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annotate diffbind.xml @ 15:194e3f2c1d86 draft

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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 515da67fc6c289aa7ea75a0819b5da3a902f87a2
author iuc
date Tue, 09 Jul 2019 18:46:09 -0400
parents c97a786e8fb5
children 163688bb8f73
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194e3f2c1d86 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 515da67fc6c289aa7ea75a0819b5da3a902f87a2
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1 <tool id="diffbind" name="DiffBind" version="2.10.0">
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2 <description> differential binding analysis of ChIP-Seq peak data</description>
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3 <requirements>
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4 <requirement type="package" version="2.10.0">bioconductor-diffbind</requirement>
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5 <requirement type="package" version="1.20.3">r-getopt</requirement>
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6 <requirement type="package" version="0.2.20">r-rjson</requirement>
9
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7 </requirements>
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8 <stdio>
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9 <regex match="Execution halted"
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10 source="both"
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11 level="fatal"
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12 description="Execution halted." />
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13 <regex match="Input-Error 01"
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14 source="both"
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15 level="fatal"
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16 description="Error in your input parameters: Make sure you only apply factors to selected samples." />
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17 <regex match="Error in"
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18 source="both"
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19 level="fatal"
12
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20 description="An undefined error occured, please check your input carefully and contact your administrator." />
9
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21 </stdio>
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22 <version_command><![CDATA[
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23 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: ")
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24 ]]></version_command>
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25 <command><![CDATA[
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26 #import re
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27 #import json
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28
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29 ## Adapted from DESeq2 wrapper
4c7ab9995f9e planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit cc4c1c4131518b9cbf986a1f252767ff73ca938e
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30 #set $temp_factor_names = list()
4c7ab9995f9e planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit cc4c1c4131518b9cbf986a1f252767ff73ca938e
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31 #set $temp_factor = list()
4c7ab9995f9e planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit cc4c1c4131518b9cbf986a1f252767ff73ca938e
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33 #for $g in $rep_group:
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35 #set $peak_files = list()
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36 #set $bam_files = list()
4c7ab9995f9e planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit cc4c1c4131518b9cbf986a1f252767ff73ca938e
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37 #set $bam_controls = list()
4c7ab9995f9e planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit cc4c1c4131518b9cbf986a1f252767ff73ca938e
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38
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39 #for $file in $g.peaks:
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40 #set $file_name = str($g.groupName) + "-" + re.sub('[^\w\-]', '_', str($file.element_identifier)) + "-peaks.bed"
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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41 ln -s '${file}' '${file_name}' &&
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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42 $peak_files.append($file_name)
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43 #end for
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45 #for $bam in $g.bamreads:
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c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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46 #set $bam_name = re.sub('[^\w\-]', '_', str($bam.element_identifier))
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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47 #set $bam_file = $bam_name + "-bamreads.bam"
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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48 #set $bam_index = $bam_name + "-bamreads.bai"
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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49 ln -s '${bam}' '${bam_file}' &&
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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50 ln -s '${bam.metadata.bam_index}' '${bam_index}' &&
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51 $bam_files.append($bam_file)
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52 #end for
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54 $temp_factor.append( {str($g.groupName): $peak_files} )
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55 $temp_factor.append( {str($g.groupName): $bam_files} )
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56
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57 #if str( $g.bamcontrol ) != 'None':
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58 #for $ctrl in $g.bamcontrol:
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59 #set $ctrl_name = re.sub('[^\w\-]', '_', str($ctrl.element_identifier))
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60 #set $ctrl_file = $ctrl_name + "-bamcontrol.bam"
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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61 #set ctrl_index = $ctrl_name + "-bamcontrol.bai"
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62 #if $ctrl_file not in json.dumps($temp_factor):
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63 ln -s '${ctrl}' '${ctrl_file}' &&
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64 ln -s '${ctrl.metadata.bam_index}' '${ctrl_index}' &&
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65 #end if
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66 $bam_controls.append($ctrl_file)
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67 #end for
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68 $temp_factor.append( {str($g.groupName): $bam_controls} )
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69 #end if
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70
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71 #end for
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72
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73 $temp_factor.reverse()
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74 $temp_factor_names.append(["Condition", $temp_factor])
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75
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77 Rscript '$__tool_directory__/diffbind.R'
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79 -i '#echo json.dumps(temp_factor_names)#'
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80 -o '$outfile'
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81 -t $th
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82 -f $out.format
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83 -p '$plots'
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84
11
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85 #if $scorecol:
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86 -n "$scorecol"
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87 #end if
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88 #if $lowerbetter:
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89 -l "$lowerbetter"
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90 #end if
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91 #if $summits:
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92 -s "$summits"
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93 #end if
