Mercurial > repos > bgruening > diffbind
diff diffbind.xml @ 23:393393c58c35 draft
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit cc4c1c4131518b9cbf986a1f252767ff73ca938e
| author | iuc |
|---|---|
| date | Sat, 07 Apr 2018 15:45:03 -0400 |
| parents | 51f0f4df83c2 |
| children | 15bbd86c6c7b |
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--- a/diffbind.xml Sun Jan 28 05:10:06 2018 -0500 +++ b/diffbind.xml Sat Apr 07 15:45:03 2018 -0400 @@ -1,10 +1,9 @@ -<tool id="diffbind" name="DiffBind" version="2.6.5.0"> +<tool id="diffbind" name="DiffBind" version="2.6.6.1"> <description> differential binding analysis of ChIP-Seq peak data</description> <requirements> - <requirement type="package" version="2.6.5">bioconductor-diffbind</requirement> + <requirement type="package" version="2.6.6">bioconductor-diffbind</requirement> <requirement type="package" version="1.20.0">r-getopt</requirement> - <!--added rmysql requirement to remove: "Warning: namespace ‘RMySQL’ is not available"--> - <requirement type="package" version="0.10.11">r-rmysql</requirement> + <requirement type="package" version="0.2.15">r-rjson</requirement> </requirements> <stdio> <regex match="Execution halted" @@ -21,130 +20,180 @@ description="An undefined error occured, please check your intput carefully and contact your administrator." /> </stdio> <version_command><![CDATA[ -echo $(R --version | grep version | grep -v GNU)", DiffBind version" $(R --vanilla --slave -e "library(DiffBind); cat(sessionInfo()\$otherPkgs\$DiffBind\$Version)" 2> /dev/null | grep -v -i "WARNING: ")," getopt version" $(R --vanilla --slave -e "library(getopt); cat(sessionInfo()\$otherPkgs\$getopt\$Version)" 2> /dev/null | grep -v -i "WARNING: ")", rmysql version" $(R --vanilla --slave -e "library(rmysql); cat(sessionInfo()\$otherPkgs\$rmysql\$Version)" 2> /dev/null | grep -v -i "WARNING: ") +echo $(R --version | grep version | grep -v GNU)", DiffBind version" $(R --vanilla --slave -e "library(DiffBind); cat(sessionInfo()\$otherPkgs\$DiffBind\$Version)" 2> /dev/null | grep -v -i "WARNING: ")", rjson version" $(R --vanilla --slave -e "library(rjson); cat(sessionInfo()\$otherPkgs\$rjson\$Version)" 2> /dev/null | grep -v -i "WARNING: ") ]]></version_command> <command><![CDATA[ - ## seems that diffbind also needs file extensions to work properly - #set $counter = 1 - #for $sample in $samples: - ln -s $sample.bamreads #echo str($counter) + "_bamreads.bam"# && - ln -s ${sample.bamreads.metadata.bam_index} #echo str($counter) + "_bamreads.bai"# && - #if str( $sample.bamcontrol ) != 'None': - ln -s $sample.bamcontrol #echo str($counter) + "_bamcontrol.bam"# && - ln -s ${sample.bamcontrol.metadata.bam_index} #echo str($counter) + "_bamcontrol.bai"# && - #end if - #set $counter = $counter + 1 +#import re +#import json + +## Adapted from DESeq2 wrapper +#set $temp_factor_names = list() +#set $temp_factor = list() + +#for $g in $rep_group: + + #set $peak_files = list() + #set $bam_files = list() + #set $bam_controls = list() + + #for $file in $g.peaks: + #set $file_name = re.sub('[^\w\-\s]', '_', str($file.element_identifier)) + ln -s '${file}' ${g.groupName}-${file_name}-peaks.bed && + $peak_files.append(str($g.groupName) + '-' + $file_name + '-peaks.bed') + #end for + + #for $bam in $g.bamreads: + #set $bam_name = re.sub('[^\w\-\s]', '_', str($bam.element_identifier)) + ln -s '${bam}' ${bam_name}-bamreads.bam && + ln -s ${bam.metadata.bam_index} ${bam_name}-bamreads.bai && + $bam_files.append($bam_name + '-bamreads.bam') + #end for + + $temp_factor.append( {str($g.groupName): $peak_files} ) + $temp_factor.append( {str($g.groupName): $bam_files} ) + + #if str( $g.bamcontrol ) != 'None': + #for $ctrl in $g.bamcontrol: + #set $ctrl_name = re.sub('[^\w\-\s]', '_', str($ctrl.element_identifier)) + ln -s '${ctrl}' ${g.groupName}-${ctrl_name}-bamcontrol.bam && + ln -s ${ctrl.metadata.bam_index} ${g.groupName}-${ctrl_name}-bamcontrol.bai && + $bam_controls.append(str($g.groupName) + '-' + $ctrl_name + '-bamcontrol.bam') #end for + $temp_factor.append( {str($g.groupName): $bam_controls} ) + #end if - Rscript '$__tool_directory__/diffbind.R' - -i $infile - -o '$outfile' - -p '$plots' - -f $format - -t $th +#end for + +$temp_factor.reverse() +$temp_factor_names.append([str($factorName), $temp_factor]) + + +Rscript '$__tool_directory__/diffbind.R' + + -i '#echo json.dumps(temp_factor_names)#' + -o '$outfile' + -t $th + -f $out.format + -p '$plots' - #if $binding_affinity_matrix: - -b - #end if + #if $scorecol: + -n "$scorecol" + #end if + #if $lowerbetter: + -l "$lowerbetter" + #end if + #if $summits: + -s "$summits" + #end if + + #if $out.binding_matrix: + -b + #end if + + #if $out.rdata: + -r + #end if + + #if $out.analysis_info: + -a + #end if + + #if $out.rscript: + && cp '$__tool_directory__/diffbind.R' '$rscript' + #end if ]]> </command> - <configfiles> -<configfile name="infile"><![CDATA[ -#set $counter = 1 -#for $sample in $samples: -#if str( $sample.bamcontrol ) != 'None' and $counter == 1: -SampleID,Tissue,Factor,Condition,Replicate,bamReads,bamControl,Peaks -#elif $counter == 1: -SampleID,Tissue,Factor,Condition,Replicate,bamReads,Peaks -#end if -#if str( $sample.bamcontrol ) != 'None': -$sample.sample_id,$sample.tissue,$sample.factor,$sample.condition,$sample.replicate,#echo str($counter) + '_bamreads.bam'#,#echo str($counter) + '_bamcontrol.bam'#,$sample.peaks -#else: -$sample.sample_id,$sample.tissue,$sample.factor,$sample.condition,$sample.replicate,#echo str($counter) + '_bamreads.bam'#,$sample.peaks -#end if -#set $counter = $counter + 1 -#end for]]></configfile> - </configfiles> <inputs> - <repeat name="samples" title="Samples" min="2"> - <param name="sample_id" type="text" value="Sample ID" label="Specify a sample id" help="e.g. BT474.1-" /> - <param name="tissue" type="text" value="Tissue" label="Specify the tissue" help="e.g. BT474" /> - <param name="factor" type="text" value="Factor Name" label="Specify a factor name" help="e.g. ER" /> - <param name="condition" type="text" value="Condition" label="Specify the condition" help="e.g. Resistent" /> - <param name="replicate" type="integer" value="1" label="Specify the replicate number" help="e.g. 1" /> - <param name="bamreads" type="data" format="bam" label="Read BAM file" help="Specify the Read BAM file, used for Peak calling."/> - <param name="bamcontrol" type="data" format="bam" optional="True" label="Control BAM file" help="If specifying a control BAM file for this sample, then all samples are required to specify one."/> - <param name="peaks" type="data" format="bed" label="Peak file" help="Result of your Peak calling experiment."/> + <param name="factorName" type="text" label="Name" help="Name of experiment factor of interest (e.g. Condition). One factor must be entered and there must be two or more groups. NOTE: Please only use letters, numbers or underscores."> + <sanitizer> + <valid initial="string.letters,string.digits"><add value="_" /></valid> + </sanitizer> + </param> + <repeat name="rep_group" title="Group" min="2" default="2"> + <param name="groupName" type="text" label="Name" + help="Name of group that the peak files belong to (e.g. Resistant or Responsive). NOTE: Please only use letters, numbers or underscores (case sensitive)."