1 JFitom v Alpha – USER MANUAL Introduction JFITOM is a
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1. Options are set according to the options file Figure 1 Graphical User I nterface of J FITOM Credits Original FITOM and xFITOM code by Ivan Erill JFITOM development by Omar Shehab and Ivan Erill Ivan Erill 2010 If using for research please cite Erill O Neill M C A reexamination of information theory based methods for DNA binding site identification BMC Bioinformatics 2009 Feb 11 10 1 57 How to Get J FITOM Visit http userpages umbc edu erill 7399 and select JFITOM project This opens the home page of JFITOM project The executable file is available on the page for download System requirements Software biojava jar jFitom core jar bytecode jar commons cli jar commons collections lt latest version gt jar commons dbcp lt latest version gt jar commons pool lt latest version gt jar jgrapht jdk1 5 jar log4j lt latest version gt jar JDK JRE 1 5 or above Operating system All operating systems supported by J DK J RE 1 5 or above Hardware All hardware configurations supported by J DK J RE 1 5 or above All required libraries except JDK JRE are shipped with JFITOM They are archived in the J FITOM executable file Getting started JFITOM s distant predecessor FITOM is a command line argument based program meaning that it is run from a DOS prompt Its more immediate predecessor xFITOM includes a Graphical User Interface GUI to select the required files2. J Fitom v Alpha USER MANUAL Introduction J FITOM is a portable and extended version of xFITOM a computer program for the detection of binding sites in DNA sequences JFITOM implements several methods described in the literature to compute an approximation of binding affinity for a particular transcription factor binding site based on a collection of binding sequences provided by the user Using these methods JFITOM scans a sequence file looking for putative binding sites across the DNA sequence in both strands and filters the results according to a user specified threshold JFITOM will also link the identified sites with annotated genes and it will infer their roles from their location in the vicinity of genes E JFitom Alpha File Help Input and output files Default path Genome file Set default path Browse Browse Sites file Current options file vV Are these sites palindromic Output file Browse Scoring methods ORi ORixRseq Olseq x RE Annotation strategy Gene search hysteresis Intergenic distance Operator distance Maximum intragenic distance Filtering strategy Maximum number of sites OReturntop 100 sites Returntop 10 of the genes Threshold site score In a normalized score scale of 0 0 to 1 0 0 5 Standard deviation from the mean 0 0 Generate results Generate log files View the result file MS Windows Generate results Restore Factory settings
3. and to set all the necessary options JFITOM provides new functionality like platform independency and the ability to create a list of regulated genes for each site JFITOM can be launched from the command line from a script file or using the GUI To run J FITOM from the command line the user has to use java with JFITOM run jar as the main argument JFITOM run jar takes three command line arguments which are as follows the options file the genome file and the site collection file A sample command to run J FITOM would be as follows java jar JFITOM run jar g genome file s site file o options OPT To run JFITOM from script files the same commands can be used JFITOM GUI facilitates saving or customizing options in an interactive way If the JFITOM command is not provided with any argument or if J FITOM run jar is double clicked J FITOM will launch the graphical user interface Main operation files in J FITOM JFITOM operates with three main files a file containing the genome sequence to be searched genome file a file containing a list of binding sites collection file and a file specifying the program options options file The sequence file Genome file The sequence file Sequence _file ext is the file containing the sequence or sequences the user wants to scan The sequence file can only be in GenBank format GB GBK or Genbank The collection file Site Collection file The collection file Collection_f
4. andard deviation from the mean 0 0 Figure 6 Filtering strategy The last section Generate results allows the user to store the options and launch the analysis The user can also decide if she wants to generate log files or view the result immediately Generate results Generate log files View the result file MS Windows Generate results Restore default options Figure 7 Generate result Input and output file processing Loading genome file The main JFITOM program loads the genome file first The file can be only in GenBank format After loading the file J FITOM uses Bio ava library to parse that file and extract the genes along with annotations For annotation the following parameters are stored name location strand locus tag protein id product and note Loading sequence file After the genome file JFITOM loads the file containing a list of known binding sites which are used to constructor model of binding site or motif The file can be in two formats FAS or TXT files Loading options file If run from the command line with an options file as parameter JFITOM functions according to the options set in the options file If no options file is specified as the command line parameter it looks for an options file in the default folder lt JFITOM home directory gt config options conf If the options file is not available in the default location it runs with factory settings If the options file is missing any parameter th
