TRANSFAC Plugin
Contents
1. e Genomic Use this mode if genomic information is available in the form of genomic regions for which you wish to search for transcription factor binding sites Genomic regions and reference selection This section will be enabled in the Genomic mode of analysis Data is selected by clicking the corresponding folder icon jy in the right hand side of the wizard e regions The regions of interest as a track of annotations e reference The reference genome as a track of symbols Sequence selection This section will be enabled in the Classic mode of analysis Data is selected by clicking the corresponding folder icon 5 in the right hand side of the wizard e DNA sequences The raw DNA sequences of interest Clicking Next now opens the wizard step shown in figure 3 2 E BIOBASE TRANSFAC re n Configure TRANSFAC 1 Select input 2 Configure TRANSFAC Configure profile matrix Selecte profile matrix to use plants x Select cut off Minimize false positives O use only high quality matrices A previous gt Next X Cancel Figure 3 2 Selecting profiles and settings This wizard window allows you to configure the profile matrix parameters Select profile matrix to use From the dropdown menu you specify the profile set of position weight matrices for a taxonomic group tissue etc with which you want to scan your sequences regions for putative transcription factor binding sites Select cut off This2. negatives is computed for every cut off ranging from minFN to minFP whereby the false positive rate at minFN 10 false negative rate is defined as 100 The score at which this sum is minimal is used for the minSUM cut off e Set Matrix similarity cut off Here it is possible to define similarity score to use in the search see Kel et al 2003 for details The range is from 0 0 to 1 0 where a score of 1 0 is given to the exact match and 0 0 will match anything e Use cutoffs from selected profile The cutoffs that were defined in the profile will be used Use only high quality matrices When enabled this will exclude highly abundant matrices which produce at minSUM see below more than 10 hits false positives per 1000 nucleotides 3 high quality matrices are defined as matrices producing less than 10 hits FP per 1000 nucleotides in sequences 10 000 to 5 000 nucleotides upstream of the transcription start sites at minSUM About 5 of the current matrices producing higher FP rate can be excluded as highly abundant low quality these 5 of matrices produce about 50 of all FP hits Chapter 4 Output of TRANSFAC TFBS Plugin Clicking Next allows you to specify the output options as shown in figure 4 1 Biobase Transfac free x Result handling 1 Select input 2 Configure TRANSFAC 3 Result handling Output options y Create annotations _ Create an output table D esult handling D Open Save Lo
3. C consensus is provided in the last column Click here for more information about the Matrix block The nucleotide frequency matrix for the reverse complement is displayed to the right e Transcription factors Lists the transcription factors from which binding sites were used for matrix construction hyperlinked to corresponding Locus Reports The count of binding sites contributed to the calculation of the positional weight matrix by each factor is displayed in the bar graph e Aligned Binding Sites Lists the transcription factor binding sites used to generate the matrix with links to the corresponding Site Reports Includes the part of the sequence within the matrix window the start of the matrix window and the displayed sequence relative to the complete sequence as given in the Site Report The aligned binding sites are not given for all matrices This depends on the availability of the sites e g not for all SELEX matrices the sites have been published Also for ChIP based matrices no site alignments are given The following information is provided for each site when available the transcription factor hyperlinked to the corresponding Locus Report the gene from which the binding site was derived hyperlinked to the corresponding Locus Report a graphical summary of the CHAPTER 4 OUTPUT OF TRANSFAC TFBS PLUGIN 12 experimental evidence supporting the TF DNA binding interaction hyperlinked to the detailed Site Report the experi
4. Q TRANSFAC Plugin USER MANUAL User manual for TRANSFAC TFBS Plugin 1 1 Windows Mac OS X and Linux July 8 2015 This software is for research purposes only CLC bio a QIAGEN Company Silkeborgvej 2 Prismet DK 8000 Aarhus C Denmark LC bio AQIAGEN Company Contents 1 Introduction to the TRANSFAC TFBS Plugin 4 2 Installation of the TRANSFAC TFBS Plugin 5 3 Searching for transcription factor binding sites 6 4 Output of TRANSFAC TFBS Plugin 9 DTM OUTOUT TABI a a A ew ee Oe i ai e a 9 4 2 Matrix REPOS ase as et we se ce em a et ee a eR ew Be ws Oe Gre 10 Bibliography 14 Chapter 1 Introduction to the TRANSFAC TFBS Plugin This manual is for the commercial non free version of the TRANSFAC plugin The differences from the free version of the plugin are that it bundles all data necessary and should work out of the box without any additional configuration The TRANSFAC plugin can be used to search for putative transcription factor binding sites in DNA sequences The binding site predictions are done by the Match tool which uses the positional weight matrix library from TRANSFAC to analyze your sequences For each of these matrices Match contains optimized parameters for minimization of error rates false negative rate and false positive rate As a special feature Match provides the option to use profiles that are specific subsets of matrices with optimized cut offs These profiles allow users