> + <sanitizer> + <valid initial="string.letters,string.digits"><add value="_" /></valid> + </sanitizer> + </param> + <param name="peaks" type="data" format="bed" multiple="true" label="Peak files" help="Result of your Peak calling experiment"/> + <param name="bamreads" type="data" format="bam" multiple="true" label="Read BAM file" help="Specify the Read BAM file used for Peak calling."/> + <param name="bamcontrol" type="data" format="bam" multiple="true" optional="True" label="Control BAM file" help="If specifying a control BAM file, all samples are required to specify one."/> </repeat> - <param name="th" type="float" value="1" min="0" max="1" - label="FDR Threshold" - help="Significance threshold; all sites with FDR less than or equal to this value will be included in the report. A value of 1 will include all binding sites in the report. Default: 1"/> - <param name="pdf" type="boolean" truevalue="" falsevalue="" checked="true" - label="Visualising the analysis results" - help="output an additional PDF file" /> - <param name="format" type="select" label="Output Format"> - <option value="bed">BED</option> - <option value="gff">GFF</option> - <option value="wig">WIG</option> - </param> - <param name="binding_affinity_matrix" type="boolean" truevalue="True" falsevalue="" checked="False" label="Output binding affinity matrix?" help="Output a table of the binding scores" /> + + <param name="scorecol" type="integer" min="0" value="8" label="Score Column" help="Column in peak files that contains peak scores. Default: 8 (narrowPeak)"/> + <param name="lowerbetter" type="boolean" truevalue="True" falsevalue="" checked="False" label="Lower score is better?" help="DiffBind by default assumes that a higher score indicates a better peak, for example narrowPeaks -log10pvalue. If this is not the case, for example if the score is a p-value or FDR, set this option to Yes. Default: No" /> + <param name="summits" type="integer" min="0" optional="True" label="Summits" help="Extend peaks Nbp up- and downstream of the summit. For punctate peaks it is advisable to extend (e.g. 250bp), see the DiffBind User Guide"/> + <param name="th" type="float" value="0.05" min="0" max="1" label="FDR Threshold" help="Significance threshold; all sites with FDR less than or equal to this value will be included in the output. A value of 1 will output all binding sites. Default: 0.05"/> + + <!-- Output Options --> + <section name="out" expanded="false" title="Output Options"> + <param name="format" type="select" label="Output Format"> + <option value="bed">BED</option> + <option value="gff">GFF</option> + <option value="wig">WIG</option> + </param> + <param name="pdf" type="boolean" truevalue="True" falsevalue="" checked="False" label="Visualising the analysis results" help="output an additional PDF file" /> + <param name="binding_matrix" type="boolean" truevalue="True" falsevalue="" checked="False" label="Output binding affinity matrix?" help="Output a table of the binding scores" /> + <param name="rdata" type="boolean" truevalue="True" falsevalue="" checked="False" label="Output RData file?" help="Output all the data used by R to construct the plots and tables, can be loaded into R. Default: No"/> + <param name="rscript" type="boolean" truevalue="True" falsevalue="False" checked="False" label="Output Rscript?" help="If this option is set to Yes, the Rscript used will be provided as a text file in the output. Default: No"/> + <param name="analysis_info" type="boolean" truevalue="True" falsevalue="False" checked="False" label="Output analysis info?" help="If this option is set to Yes, information from the dba.count and dba.analyze commmands will be output in a text file. Default: No"/> + </section> </inputs> + <outputs> - <data name="outfile" format="bed" label="Differential binding sites on ${on_string}"> + <data name="outfile" format="bed" label="${tool.name} on ${on_string}: Differentially bound sites"> <change_format> <when input="format" value="wig" format="wig" /> <when input="format" value="gff" format="gff" /> </change_format> </data> - <data name="plots" format="pdf" label="Differential binding sites on ${on_string}"> - <filter>pdf == True</filter> + <data name="plots" format="pdf" label="${tool.name} on ${on_string}: Plots"> + <filter>out['pdf']</filter> + </data> + <data name="binding_matrix" format="tabular" from_work_dir="bmatrix.tab" label="${tool.name} on ${on_string}: Binding matrix"> + <filter>out['binding_matrix']</filter> </data> - <data name="binding_matrix" format="tabular" from_work_dir="bmatrix.tab" label="Differential binding sites on ${on_string}"> - <filter>binding_affinity_matrix == True</filter> + <data name="rdata" format="rdata" from_work_dir="DiffBind_analysis.RData" label="${tool.name} on ${on_string}: RData file"> + <filter>out['rdata']</filter> + </data> + <data name="rscript" format="txt" label="${tool.name} on ${on_string}: Rscript"> + <filter>out['rscript']</filter> + </data> + <data name="analysis_info" format="txt" from_work_dir="DiffBind_analysis_info.txt" label="${tool.name} on ${on_string}: Analysis info"> + <filter>out['analysis_info']</filter> </data> </outputs> + <tests> - <test> - <repeat name="samples"> - <param name="sample_id" value="BT4741" /> - <param name="tissue" value="BT474" /> - <param name="factor" value="ER" /> - <param name="condition" value="Resistant" /> - <param name="replicate" value="1" /> - <param name="bamreads" ftype="bam" value="BT474_ER_1.bam" /> - <param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz" /> + <test expect_num_outputs="6"> + <param name="factorName" value="Condition"/> + <repeat name="rep_group"> + <param name="groupName" value="Resistant"/> + <param name="peaks" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/> + <param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" /> </repeat> - <repeat name="samples"> - <param name="sample_id" value="BT4742" /> - <param name="tissue" value="BT474" /> - <param name="factor" value="ER" /> - <param name="condition" value="Resistant" /> - <param name="replicate" value="2" /> - <param name="bamreads" ftype="bam" value="BT474_ER_2.bam" /> - <param name="peaks" ftype="bed" value="BT474_ER_2.bed.gz" /> + <repeat name="rep_group"> + <param name="groupName" value="Responsive"/> + <param name="peaks" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/> + <param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" /> </repeat> - <repeat name="samples"> - <param name="sample_id" value="MCF71" /> - <param name="tissue" value="MCF7" /> - <param name="factor" value="ER" /> - <param name="condition" value="Responsive" /> - <param name="replicate" value="1" /> - <param name="bamreads" ftype="bam" value="MCF7_ER_1.bam" /> - <param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz" /> - </repeat> - <repeat name="samples"> - <param name="sample_id" value="MCF72" /> - <param name="tissue" value="MCF7" /> - <param name="factor" value="ER" /> - <param name="condition" value="Responsive" /> - <param name="replicate" value="2" /> - <param name="bamreads" ftype="bam" value="MCF7_ER_2.bam" /> - <param name="peaks" ftype="bed" value="MCF7_ER_2.bed.gz" /> - </repeat> + <param name="scorecol" value="5" /> <param name="pdf" value="True" /> - <param name="binding_affinity_matrix" value="True" /> + <param name="binding_matrix" value="True" /> + <param name="rdata" value="True" /> + <param name="rscript" value="True"/> + <param name="analysis_info" value="True"/> <output name="outfile" value="out_diffbind.bed" /> + <output name="plots" value="out_plots.pdf" compare="sim_size" /> <output