way describing how well they conform to isostericity rules inferred from structure 38 Ribostral Alignment Viewer Microsoft Internet Explorer Painia File Edit View Favorites Tools Help kid Q Back y amp Y x E A J Search 5 Favorites a F Address B C Documents and Settings Ali Desktop Ribostral example sm Go Links Alignment file t3H tWH tHS_before fasta BP list tSH tWH tHS xls BP family symbols used in 13D mask non symmetric BPs must be read from UPPERCASE to lowercase to reflect edges in the order W H 3 cWW ab tWW cd cWH ef tWH gh cWS y tWS kl cHH mn tHH op cHS qr tHS st cSS uv tSs wx biER yz unspecified 7 Color Codes no info one gap both gaps Isosteric Nearlsostenic Heterosteric Forbidden UG oar PARTS structure Local Numbers N E RC aa EAT 3D Directional Mask M1 bthTb 3D Mas j UAAGG AAAUG AAGGC GGGUC GGGUG CAGUG CGCUG GGAUA UAUGA eo e w N D w O wo 4
34. irst sequence in the alignment if it is a structure mask for example or if it is a reference sequence that does not belong to the subgroup 1 can be placed instead of 0 in the third column In that case the program still reads the first sequence but does not include it in the analysis The main GUI then displays any known details associated with the FASTA file and also unlocks some buttons or check boxes that permit the user to analyze the alignment Figure 6 Note that if this step loading a new FASTA file is repeated at any stage during the analysis all previous data that corresponds to previously read alignments will be erased from memory and the GUI will be reinitialized ribostral File Tools Help Vv 5 _ABE_2004_UNIQUE fasta Fy Beera A curva Figure 6 GUI changes when an alignment file is successfully loaded The text box in the middle displays the name of the sequence file in memory At this stage two options are possible for carrying out sequence and optionally structure analysis analyzing a list of nucleotides with or without structure data for the complete sequence analysis of all positions listed in it or interactively and directly analyzing individual positions on screen The first option is covered first 2 3 Preparations for the analysis of a list of nucleotides After loading a FASTA file the user can load an Excel file containing a list of the nucleotides of interest This is done by activating
35. ith descriptive suffixes as the original FASTA files they are created from If more than one original file was inputted into this tool a merged file containing all of them is also created 36 6 5 Tool 5 Create fasta from mat This is a simple tool that allows the user to open a mat file representing a sequence alignment in MATLAB data format and recreate the fasta file it was originally created from If the file contains more than one subgroup or phylogenetic domain separate FASTA files containing the sequence of each of them in addition to one containing them all together are created and saved in the same directory as the input mat file 7 Help menu Under the help menu the manual website and version information of Ribostral can be found To check if any updated version of the program is available the user can compare the local version number to the version number listed on Ribostral website 8 Sample study In this section a simple example of how Ribostral can be used to improve a sequence alignment based on structure is given We will do this exercise on a small part of an alignment to keep things simple and clear This is the starting alignment gt Structure CGCCA UAAGG gt Seq2 CGCCU AAAUG gt Seq3 GGCAU AAGGC gt Seq4 GGCAC GGGUC gt Seq5 CACAC GGGUG gt Seq 6 CGCAG CAGUG gt Seq7 AGUCG CGCUG gt Seq8 UACAC GGAUA gt Seq9 UGUCA
36. m that dynamically displays parts of structure creates full 2D annotations of them and shows corresponding positions in sequence alignments in an alignment editor 3 However it too does not evaluate alignments based on isostericities of basepairs formed in structure the way Ribostral does Ribostral which runs under multiple platforms can either be used like other sequence analysis programs i e to simply provide substitution patterns of basepairs in a sequence alignment without any relation to structure or can be used as the much more powerful tool that it is designed to be to provide substitution patterns and at the same time superimpose structural information on top of the substitution patterns to make sense of it Ribostral does that by coloring substitution patterns of each basepair in a way that reflects the edge to edge family and the isosteric subfamily it belongs to 4 Even in its more simple usage as a program that provides sequence substitution analysis without the use of 3D structural information Ribostral is more convenient than other programs because it allows for simultaneous analysis of lists of basepairs and produces a single and easily portable HTML output without having to input nucleotide numbers of interest one position at a time Interactive position by position analysis is also possible where in addition to what has been described above an integrated structure viewer is also available In addition to providing subs