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94
11
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95 #if $out.binding_matrix:
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96 -b
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97 #end if
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98
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99 #if $out.rdata:
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100 -r
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101 #end if
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102
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103 #if $out.analysis_info:
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104 -a
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105 #end if
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106
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107 #if $out.rscript:
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108 && cp '$__tool_directory__/diffbind.R' '$rscript'
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109 #end if
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110 ]]>
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111 </command>
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112 <inputs>
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113 <repeat name="rep_group" title="Group" min="2" max="2" default="2">
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114 <param name="groupName" type="text" label="Name"
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115 help="Name for the Group that the peak and BAM files belong to e.g. Resistant/Responsive (two Groups must be specified for DiffBind). NOTE: Please only use letters, numbers or underscores.">
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116 <sanitizer>
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117 <valid initial="string.letters,string.digits"><add value="_" /></valid>
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118 </sanitizer>
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119 <validator type="empty_field" />
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120 </param>
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121 <param name="peaks" type="data" format="bed" multiple="true" label="Peak files" help="Result of your Peak calling experiment"/>
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122 <param name="bamreads" type="data" format="bam" multiple="true" label="Read BAM files" help="Specify the Read BAM files used in the Peak calling. The input order of the BAM files for the samples MUST match the input order of the peaks files."/>
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123 <param name="bamcontrol" type="data" format="bam" multiple="true" optional="True" label="Control BAM files" help="If specifying a control BAM file, all samples are required to specify one, see Help section below. The input order of the BAM files for the samples MUST match the input order of the peaks files."/>
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124 </repeat>
11
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125
12
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126 <param name="scorecol" type="integer" min="0" value="8" label="Score Column" help="Column in peak files that contains peak scores. Default: 8 (narrowPeak)">
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127 <sanitizer>
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128 <valid initial="string.digits"/>
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129 </sanitizer>
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130 </param>
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131 <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" />
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132 <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">
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133 <sanitizer>
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134 <valid initial="string.digits"/>
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135 </sanitizer>
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136 </param>
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137 <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"/>
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138
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139 <!-- Output Options -->
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140 <section name="out" expanded="false" title="Output Options">
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141 <param name="format" type="select" label="Output Format">
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142 <option value="interval" selected="True">Interval</option>
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143 <option value="bed">BED</option>
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144 <option value="tabular">Tabular (tab-separated)</option>
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145 </param>
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146 <param name="pdf" type="boolean" truevalue="True" falsevalue="" checked="False" label="Visualising the analysis results" help="output an additional PDF file" />
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147 <param name="binding_matrix" type="boolean" truevalue="True" falsevalue="" checked="False" label="Output binding affinity matrix?" help="Output a table of the binding scores" />
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148 <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"/>
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149 <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"/>
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150 <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"/>
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151 </section>
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152 </inputs>
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153
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154 <outputs>
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155 <data name="outfile" format="interval" label="${tool.name} on ${on_string}: Differentially bound sites">
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156 <change_format>
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157 <when input="out.format" value="bed" format="bed" />
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158 <when input="out.format" value="tabular" format="tabular" />
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159 </change_format>
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160 </data>
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161 <data name="plots" format="pdf" label="${tool.name} on ${on_string}: Plots">
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162 <filter>out['pdf']</filter>
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163 </data>
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164 <data name="binding_matrix" format="tabular" from_work_dir="bmatrix.tab" label="${tool.name} on ${on_string}: Binding matrix">
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165 <filter>out['binding_matrix']</filter>
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166 </data>
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167 <data name="rdata" format="rdata" from_work_dir="DiffBind_analysis.RData" label="${tool.name} on ${on_string}: RData file">
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168 <filter>out['rdata']</filter>
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169 </data>
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170 <data name="rscript" format="txt" label="${tool.name} on ${on_string}: Rscript">
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171 <filter>out['rscript']</filter>
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172 </data>
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173 <data name="analysis_info" format="txt" from_work_dir="DiffBind_analysis_info.txt" label="${tool.name} on ${on_string}: Analysis info">
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174 <filter>out['analysis_info']</filter>
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175 </data>
9
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176 </outputs>
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177
9
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178 <tests>
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179 <!-- Ensure outputs work -->
11
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180 <test expect_num_outputs="6">
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181 <repeat name="rep_group">
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182 <param name="groupName" value="Resistant"/>
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183 <param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