name="binding_matrix" value="out_binding.matrix" /> + <output name="rdata" value="DiffBind_analysis.RData" compare="sim_size"/> + <output name="rscript" value="out_rscript.txt"/> + <output name="analysis_info" value="out_analysis_info.txt" compare="sim_size" > + <assert_contents> + <has_text text="SessionInfo"/> + </assert_contents> + </output> </test> </tests> <help><![CDATA[ @@ -166,7 +215,7 @@ in peak sets, and identifying statistically significantly differentially bound sites based on evidence of binding affinity (measured by differences in read densities). To this end it uses statistical routines developed in an RNA-Seq context (primarily the Bioconductor packages -edgeR and DESeq2 ). Additionally, the package builds on Rgraphics routines to provide a +edgeR and DESeq2). Additionally, the package builds on Rgraphics routines to provide a set of standardized plots to aid in binding analysis. The `DiffBind User Guide`_ includes a brief overview of the processing flow, followed by four sections of @@ -176,11 +225,9 @@ as well as comparing the results of an occupancy-based analysis with an affinity-based one. Finally, certain technical aspects of the how these analyses are accomplished are detailed. -Note DiffBind requires a minimum of four samples (two groups with two replicates each). +Note this DiffBind tool requires a minimum of four samples (two groups with two replicates each). -.. _DiffBind: https://bioconductor.org/packages/release/bioc/html/DiffBind.html -.. _`Bioconductor package`: https://bioconductor.org/packages/release/bioc/html/DiffBind.html -.. _`DiffBind User Guide`: https://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf +----- **Inputs** @@ -194,56 +241,29 @@ **Sample Information** -You have to specify your sample information in the tool form above. +You have to specify your sample information in the tool form above, where Factor is the groups you want to compare (e.g Resistant and Responsive). Example: - ============= ========== ========== ============= ============= - **SampleID** **Tissue** **Factor** **Condition** **Replicate** - ------------- ---------- ---------- ------------- ------------- - BT4741 BT474 ER Resistant 1 - BT4742 BT474 ER Resistant 2 - MCF71 MCF7 ER Responsive 1 - MCF72 MCF7 ER Responsive 2 - MCF73 MCF7 ER Responsive 3 - T47D1 T47D ER Responsive 1 - T47D2 T47D ER Responsive 2 - MCF7r1 MCF7 ER Resistant 1 - MCF7r2 MCF7 ER Resistant 2 - ZR751 ZR75 ER Responsive 1 - ZR752 ZR75 ER Responsive 2 - ============= ========== ========== ============= ============= - -Or provide a sample sheet tabular file such as below. - -Example: - - ======== ====== ====== ========== ========== ========= ==================== ========= ===================== ================= ========== - SampleID Tissue Factor Condition Treatment Replicate bamReads ControlID bamControl Peaks PeakCaller - ======== ====== ====== ========== ========== ========= ==================== ========= ===================== ================= ========== - BT4741 BT474 ER Resistant Full-Media 1 Chr18_BT474_ER_1.bam BT474c Chr18_BT474_input.bam BT474_ER_1.bed.gz bed - BT4742 BT474 ER Resistant Full-Media 2 Chr18_BT474_ER_2.bam BT474c Chr18_BT474_input.bam BT474_ER_2.bed.gz bed - MCF71 MCF7 ER Responsive Full-Media 1 Chr18_MCF7_ER_1.bam MCF7c Chr18_MCF7_input.bam MCF7_ER_1.bed.gz bed - MCF72 MCF7 ER Responsive Full-Media 2 Chr18_MCF7_ER_2.bam MCF7c Chr18_MCF7_input.bam MCF7_ER_2.bed.gz bed - MCF73 MCF7 ER Responsive Full-Media 3 Chr18_MCF7_ER_3.bam MCF7c Chr18_MCF7_input.bam MCF7_ER_3.bed.gz bed - T47D1 T47D ER Responsive Full-Media 1 Chr18_T47D_ER_1.bam T47Dc Chr18_T47D_input.bam T47D_ER_1.bed.gz bed - T47D2 T47D ER Responsive Full-Media 2 Chr18_T47D_ER_2.bam T47Dc Chr18_T47D_input.bam