5. e value of the parameter is used from factory settings When the user is running the GUI the options are saved in the default path before generating the result The following options are defined in the file latestGenomeFile file path in standard format latestSitesFile file path in standard format isPalindrome Y if the binding sites are palindromic else N latestOutputFile file path in standard format scoreMethod 0 for Rj 1 for Ri X Rseg 2 for Iseq and 3 for Iseq X RE more details in following sections maxHysteresisLimit maximum number of base pairs to be scanned looking for a gene upstream or downstream located binding site maxIntergenicDistance maximum number of base pairs between genes to be reported as part of an operon maxOperatorDistanceOut number of base pairs upstream of a gene translational start site for a site to be considered operator If maxed the site will be labeled intergenic maxOperatorDistanceIn maximum number of base pairs downstream of a gene translational start site for the site to be considered operator IF maxed the site will be labeled intragenic resultSizeMethod 0 if the absolute size or 1 if relative size is specified resultSizeMethodValue the size of the list thresholdScoreMethod 0 if the threshold is an SD band and 1 if the threshold is normalized thresholdScoreValue the threshold score saveOptions Y if the user wants to save the options t
6. ed by the user input and used as the threshold value please refer to Figure 5 Annotating the genes After building the list of site J FITOM annotates each site with the following information category whether the site is intergenic intragenic operator isolated or none of these relative position distance from the first Gene genes a LinkedList of co regulated Genes J FITOM allows the user to set following parameters for annotation Gene search hysteresis JFITOM uses this value as the highest limit up to which it will scan for the first forward Gene for downstream search or the first reverse Gene for upstream search around a site If no gene is found within the hysteresis in either direction the site will be marked as isolated Intergenic distance On upstream or downstream region when J FITOM finds the first gene it looks for the co regulated genes in an operon configuration These genes may be separated at most by this distance If no gene found FITOM stops searching for the site for that region Operator distance When a site is found in an intergenic region JFITOM further checks if it is within the operator distance from the start of the closest gene If it is the site is an operator site otherwise it is an intergenic site Maximum intragenic distance When a site is found inside a gene JFITOM further checks if it is within the maximum intragenic distance from the s
7. ile ext is the file containing the collection of known binding sites that the user provides the program with in order to construct its model of binding site or motif Collection files can be either bare site files plain text with aligned sites on consecutive lines or FASTA files in which each site line is preceded by an identification line beginning with gt Accepted extensions are FAS FNA for FASTA files and TXT for bare site files The options file Options file The options file OPT stores different operational strategies and information If no such file is specified in the command line argument the software operates with factory defaults Main functionalities JFITOM provides the following functionalities dealing with different aspects of program operation The GUI The graphical user interface is very simple and intuitive All the operations are done on a single window Figure 1 The window contains a form for user input which is divided into six sections The first section is Input and output files This section takes the genome file and the sites file as user input If the user wants to specify that the sites are palindromic she can click the check box The user can also specify the name for the output file where the result will be stored Input and output files Genome file Browse Sites file Are these sites palindromic Output File Browse Figure 2 Input and output files The second section Defau