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184 <param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
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185 </repeat>
11
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186 <repeat name="rep_group">
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187 <param name="groupName" value="Responsive"/>
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188 <param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
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189 <param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
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190 </repeat>
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191 <param name="scorecol" value="5" />
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192 <param name="format" value="interval"/>
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193 <param name="pdf" value="True" />
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194 <param name="binding_matrix" value="True" />
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195 <param name="rdata" value="True" />
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196 <param name="rscript" value="True"/>
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197 <param name="analysis_info" value="True"/>
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198 <output name="outfile" ftype="interval" value="out_diffbind.interval" />
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199 <output name="plots" value="out_plots.pdf" compare="sim_size" />
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200 <output name="binding_matrix" value="out_binding_matrix.tab" />
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201 <output name="rdata" value="DiffBind_analysis.RData" compare="sim_size"/>
13
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202 <output name="rscript">
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203 <assert_contents>
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204 <has_text text="write.table"/>
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205 </assert_contents>
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206 </output>
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207 <output name="analysis_info" compare="sim_size" >
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208 <assert_contents>
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209 <has_text text="SessionInfo"/>
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210 </assert_contents>
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211 </output>
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212 </test>
14
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213 <!-- Ensure control BAMs input works -->
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214 <test expect_num_outputs="1">
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215 <repeat name="rep_group">
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216 <param name="groupName" value="Resistant"/>
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217 <param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
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218 <param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
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219 <param name="bamcontrol" ftype="bam" value="input1.bam,input2.bam" />
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220 </repeat>
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221 <repeat name="rep_group">
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222 <param name="groupName" value="Responsive"/>
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223 <param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
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224 <param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
c97a786e8fb5 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 1fe103338548dd65731a5a0887be59745370debd
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225 <param name="bamcontrol" ftype="bam" value="input1.bam,input2.bam" />
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226 </repeat>
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227 <param name="scorecol" value="5" />
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228 <param name="format" value="interval"/>
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229 <output name="outfile" ftype="interval" value="out_diffbind_ctrl.interval" />
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230 </test>
13
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231 <!-- Ensure BED output works -->
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232 <test expect_num_outputs="1">
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233 <repeat name="rep_group">
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234 <param name="groupName" value="Resistant"/>
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235 <param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
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236 <param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
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237 </repeat>
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238 <repeat name="rep_group">
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239 <param name="groupName" value="Responsive"/>
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240 <param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
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241 <param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
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242 </repeat>
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243 <param name="scorecol" value="5" />
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244 <param name="format" value="bed"/>
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245 <output name="outfile" ftype="bed" value="out_diffbind.bed" />
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246 </test>
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247 <!-- Ensure tabular output works -->
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248 <test expect_num_outputs="1">
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249 <repeat name="rep_group">
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250 <param name="groupName" value="Resistant"/>
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251 <param name="peaks" ftype="bed" value="BT474_ER_1.bed.gz,BT474_ER_2.bed.gz"/>
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252 <param name="bamreads" ftype="bam" value="BT474_ER_1.bam,BT474_ER_2.bam" />
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253 </repeat>
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254 <repeat name="rep_group">
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255 <param name="groupName" value="Responsive"/>
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256 <param name="peaks" ftype="bed" value="MCF7_ER_1.bed.gz,MCF7_ER_2.bed.gz"/>
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257 <param name="bamreads" ftype="bam" value="MCF7_ER_1.bam,MCF7_ER_2.bam" />
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258 </repeat>
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259 <param name="scorecol" value="5" />
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260 <param name="format" value="tabular"/>
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261 <output name="outfile" ftype="tabular" file="out_diffbind.tab" />
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262 </test>
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263 </tests>
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264 <help><![CDATA[
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265
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266 .. class:: infomark
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267
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268 **What it does**
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269
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270 DiffBind_ is a `Bioconductor package`_ that provides functions for processing ChIP-Seq data enriched for genomic loci where specific
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271 protein/DNA binding occurs, including peak sets identified by ChIP-Seq peak callers and
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272 aligned sequence read datasets. It is designed to work with multiple peak sets simultaneously,
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273 representing different ChIP experiments (antibodies, transcription factor and/or histone
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274 marks, experimental conditions, replicates) as well as managing the results of multiple peak
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275 callers.