T47D_ER_2.bed.gz bed - MCF7r1 MCF7 ER Resistant Full-Media 1 Chr18_TAMR_ER_1.bam TAMRc Chr18_TAMR_input.bam TAMR_ER_1.bed.gz bed - MCF7r2 MCF7 ER Resistant Full-Media 2 Chr18_TAMR_ER_2.bam TAMRc Chr18_TAMR_input.bam TAMR_ER_2.bed.gz bed - ZR751 ZR75 ER Responsive Full-Media 1 Chr18_ZR75_ER_1.bam ZR75c Chr18_ZR75_input.bam ZR75_ER_1.bed.gz bed - ZR752 ZR75 ER Responsive Full-Media 2 Chr18_ZR75_ER_2.bam ZR75c Chr18_ZR75_input.bam ZR75_ER_2.bed.gz bed - ======== ====== ====== ========== ========== ========= ==================== ========= ===================== ================= ========== + ============= ============= + **SampleID** **Group** + ------------- ------------- + BT4741 Resistant + BT4742 Resistant + MCF71 Responsive + MCF72 Responsive + ============= ============= **Peak files** -Result of your Peak calling experiment in bed format, one file for each sample is required. +Result of your Peak calling experiment in bed format, one file for each sample is required. The peak caller, format and score column can be specified in the tool form above. The default settings expect narrowPeak bed format, which has the score in the 8th column (-log10pvalue), and can be output from MACS2. -Example: +Example (MACS.xls file in bed format): - ======= ======= ======= =============== ======= - 1 2 3 4 **5** - ======= ======= ======= =============== ======= + ======= ======= ======= =============== ============== + 1 2 3 4 **5 (Score)** + ======= ======= ======= =============== ============== chr18 215562 216063 MACS_peak_16037 56.11 chr18 311530 312105 MACS_peak_16038 222.49 chr18 356656 357315 MACS_peak_16039 92.06 @@ -254,42 +274,56 @@ chr18 503518 504552 MACS_peak_16044 818.30 chr18 531672 532274 MACS_peak_16045 159.30 chr18 568326 569282 MACS_peak_16046 601.11 - ======= ======= ======= =============== ======= + ======= ======= ======= =============== ============== -* BAM file which contains the mapped sequencing reads can be associated with each peakset -* Control BAM file represents a control dataset and are optional, but have to specified for all when used. +* BAM file which contains the mapped sequencing reads associated with each peakset, one file for each sample is required. +* Optional: Control BAM file representing a control dataset. If used, has to be specified for all samples. Note that the DiffBind authors say control reads are best utilized prior to running DiffBind, at the peak calling stage (e.g. with MACS2) and in blacklists, see this `Bioconductor post`_. +----- **Outputs** +This tool outputs + + * differentially bound sites in BED, WIG or GFF format + +Optionally, under **Output Options** you can choose to output + + * a PDF of plots (Heatmap, PCA, MA, Volcano, Boxplots) + * a binding affinity matrix + * the R script used by this tool + * an RData file of the R objects generated + * a text file with information on the analysis (number of Intervals, FriP scores, method used) + +**Differentially Bound Sites** + As output format you can choose BED, GFF, WIG. -Example: - -======== ====== =======+ -seqnames ranges strand Conc Conc_Resistant +Example - BED format: -2452 chr18 [64490686, 64491186] * | 6.36 1.39 -1291 chr18 [34597713, 34598213] * | 5.33 0.22 -976 chr18 [26860997, 26861497] * | 7.3 3.13 -2338 chr18 [60892900, 60893400] * | 7.13 1.84 -2077 chr18 [55569087, 55569587] * | 5.52 1.89 + ======== ====== ====== ===== ====== ===== =============== ============== ======= ======== ======== + seqnames start end width strand Conc Conc_Responsive Conc_Resistant Fold p.value **FDR** + ======== ====== ====== ===== ====== ===== =============== ============== ======= ======== ======== + chr18 394600 396513 1914 * 7.15 5.55 7.89 -2.35 7.06e-24 9.84e-21 + chr18 111567 