5. available e Related family matrices Lists related matrices that are generally applicable to a family of transcription factors to which the factors used to construct this positional weight matrix belong e Related factor specific matrices Lists other matrices that have been constructed using the factors used to construct this positional weight matrix Identifiers Displays the identifiers associated with the matrix e BKL Accessions Lists the identifier for the matrix of the page e Similar matrix in Lists closely related matrices in public databases including JASPAR and UnIPROBE References Lists references from which whole sets of sequences or complete matrix was derived including for the individual binding site sequences Matrices constructed by us typically have a link to a TRANSFAC Report which describes the way we used for site alignment and matrix construction This block gives the full citations with titles that correspond to the PubMed identifiers displayed When a PubMed identifier is not available the Medline identifier is displayed When neither a PubMed nor a Medline identifier is available a BIOBASE specific number is assigned preceded by a P Nearly all of the reference numbers are hyperlinked to the PubMed database where the abstracts may be read All references cited in the annotations and properties section are listed and other references known to contain information about the protein may also be listed The fi
6. g handling Open log A Previous gt Next X Cancel Figure 4 1 Selecting profiles and settings Based on the settings in figure 3 2 the Workbench uses the Match program from BIOBASE to search for transcription factor binding sites The binding sites can be reported as annotations that are either added to the input sequences or reported as a track of annotations depending on the analysis type Classic or Genomic see Chapter 3 and or as an output table as shown in figure 4 4 4 1 Output table The table includes the following information e Matrix ID Identifier for the matrix with which the putative binding site was found e Factor name Name of the binding factor represented by the matrix If a group of factors is assigned to a matrix only representative factor s are given For a complete list of linked binding factors please see the Matrix Report in TRANSFAC 9 CHAPTER 4 OUTPUT OF TRANSFAC TFBS PLUGIN 10 E bxtracted ann x Extracted annotations subset TRANSFAC table CLC Genomics Workbench 7 9 2 Beta 1 Transcription factor binding sites BREA Bower 15s Profile vertebrate_non_redundant_minFP Liter column width Manual e MatrixiD Factor name Region Strand Match sequence V GRE_C GR complement 18 33 minus cAGGACaaagcagect Show column V RFX1_O1 RDA 34 50 plus ttggagecet t Z Matri ID V MUSCLEINI_B Muscle initiator 93 113 plus gctggacagCAccCa
7. gegcct i 26 32 plus AGCA ld Factor name DBP 67 73 plus Region DBP 70 76 plus DBP 100 106 plus mi Strand Pax 42 52 plus Y Match sequence V Pax complement 51 61 minus ar Vra oe Pax 60 78 plus Core similarity V PAX_6 Pax complement 79 89 minus ctcaggCCCAG Z Matrix similarity V PAX_Q6 Pax 94 104 plus CTGGAcagcac I Matrix report 06 Pax complement 110 120 minus gectecTCCAG 7 Pa complement 145 155 minus ttcactTTCAG Select All v Pax 157 167 plus CCGG Deselect All Pa complement 159 169 minus lea VS Pax complement 170 180 minus LKR PXR CAR COUP RAR complement 118 134 minu Churchill 158 163 plus Helios A 16 26 plus Helios A complement 112 122 minu V HELIOSA Helios A complement 121 131 minu V AP2ALPHA_03 AP 2alphaA 27 41 plus V AP2ALPHA_03 AP 2alphaA complement 27 41 minus V AP2ALPHA_03 AP 2alphaa 6 plus V AP2ALPHA 03 AP 2alphaA complement 68 82 minus V AP2ALPHA03 AP 2alphaA 74 88 plus V AP2ALPHA 03 A complement 74 88 minus V AP2ALPHA 03 plus V AP2ALPHA 03 complement 80 94 minus V CTCF_01 complement 106 125 minu V BEN_01 complement 13 20 minus V BEN_01 106 113 plus V NANOG_02 at 14 33 plus aggACA Agcagect V SIX1_01 be complement 47 63 minu Gtecagag v sba_01 S 56 72 plus gtccagaGATCAgcage V SIX1_01 Shel 155 171 plus gecegggAATcTetgtg V HOXC13_01 HOXC1 complement 44 59 minus ggaaCTTAAgatgtcc V HDX_01 Hdx 18 34 plus caggacaAAGcAagectt IV HDX_01 Hdx complement 47 63 min