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276
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277 The primary emphasis of DiffBind is on identifying sites that are differentially bound
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278 between two sample groups. It includes functions to support the processing of peak sets,
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279 including overlapping and merging peak sets, counting sequencing reads overlapping intervals
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280 in peak sets, and identifying statistically significantly differentially bound sites based on
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281 evidence of binding affinity (measured by differences in read densities). To this end it uses
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282 statistical routines developed in an RNA-Seq context (primarily the Bioconductor packages
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283 edgeR and DESeq2). Additionally, the package builds on Rgraphics routines to provide a
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284 set of standardized plots to aid in binding analysis.
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285
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286 The `DiffBind User Guide`_ includes a brief overview of the processing flow, followed by four sections of
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287 examples: the first focusing on the core task of obtaining differentially bound sites based on
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288 affinity data, the second working through the main plotting routines, the third discussing the
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289 use of a blocking factor, and the fourth revisiting occupancy data (peak calls) in more detail,
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290 as well as comparing the results of an occupancy-based analysis with an affinity-based one.
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291 Finally, certain technical aspects of the how these analyses are accomplished are detailed.
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292
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293 **Note this DiffBind tool requires a minimum of four samples (two groups with two replicates each).**
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294
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295 -----
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296
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297 **Inputs**
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298
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299 DiffBind works primarily with peaksets, which are sets of genomic intervals representing
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300 candidate protein binding sites. Each interval consists of a chromosome, a start and end
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301 position, and usually a score of some type indicating confidence in, or strength of, the peak.
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302 Associated with each peakset are metadata relating to the experiment from which the peakset
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303 was derived. Additionally, files containing mapped sequencing reads (BAM files) need to
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304 be associated with each peakset (one for the ChIP data, and optionally another representing
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305 a control sample)
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306
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307 **Groups**
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308
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309 You have to specify the name of the Group and the peak and BAM files for the two Groups you want to compare (e.g Resistant and Responsive) in the tool form above.
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310
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311 Example:
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312
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313 ============= =============
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314 **Sample** **Group**
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315 ------------- -------------
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316 BT4741 Resistant
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317 BT4742 Resistant
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318 MCF71 Responsive
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319 MCF72 Responsive
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320 ============= =============
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321
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322
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323 **Peak files**
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324
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325 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.
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326
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327 Example:
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328
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329 ======= ======= ======= =============== ==============
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330 1 2 3 4 **5 (Score)**
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331 ======= ======= ======= =============== ==============
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332 chr18 215562 216063 peak_16037 56.11
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333 chr18 311530 312105 peak_16038 222.49
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334 chr18 356656 357315 peak_16039 92.06
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335 chr18 371110 372092 peak_16040 123.86
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336 chr18 395116 396464 peak_16041 1545.39
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337 chr18 399014 400382 peak_16042 1835.19
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338 chr18 499134 500200 peak_16043 748.32
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339 chr18 503518 504552 peak_16044 818.30
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340 chr18 531672 532274 peak_16045 159.30
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341 chr18 568326 569282 peak_16046 601.11
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342 ======= ======= ======= =============== ==============
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343
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344 * BAM file which contains the mapped sequencing reads associated with each peakset, one file for each sample is required.
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345 * 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`_.