112005 439 * 5.71 6.53 3.63 2.89 1.27e-08 8.88e-06 + chr18 346464 347342 879 * 5 5.77 3.24 2.52 6.51e-06 0.00303 + chr18 399014 400382 1369 * 7.62 7 8.05 -1.04 1.04e-05 0.00364 + chr18 371110 372102 993 * 4.63 3.07 5.36 -2.3 8.1e-05 0.0226 + ======== ====== ====== ===== ====== ===== =============== ============== ======= ======== ======== -Conc_Responsive Fold p-value FDR -<numeric> <numeric> <numeric> <numeric> -2452 7 -5.61 3.57e-10 1.02e-06 -1291 5.97 -5.75 1.1e-09 1.57e-06 -976 7.92 -4.79 1.1e-08 1.05e-05 -2338 7.77 -5.93 1.68e-08 1.17e-05 -2077 6.13 -4.23 2.36e-08 1.17e-05 + Columns contain the following data: -The value columns show the -Conc mean read concentration over all the samples (the default calculation uses log2 normalized ChIP read counts with control read counts subtracted) -Conc_Resistant mean concentration over the first (Resistant) group -Conc_Responsive mean concentration over second (Responsive) group -Fold column shows the difference in mean concentrations between the two groups (Conc_Resistant - Conc_Responsive), with a positive value indicating increased binding affinity in the Resistant group and a negative value indicating increased binding affinity in the Responsive group. -p-value confidence measure for identifying these sites as differentially bound -FDR a multiple testing corrected FDR p-value +* **1st**: Chromosome name +* **2nd**: Start position of site +* **3rd**: End position of site +* **4th**: Length of site +* **5th**: Strand +* **6th**: Mean read concentration over all the samples (the default calculation uses log2 normalized ChIP read counts with control read counts subtracted) +* **7th**: Mean concentration over the first (e.g. Resistant) group +* **8th**: Mean concentration over second (e.g. Responsive) group +* **9th**: Fold shows the difference in mean concentrations between the two groups (e.g. Resistant - Responsive), with a positive value indicating increased binding affinity in the first group and a negative value indicating increased binding affinity in the second group. +* **10th**: P-value confidence measure for identifying these sites as differentially bound +* **11th**: a multiple testing corrected FDR p-value **Binding Affinity Matrix** @@ -299,23 +333,22 @@ Example: - ====== ====== ====== ========== ========== ========= ====== ========= ==== - ID Tissue Factor Condition Treatment Replicate Caller Intervals FRiP - ====== ====== ====== ========== ========== ========= ====== ========= ==== - BT4741 BT474 ER Resistant Full-Media 1 counts 2845 0.16 - BT4742 BT474 ER Resistant Full-Media 2 counts 2845 0.15 - MCF71 MCF7 ER Responsive Full-Media 1 counts 2845 0.27 - MCF72 MCF7 ER Responsive Full-Media 2 counts 2845 0.17 - MCF73 MCF7 ER Responsive Full-Media 3 counts 2845 0.23 - T47D1 T47D ER Responsive Full-Media 1 counts 2845 0.10 - T47D2 T47D ER Responsive Full-Media 2 counts 2845 0.06 - MCF7r1 MCF7 ER Resistant Full-Media 1 counts 2845 0.20 - MCF7r2 MCF7 ER Resistant Full-Media 2 counts 2845 0.13 - ZR751 ZR75 ER Responsive Full-Media 1 counts 2845 0.32 - ZR752 ZR75 ER Responsive Full-Media 2 counts 2845 0.22 - ====== ====== ====== ========== ========== ========= ====== ========= ==== + ===== ====== ====== ================ ================ ================ ================ + CHR START END MCF7_ER_1.bed MCF7_ER_2.bed BT474_ER_1.bed BT474_ER_2.bed + ===== ====== ====== ================ ================ ================ ================ + chr18 111567 112005 137.615208000375 59.878372946728 29.4139375878664 19.9594576489093 + chr18 189223 189652 19.9594576489093 12.6059732519427 11.5554754809475 23.110950961895 + chr18 215232 216063 11.5554754809475 15.7574665649284 31.5149331298568 72.4843461986707 + chr18 311530 