8. ing the administrator privileges you were using so that you could install the plugin tin order to install plugins the Workbench must be run with administrator privileges 5 Chapter 3 Searching for transcription factor binding sites Once the plugin is installed you can start searching for transcription factor binding sites Depending on the workbench product you have installed Toolbox Epigenomics Analyses k Transfac TFBS A This opens the wizard the first step shown in figure 3 1 and explained below ES Biobase Transfac free ae 3 Select input 1 Select input Type of analysis Classic 2 Genomic Genomic regions and reference selection Sequence selection DNA sequences Dp Inconsistent input Please provide sorne sequences 2 S X cancel Figure 3 1 Select type of analysis and input data to search for transcription factor binding sites The first wizard step allows you to specify the input data Type of analysis First you need to select between the two modes of the analysis each explained below e Classic This is the legacy mode of analysis where you have raw sequences without genomic information and wish to search for transcription factor binding sites in these 1In the CLC Main Workbench the Transfac TFBS tool is found here Toolbox in the Menu Bar Nucleotide Sequence Analyses A Transfac TFBS Ay 6 CHAPTER 3 SEARCHING FOR TRANSCRIPTION FACTOR BINDING SITES 7 sequences
9. iption of what the matrix report contains You can Export the table in csv or Excel format 4 2 Matrix Reports Matrix reports can be accessed by clicking the link in an entry in the Matrix Report column of the output table It will load a html page in the default web browser of your system see figure 4 3 In the following we will describe the sections that exists in a Matrix Report CHAPTER 4 OUTPUT OF TRANSFAC TFBS PLUGIN 11 BIO BASE P DOF1_01 Dof1 Table of Contents y Matrix Overview unat is tis Consensus sequence logo __xAAAG_ a RERCIIIAHES neces Nucleotide position frequency S Nucleotide position frequency c l 2 3 wot ft oN N D gt Hf eo eo oNW DN O g Boe ous a 4 Y Nbweo oO oO ou NNSA Boe oR ACC FC OOM HHO N zzzarr gt re 22 gt NNULCSC CO OU ND nNunoooo uuaoooooamas a o xn a 4228545407222 Figure 4 3 A Matrix Report in a browser window Positional Weight Matrix Describes the characteristics of the matrix Matrix overview e Sequence logo A matrix logo which displays the consensus sequence graphically is shown The reverse complement consensus sequence is displayed to the right ca as cIAGG_cAsAGGTcA Figure 4 4 Example matrix logo e Nucleotide position frequency Displays the nucleotide frequency matrix Read down to identify the binding site and across to identify the nucleotide frequency at that particular binding site position The derived IUPA
10. mental source and the references supporting the TF DNA binding interaction The experimental evidence categories are organized as CI chromatin immunoprecipitation DM DNA modification methylation etc FA functional analysis FO footprinting GS simple gel shift gel retardation IP immunoprecipitation SE SELEX SS supershift competitive gel shift OT other All experimental methods that do not fall into one of the other categories will be assigned to the OT other category e Matrix type Specifies whether a matrix is specific for a factor or more generally representative of a family of related factors e Matrix classification For matrices built from vertebrate transcription factors specifies the class that the matrix has been assigned to based on a matrix clustering algorithm submitted for publication As of the 2012 3 release there are 44 classes AP2 EREBP ARID ATHOOK BHLH BHSH BZIP CHCH CU FIST DM E2 E2F ETS FORKHEAD GCM GENINI GRAINY HISTONE HMG HOX HSF IRF MADS MYB NAM P53 REL RFX RUNT SAND SMAD STAT SWI4 TBP TBX TCP TEA WRKY ZFC2H2 ZFC4 NR ZFC6 ZFDOF ZFGATA ZFPHD ZFRING Matrices that do not fall into one of these classes are classified as unclassified e Matrix category Describes which method was used to create the matrix Possible methods include matrix compiled from individual genomic sites SELEX CASTing SAAB Target Detection Assay and more e Applica
11. menu lets you configure the cut off to use for the selected set of matrices the options are e Minimize false positives minFP The minFP cutoff can be used to reduce the number of false positives The false positive rate is estimated by applying the Match algorithm to upstream sequences The minFP cut off is defined as the score that gives one percent of hits in the used sequences relative to the number of hits received at the minFN cut off For example if you have done peak detection from a ChIP seq analysis the regions where peaks are found then are the genomic regions you wish to investigate further CHAPTER 3 SEARCHING FOR TRANSCRIPTION FACTOR BINDING SITES 8 e Minimize false negatives minFN The minFN cut off can be used to reduce the number of false negatives The false negative rate is measured as far as available on known genomic binding sites for the transcription factors In case not sufficient less than 10 genomic binding sites are available SELEX sites or sets of oligonucleotides based on the nucleotide distribution in the weight matrix are used for estimating the minFN cut off The minFN cut off is defined as that score at which at least 90 of the positive test set are recognized i e it equals a false negative rate of 10 e Minimize the sum of both error rates minSUM The minSUM cut off can be used to minimize the sum of both error rates The sum of corresponding percentages for false positives and false