37. ole table to be in one neutral color 2 It has been shown that the colored GUI buttons may display the results of either a whole BP family or an individual position To prevent any confusion a thin white box appears everytime one of these two types of analyses is requested This line surrounds the output buttons as well as the input buttons giving rise to them The difference can be clearly seen between Figures 20 and 21 on the one hand and Figures 22 and 23 on the other 5 Ribostral preferences Ribostral is a diverse program capable of dealing with data in a variety of ways Through its preferences the user can enhance his experience with the program and make it do what is best for the investigation in hands Ribostral preferences can be accessed from the File menu of the main GUI They are then saved as a data file on the users disk for easy passing between all subordinate Ribostral GUIs Last positions of GUIs before exiting them are also saved in this file so that GUIs always open in the last known position they were closed in Figure 24 shows the default preferences of the program ee conee rm ra ae is Round to closest integer EE SZ Ul Figure 24 Ribostral default preferences The preferences are divided into several categories Round radio buttons belonging to the same category are mutually exclusive The first two categories Display Totals and Display Expected affect only the list output files and do no
38. positions simultaneously Fd Microsoft Excel rr0033_55_AllClasses aa o x GS File Edit view Insert Format Tools Data nunma Window Help sre porie 7 Beil O A 2 AB X f Universal Interaction 26 tSVV 58 2115S 28 4 28 tws 53 cv 29 cWS 50 cv 30 cWS rh rT Figure 9 Basepair list for analysis This Excel list from lt at includes structural data about some of the basepairs to show that structural data is optional LB2 10 As seen in Figures 8 and 9 the first column of the Excel sheet contains the name of the reference organism for that row and subsequent columns contain the nucleotide numbers to be analyzed The name must be the full name or part of a name case insensitive present in the loaded FASTA alignment file It does not necessarily need to be the beginning of the name e g Halomari can be used if the actual name in the FASTA alignment is Eu_halomarv If several FASTA comment lines share this name the first of the occurrences will be considered as the reference organism e g if halo is used and it is present three times in the sequence alignment first as EU_HALOJAPO then as Eu_halomari and then as Eu_halomedy the first will be used It is advisable for the user to manually check sequence names in the FASTA file to prevent inadvertent reference to unintended organisms The most common error is caused by incorrect spelling of the source org
39. rmatics 21 3320 3321 4 Leontis N B Stombaugh J and Westhof E 2002 The non Watson Crick base pairs and their associated isostericity matrices Nucleic Acids Res 30 3497 3531 5 Lemieux S and Major F 2002 RNA canonical and noncanonical base pairing types a recognition method and complete repertoire Nucleic Acids Res 30 4250 4263 6 Mokdad A Krasovska M V Sponer J and Leontis N B 2006 Structural and evolutionary classification of G U wobble basepairs in the ribosome Nucleic Acids Res 34 1326 1341 7 Sarver M Zirbel C Stombaugh J Mokdad A Leontis N 2006 Finding Local and Composite Recurrent Structural Motifs in RNA 3D Structure Journal of Mathematical Biology accepted into special RNA issue Citation Please cite the following work if you use any component of Ribostral or the nomenclatures proposed in it Mokdad A and Leontis N 2006 Ribostral An RNA 3D alignment analyzer and viewer based on basepair isostericities Bioinformatics 22 17 2168 70
40. s the legend of all codes and colors used Following this is the listing of all organism names and their sequences exactly as they appear in the original FASTA alignment Organism names change their color gradually as they approach the limit of the subgroup or domain they belong to This is how it is made clear that sequence 39 for example is the end of the archaea domain The first five sequences those on black background represent the universal numbers local numbers and three types of structural masks that describe basepairing patterns Universal numbers are assigned to each character seen in the sequence including all indels Local numbers are the numbers that correspond to the reference sequence unlike universal numbers local numbers are not assigned to indels in this sequence In the Alignment Viewer 24 only decimal representatives of local numbers are listed i e the first 1 stands for 10 the first 2 stands for 20 and so on until the first 0 is seen which stands for 100 After that the numbering cycle is repeated so the second 1 stands for 110 the second 2 stands for 120 and so on The structural masks describe basepairing patterns reported in the Excel BP list directly on top of the sequences Figure 18 is a schematic description of the information represented by structural masks A 2697 A 2697 G2608 G2e08 2696 G 10 A 2699 2606 G GD A 2699 2605 G27 2695 C G2700 2694 A CH gt G