312172 17.8584621069189 11.5554754809475 54.6258840917518 43.0704086108043 + chr18 346464 347342 75.6358395116564 40.9694130688139 21.0099554199046 16.8079643359236 + chr18 356560 357362 11.5554754809475 14.7069687939332 57.7773774047375 53.5753863207566 + chr18 371110 372102 8.40398216796182 9.45447993895705 81.9388261376278 82.989323908623 + chr18 394600 396513 56.7268796337423 43.0704086108043 510.541916703681 438.05757050501 + chr18 399014 400382 156.524167878289 117.655750351465 558.864814169461 496.885445680743 + chr18 498906 500200 767.913870597511 278.381909313735 196.443083176108 181.736114382174 + ===== ====== ====== ================ ================ ================ ================ - +----- **More Information** @@ -328,21 +361,18 @@ #. Plotting and reporting - * **Reading in peaksets**: +**Reading in peaksets**: The first step is to read in a set of peaksets and associated -metadata. Peaksets are derived either from ChIP-Seq peak callers, such as MACS -([1]), or using some other criterion (e.g. genomic windows, or all the promoter regions -in a genome). The easiest way to read in peaksets is using a comma-separated value -(csv) sample sheet with one line for each peakset. (Spreadsheets in Excel® format, with -a .xls or .xlsx suffix, are also accepted.) A single experiment can have more than +metadata. Peaksets are derived either from ChIP-Seq peak callers, such as **MACS2**, or using some other criterion (e.g. genomic windows, or all the promoter regions +in a genome). A single experiment can have more than one associated peakset; e.g. if multiple peak callers are used for comparison purposes each sample would have more than one line in the sample sheet. Once the peaksets are read in, a merging function finds all overlapping peaks and derives a single set of unique genomic intervals covering all the supplied peaks (a consensus peakset for the experiment). - * **Occupancy analysis**: +**Occupancy analysis**: Peaksets, especially those generated by peak callers, provide an insight into the potential occupancy of the protein being ChIPed for at specific @@ -356,7 +386,7 @@ a consensus peakset, representing an overall set of candidate binding sites to be used in further analysis. - * **Counting reads**: +**Counting reads**: Once a consensus peakset has been derived, DiffBind can use the supplied sequence read files to count how many reads overlap each interval for each @@ -368,7 +398,7 @@ data. The binding affinity matrix is used for QC plotting as well as for subsequent differential analysis. - * **Differential binding affinity analysis**: +**Differential binding affinity analysis**: The core functionality of DiffBind is the differential binding affinity analysis, which enables binding sites to be identified that @@ -378,7 +408,7 @@ This will assign a p-value and FDR to each candidate binding site indicating confidence that they are differentially bound. - * **Plotting and reporting**: +**Plotting and reporting**: Once one or more contrasts have been run, DiffBind provides a number of functions for reporting and plotting the results. MA plots give an @@ -389,11 +419,18 @@ bound sites to be extracted for further processing, such as annotation, motif, and pathway analyses. +----- + **References** DiffBind Authors: Rory Stark, Gordon Brown (2011) Wrapper authors: Bjoern Gruening, Pavankumar Videm +.. _DiffBind: https://bioconductor.org/packages/release/bioc/html/DiffBind.html +.. _`Bioconductor package`: https://bioconductor.org/packages/release/bioc/html/DiffBind.html +.. _`DiffBind User Guide`: https://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf +.. _`Bioconductor post`: https://support.bioconductor.org/p/69924/ + ]]> </help> <citations>