8. lt path allows the user to set the default path for present session After setting the default path all other file browsing controls of J FITOM sets their current directory to this path After setting the default path if the directory does not contain any OPT file it requests the user to give a file name OPT where the options will be save Instead of creating a new file a user may also select an old OPT file In that case J FITOM GUI controls are set to the values stored in the options file If the user chooses not to select the options file at that time she is asked again for the file name during computing and storing options and result Default path Set default path Browse Current options File Figure 3 Default path The third section Scoring methods allows the user to choose scoring method There are four options By default J FITOM scores using the R method Scoring methods Ri Rix Rseq Iseq Iseq x RE Figure 4 Scoring methods The fourth section Annotation strategy allows the user to set the annotation strategy While J FITOM scans a genome it needs to know the hysteresis limit the maximum number of base pairs JFITOM scans to determine the first gene with appropriate orientation intergenic the maximum number of base pairs between genes of same orientation regulated by the same binding site operator the maximum number of base pairs within which the regulating site is located before a gene seque
9. mum intergenic distance Generating output After running from the command line or from a script JFITOM saves the scored and annotated binding sites as a CSV file in the lt JFITOM home directory gt output directory and terminates If the user is running the GUI JFITOM will save the options before starting the main operation If the user wants to see the details in a log file she has to select the Generate log files check box To view the result instantly she can select View the result file before clicking the Generate result button The user can also use the default options by clicking the Restore default options button In the GUI the user can also specify the output file destination The first column of the CSV file contains symbol to distinguish each result site For each of them the columns are as follows Position the position of the site in the Genome Score the score of the site Strand on which strand does the site resides 11 Site the sequence Up category upstream category Down category downstream category Up relative position relative position regarding the first upstream gene Down relative position relative position regarding the first downstream gene Genes annotated list of upstream and downstream genes ordered according to their position 12
10. nce starts and intragenic distances the maximum number of base pairs within which the regulating site is located after a gene sequence starts The user can specify the values in terms of base pairs The GUI validates the inputs before using them Annotation strategy Gene search hysteresis 200 Intergenic distance lso E Operator distance 300 Maximum intragenic distance 50 Figure 5 Annotation strategy The fifth section Filtering strategy allows the user to set the strategy to filter the result If the user sets the maximum size of the list aS Return top N sites JFITOM returns N number of sites at most If the user sets the maximum size of the list as Return top N of the genes the number of sites JFITOM returns is N of the total number of genes If the user sets a normalized threshold JFITOM computes the maximum and minimum score of the given binding sites Then this maximum to minimum range is converted to a normalized range of 1 0 to 0 0 Finally JFITOM converts the user given threshold back to the original scale If the user sets the threshold as standard deviation JFITOM computes the mean and standard deviation of the scores of the given collection of sites Then the standard deviation is multiplied by the user input and used as the threshold score Filtering strategy Maximum number of sites Returntop 100 sites Return top 50 of the genes Threshold site score In a normalized score scale of 0 0 to 1 0 0 5 St
11. ncy of each position the information from the rest of bases at that position discard by this method is not used To correct this O Neill proposed averaging this kind of methods with the known redundancy index of the collection O Neill 1989 so that the final score was given by L R AR oiie gt R D R sequence J j Another ranking method can also be L I RE I 1 RE J based on the RE formula l 1 Sequence Figure 3 demonstrates how to choose different methods from the GUI The user can also specify the ranking method in the options file Filtering the search results JFITOM allows the user to generate a selective list of results To limit the list by size the user can set the maximum size by a number like N sites or a percentage of the number of genes for example N of the total number of genes in the genome please refer to Figure 5 JFITOM allows the user to specify the threshold score in two different ways If the user specifies a normalized threshold JFITOM computes the maximum and minimum score of the given collection of binding sites Then this maximum to minimum range is converted to a range of 1 0 to 0 0 Finally from the user given threshold the effective threshold is calculated back in the original scale If the user specifies the threshold as standard deviation from mean JFITOM computes the standard deviation of the scores of the binding sites from the user given collection Then this value is multipli