12. rst five references are shown click on the button labeled more to view all references Bibliography Kel et al 2003 Kel A E ling E G Reuter l Cheremushkin E Kel Margoulis O V and Wingender E 2003 MATCHTM a tool for searching transcription factor binding sites in DNA sequences Nucleic Acids Research 31 13 3576 3579 14
13. tion details Provides additional information about the experimental source of the binding sites used for matrix construction the experimental approach applied to obtain this set etc e Number of sequences used Provides information about the number of binding sites used for matrix construction e Additional transcription factors linked to the matrix Lists those transcription factors which did not contribute binding sites to the construction of the matrix but which are linked to the matrix usually through homology e Profile Membership Displays information about the profiles that the matrix is a part of e Profiles which include this matrix When relevant lists whether the matrix of the page is part of the vertebrate non redundant VNR or other profile for use in Match analysis e Other vertebrate non redundant VNR matrices that this matrix represents When relevant lists the group of matrices that the matrix of the page represents in the vertebrate non redundant profile VNR for use in Match analysis CHAPTER 4 OUTPUT OF TRANSFAC TFBS PLUGIN 13 Related matrices Displays related matrices e This matrix is an older version of When relevant lists any newer versions of this matrix such as a version that was created after additional binding sites became available e This matrix is a newer version of When relevant lists any older versions of this matrix such as a version that was created before additional binding sites became
14. to adapt Match to their specific interests For example promoters of a certain tissue can be searched with tissue specific profiles In order to be able to use this TRANSFAC plugin you need to install the TRANSFAC plugin into your Workbench We cover the above topics first in this manual and following that provide information on carrying out searches of TRANSFAC via the CLC Workbench TRANSFAC is a registered trademark of BIOBASE Chapter 2 Installation of the TRANSFAC TFBS Plugin The TRANSFAC TFBS Plugin is installed as a plugin Plugins are installed using the plugin manager Help in the Menu Bar Plugins and Resources E or Plugins E in the Toolbar The plugin manager has three tabs at the top e Manage Plugins This is an overview of plugins that are installed e Download Plugins This is an overview of available plugins on CLC bio s server e Manage Resources This is an overview of resources that are installed Install the plugin by clicking the Download Plugins button at the top of the dialog This will open a dialog where you can browse for the plugin The plugin is called TRANSFAC TFBS When you close the dialog you will be asked whether you wish to restart the CLC Workbench The plugin will not be ready for use before you have restarted Note that you may wish to click on the button labelled no and restart the Workbench yourself If you choose yes at this point the Workbench restarts us
15. us acttaAGATI V HDX_01 Hdx 56 72 plus gtecal 9 V HDX_01 Hdx 155 171 plus gecegggAATCTetgi V HDX_01 Hdx complement 165 181 minu ctetgTGAAGtcctage V RHOX11_01 Rhoxl1 48 64 plus cttaaGATGTecagaga V RHOX11_01 Rhox11 88 104 plus aggcaGCTGGacagcac gt Beg z as Idle 1 row selected Figure 4 2 Result table e Region Position of the matrix match putative binding site within the analyzed sequence e Strand Plus minus The strand on which the putative site was found depends on the orientation in which the matrix is given in TRANSFAC e Match sequence Shows the matching sequence Capital letters indicate the positions in the sequence that match with the core sequence of the matrix while the lower case letters refer to positions that match to the remaining part of the matrix e Core similarity The core similarity score for the matrix match The matrix core is defined as the five consecutive most conserved nucleotides within the matrix e Matrix similarity The matrix similarity score for the matrix match The Match score can vary from O to 1 with O for the lowest similarity and 1 for the highest similarity of the match to the matrix Only those matches are listed in the result for which the core and matrix similarity are higher than the chosen cut offs e Matrix Report Matrix accession with a hyperlink to a detailed build in matrix report that will open in a default browser on your system See Section 4 2 for a detailed descr
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