41. so printed out in some output files every time sequence analysis is carried out 6 Supporting tools Ribostral provides an expandable sequence and structure analysis platform Additional tools can be easily and smoothly integrated into the main program There are currently five additional tools that allow Ribostral to do operations beyond structurally analyzing sequence alignments Figure 26 Each of these tools is discussed separately below LIM icix File Tools Help I Generate BP lists from PDB 4lign sequences Extract parts of a FASTA File Merge amp remove repeats from FASTA files Create fasta from mat od era S waa rr0033_55_AllClasses xls Figure 26 Ribostral tools 6 1 Tool 1 Generate BP list from PDB This tool currently works only on PC platforms PDB files are text files containing all the atomic coordinates and related information about a structure By analyzing this data interacting bases can be identified and classified into geometric families 7 Upon activation of this tool the user first chooses a PDB file browsing starts from the default PDB directory associated with 34 Ribostral lt installation directory gt PDB_structures Then the user is prompted to enter the sequence name in the alignment that corresponds to this 3D structure Ribostral then creates an Excel BP list for each of the basepair families found in the crystal structure It also creates a separate E
42. t sign Once again if the user clicks on any one of the nucleotide patterns the names of sequences giving rise to them will be displayed in the lower portion of the GUI Figure 23 InteractiveAnalysis po Heloners Comes elfarcnsea BP Family X 2 2 ol 4 l ol ol 4 ol Eu_Halomari Figure 23 Motif interactive analysis Individual nucleotides in the motif are arranged in the order of the positions entered Since the query here is 26 22 37 the letters that correspond to local number 26 of source organism is displayed first then those that correspond for 25 then 24 23 22 and finally 37 If instead of this the query was 22 26 37 the result would be displayed differently Finally the interactive GUI provides a quick link to a basic structure viewer through the activation of the button Display 3D When this button is clicked the first time it prompts the user to choose the PDB file corresponding to the sequence studied and atomic coordinates are read the bigger the PDB file the slower this process The structure of the input nucleotides is then shown Subsequent activations of the button within the same session display the structure immediately 31 4 3 Notes 1 The user can change the color distribution of the substitution table to be similar to another basepair family This may be important in case mistakes in the BP list are suspected or in case no BP family is stated causing the wh
43. t affect the interactive analysis GUL If Display Totals is enabled the totals of each row and column of the BP list output are printed with the output as in Figure 13 Otherwise the totals will be left out If Display Expected is enabled the expected value for each substitution is printed in parentheses after the observed substitution value as in Figure 25 32 10 x Fie Edit View Favorites Tools Help Q ee x F A 72 Search Se Favorites E2 x ki Go Links gt Address sses_demo xls_55_ABE_2004_UNIQUE mat_COV_Counts html Source of numbers Halomari Archaea 39 seq OF 33 1 1 NI Bacteria 351 seq OF 3 3441 5NI Eukarya 277 seq OF 2731 ONI 0 0 28 0 0 312 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 Done E My Computer A Figure 25 Substitution counts with expected values in parentheses The expected value for each box in the table is obtained by applying the formula Expected of box in row a column b Sum of boxes in row a Sum of boxes in columns b Table total This value is a measure of the likelihood of observing substitutions in the specific box based on the values observed in the neighboring boxes Enabling the Audio option allows the program to play specific musical chimes every time an operation is completed or an error occurs This is useful in case a large file is being read or an
44. t_SEQ html Ejo Links on gt 3 Eukaryaj277 seq 190 F EN 39 seq 351 seq 277 seq Archaea Bacteria Figure 14 Matching sequence values and names The _COV_Counts html output file left panel displays the sequence counts and the corresponding _SEQ html right panel displays the names of sequences Note the value indicated by the black arrow in both outputs The left panel shows that there are three bacterial counts for AA occupying the position A3 G21 halomari local numbers The right panel shows that these three counts come from the sequences of G_complan P_palmata and S_vulgare The output files have two purposes First to display substitution patterns corresponding to each BP position analyzed and second to provide information about how well each of these positions in alignments agrees with 3D structure Based on this if there are any potential mistakes in the alignment they are easily pinpointed for their manual correction with available sequence editors All three HTML output files mentioned start with a total score assigned for each phylogenetic subgroup in the alignment in this case the subgroups are the three domains archaea bacteria and eukarya Each subgroup will have its own results printed on a separate line in the substitution tables The areas in Figure 14 indicated by the black arrows for example represent the substitutions in eukaryal sequences of the basepair A3 G21 using halomari