12. o the file before generating result else N generateLog Y if the user wants to generate log messages else N viewResult Y if the user wants to view the result immediately else N applicable only if Microsoft Excel is installed Set output path The user can specify the path where the output file should be generated Main operation Here we describe the main modus operandi of the program As mentioned above J FITOM loads the sequence and site files before start processing Parsing the genome After loading the genome file JFITOM parses it and extracts the genes The genes are stored in an annotated list in the memory Position specific weight matrix and information content The site file is then used to compute the motif position specific frequency matrix PSFM This is a matrix of the relative frequencies of each nucleotide at each position in the motif If the user specifies that the sites are palindromic then JFITOM reverse complements the sequences of all sites and appends them to the site collection before generating the PSFM The following is a demonstration of consensus computed from the frequency of nucleotide 1 2 3 4 5 6 A 0 031 0 055 0 650 0 349 0 309 0 007 C 0 928 0 015 0 015 0 071 0 158 0 007 G 0 007 0 206 0 166 0 031 0 079 0 976 T 0 031 0 722 0 166 0 547 0 452 0 007 Consensus C T A T T G From the PSFM the information content of the motif can be computed acc
13. ording to the following formula Rygere X10 X Hrg D H ge O 3 el l i l Y F S dog f S SEQ f S frequency of base S in the genome p S frequency of base S in the motif PSFM H yore 7 a Priori entropy H after entropy after observing binding H E 5X pS ost SEQ as described by Schneider et al Schneider Stormo et al 1986 and based on the assumption of positional independency among the different positions of a binding site The information content of a motif tells us about the reduction in uncertainty we experience once we know that a protein or other element binds to a sequence Schneider Stormo et al 1986 Erill and O Neill 2009 Prior to binding our uncertainty about what bases occupy the different positions of a sequence is maximal and dictated by the base composition of the genome Once we know that the protein associated with the provided motif binds that sequence however we have much less uncertainty about what bases occupy the different positions We still have uncertainty because protein binding is a noisy issue but we have decreased our uncertainty and thus we can say we have gained information Conversely seen from the point of view of a genome the information content can also be seen as the loss of entropy at certain regions in the genome from an initial random state to a state of fixation of conserved binding sites Thus motif information content can also be as an index of
14. tart of the gene If it is the site is an operator site otherwise it is an intragenic site Annotation information is determined both for the upstream and downstream area of the site A site can be one of the following categories Operator if the site is within the maximum operator distance of a gene on either side The user can set this limit for the intergenic and intragenic sites individually Intergenic if the site is between two genes and beyond their operator limit Intragenic if the site is inside a gene but after the maximum operator distance None if no gene is found during a search Isolated if the category of the site is None for both up and downstream ee 1 l Reverse Forward EE Forward EEE I la lamn Figure 7 Binding site categories based on relative distance to genes The relative position for a site is determined as the distance between starting position of the site and the annotated starting position of the closest regulated gene 10 Special cases of annotation Maximum hysteresis limit Correct Genes but beyond hysteresis limit lt lt lt 1 gt i J Reverse x il X ee Figure 8 For this site J FI TOM found no genes during downstream search Maximum intergenic distance i Se a 4 J 1 li hH ee a Forward iri Forward ha i a l gt Figure 9 For this site J FI TOM ignores correct genes beyond maxi
15. the level of redundancy RI in the different positions of the motif O Neill 1998 Even though without a complete theoretical justification a different index termed relative entropy RE has been proposed to substitute the RI in cases of heavily skewed genome RE 1 S log zan D yfr log as SEQ Relative entropy Schneider Stormo et al 1986 Erill and O Neill 2009 is also computed by J FITOM and can be used in different ranking methods Ranking methods Rsequence tells us how much information our motif conveys but it does not provide answers to how well a particular sequence fits in the motif profile which is what is required to scan for and rank putative binding sites Several ranking methods have been proposed with diverse degrees of theoretical justification JFITOM provides a basic scoring method that can be used to rank putative binding sites The sequence information content Ri Schneider 1997 is a method derived from the information content Rsequence formula that scores each position of a particular site j based on ratio of frequency in the motif with respect to genomic frequency for the particular base observed in the site R D EO log p 5 SEQ This ranking method discards information from other motif base frequencies As explained in O Neill 2003 this can lead to erroneous scoring where the same score may be given to little or heavily conserved positions since information about the redunda
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