45. th _SEQ html displays the names of organisms giving rise to the values in the first two output files The positions of the names correspond to the values observed as clarified in Figure 14 Note that the basepair identity observed in structure is indicated by bold font in the corresponding cell ve tar Ye we fees ieee Qmo ol 2G Dam Sime O Ss Alignment file 5S ABE 2004 UNIQUE mat BP list rr0033_ 5S AllClasses_demo xls Score Formula 50 2H NI 2F 2G1 3G2 num of seqs Scores J Archaea Bacteria Eukarya Counts Source of numbers halomari Archaea 39 seq OF 0G 391 ONI S 100 Loree 351 seq 13 F 0G 3121 ONI S 85 277 seq 132F 0G 1311 ONI 0 e IA EITA a eee E Archaea 39 seq Bacteria 351 seq a 277 seq 0 39 1 70 2 1 79 351 19 31 222 276 oe EE r a Figure 13 Percent output HTML file created upon phylogenetic analysis of an basepair list A similar output file with counts instead of percents is also created This analysis is for the NT list presented in Figure 8 above F 17 F Ribostro Mutual Counter Microsolt trennet i l jaj x Fle Edt View Favores Tools Hep 2 De ee eg Q O d 307 pia o Address E _55_ABE_2004_UNIQUE mat COV _ Fle ede View Favortes Tools Help e E iene ae Fer Aschaeal 39 seq 25F 0G A x Bia E K ca w zi LJS pose fie 3 9 Address E _Analysis Output r0033_5S_AlClasses_demo xls_55_ABE_2004_UNIQUE ma
46. titution information for basepairs Ribostral is also capable of analyzing substitution patterns for more than two nucleotides at once such as base triples quadruples and so on The sequences of whole motifs can be analyzed this way as will be shown below 1 1 Supported platforms and deployment process Ribostral is designed and fully tested under Windows XP It is free and can be obtained from http rna bgsu edu Ribostral The program is distributed in two forms MATLAB source files capable of running on any PC or MAC platform with MATLAB version 7 SP3 or higher with loss of some noressential options on MAC or a stand alone program capable of running under the PC platform after installation of a free compiler provided by Mathworks details can be found here Figures used in this text are based on the Windows XP version with system appearance set to Windows Classic style Upon downloading the MATLAB source files or the stand alone version the user will end up with the specific hierarchy of folders shown in Figure 1 amp Installation Directory o x Fie Edt View Favorites Tools Help a amp Agai j S l ee eek O Jo seach p Fors 777 i Audio FASTA_algnments Put your FASTA alignments here Help NT_ists lt Put your BP or NT excel lists here Output Output files will be created here PDB_structures lt Put your PDB files here Rbostral INcludes all programs
47. xcel BP list containing all of them and another Excel BP list containing all the non cWW ones among them together These lists are created in the proper format to be read directly by Ribostral and are saved in the default lt installation directory gt NT_lists folder Note that the automatic classification of basepairs is not 100 accurate so the resulting automatic lists should be visually compared to the 3D structure and corrected where needed 6 2 Tool 2 Align sequences This tool opens a GUI that interfaces with automatic motif alignment programs based on a hybrid Stochastic Context Free Grammars Markov Random Field SCFG MRF model The GUI facilitates some of the steps in running these programs and allows the user to parse and align motifs that have their SCFG MREF nodes already characterized based on known 3D structure Currently MATLAB script files still need to be modified in order to parse and align new motifs This tool mainly provides an easily expandable user friendly platform for applying automatic sequence alignment 6 3 Tool 3 Extract parts of a FASTA file This tool shown in Figure 27 requires loading a FASTA file from Ribostral main GUI before it can be activated mFastaExtractor_multipleGui Type NT numbers separated by if continuous or if not Borer o Figure 27 GUI for extracting parts of a FASTA file The user then simply needs to indicate the motif of interest by entering its nucleotide numbers
48. y changing the program preferences 3 1 Output files for a non basepair list If the input NT list is not a list of basepairs but a list of nucleotides forming distinct motids such as the one shown in Figure 8 two text output files will be created one giving counts and the other percentages of sequences that share a common pattern Figure 12 The patterns are listed in alphabetical order To clarify this the following example from Figure 12 is considered The first entry in the Figure highlighted shows that in the archaeal part of the alignment nucleotides corresponding to Haloarcula_marismortui local numbers 545 611 529 and 14 in this order are 88 GUGC and 12 GUGo From this one can deduce that Nucleotides 545 611 and 529 are GUG in 100 of the cases and so on The values in the percent output file are rounded according to the preferences of the user Ribostral preferences will be discussed in detail later 14 235_4_P_correction_trial xls_235 ABE_2004 UNIQUE mat COVAR F File Edit View Insert Format Help oela Sila al sale Fasta Alignment File 235_ ABE 2004_UNIQUE mat Positions Excel File 235_4 P_correction trial xls ij Haloarcula marismortui 545 611 Number of sequences Bacteria Number of sequences 184 4ucG GCUG 3 GUCG 79 GUCo L Eukarya Number of sequences 137 GC 3 AUAU 19 GCAU 7 GCGC ap GCGo 48 GUGC 3 UAU 3 2 Organism Escherichia coli
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