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346
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347 -----
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348
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349 **Outputs**
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350
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351 This tool outputs
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352
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353 * a table of differentially bound sites in Interval, BED or Tabular 0-based format
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354
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355 Optionally, under **Output Options** you can choose to output
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356
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357 * a PDF of plots (Heatmap, PCA, MA, Volcano, Boxplots)
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358 * a binding affinity matrix
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359 * the R script used by this tool
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360 * an RData file of the R objects generated
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361 * a text file with information on the analysis (number of Intervals, FriP scores, method used)
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362
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363 **Differentially Bound Sites**
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364
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365 The default output is Interval format, for information on Interval format see here_. Alternatively, you can choose to output BED or Tabular 0-based format as below. For an explanation of the 0-based and 1-based coordinate systems see this `Biostars post`_.
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366
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367 Example - **Interval format**:
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368
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369 ====== ====== ====== ======== ===== ====== ===========================================
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370 Chrom Start End Name Score Strand **Comment**
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371 ====== ====== ====== ======== ===== ====== ===========================================
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372 chr18 394599 396513 DiffBind 0 \. 1914|7.15|5.55|7.89|-2.35|7.06e-24|9.84e-21
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373 chr18 111566 112005 DiffBind 0 \. 439|5.71|6.53|3.63|2.89|1.27e-08|8.88e-06
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374 chr18 346463 347342 DiffBind 0 \. 879|5|5.77|3.24|2.52|6.51e-06|0.00303
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375 chr18 399013 400382 DiffBind 0 \. 1369|7.62|7|8.05|-1.04|1.04e-05|0.00364
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376 chr18 371109 372102 DiffBind 0 \. 993|4.63|3.07|5.36|-2.3|8.1e-05|0.0226
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377 ====== ====== ====== ======== ===== ====== ===========================================
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378
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379 Columns contain the following data:
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380
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381 * **Chrom**: Chromosome name
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382 * **Start**: Start position of site
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383 * **End**: End position of site
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384 * **Score**: 0
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385 * **Name**: DiffBind
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386 * **Strand**: Strand
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387 * **Comment**: The pipe ("|") separated values in this column correspond to:
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388
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389 * *width*: Length of site
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390 * *Conc*: Mean read concentration over all the samples (the default calculation uses log2 normalized ChIP read counts with control read counts subtracted)
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391 * *Conc_Group1*: Mean concentration over the first group (e.g. Responsive)
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392 * *Conc_Group2*: Mean concentration over second group (e.g. Resistant)
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393 * *Fold*: Fold shows the difference in mean concentrations between the two groups (e.g. Responsive - Resistant), with a positive value indicating increased binding affinity in the first group and a negative value indicating increased binding affinity in the second group.
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394 * *p.value*: P-value confidence measure for identifying these sites as differentially bound
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395 * *FDR*: a multiple testing corrected FDR p-value
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396
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397 Example - **BED format**:
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398
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399 ===== ====== ====== ======== ===== ======
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400 Chrom Start End Name Score Strand
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401 ===== ====== ====== ======== ===== ======
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402 chr18 394599 396513 DiffBind 0 \.
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403 chr18 111566 112005 DiffBind 0 \.
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404 chr18 346463 347342 DiffBind 0 \.
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405 chr18 399013 400382 DiffBind 0 \.
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406 chr18 371109 372102 DiffBind 0 \.
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407 ===== ====== ====== ======== ===== ======
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408
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409 Example - **Tabular format**:
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410
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411 ===== ====== ====== ======== ===== ====== ==== =============== ============== ===== ======== ========
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412 Chrom Start End Name Score Strand Conc Conc_Responsive Conc_Resistant Fold p.value FDR
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413 ===== ====== ====== ======== ===== ====== ==== =============== ============== ===== ======== ========
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414 chr18 394599 396513 DiffBind 0 \. 7.15 5.55 7.89 -2.35 7.06E-24 9.84E-21
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415 chr18 111566 112005 DiffBind 0 \. 5.71 6.53 3.63 2.89 1.27E-08 8.88E-06
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416 chr18 346463 347342 DiffBind 0 \. 5 5.77 3.24 2.52 6.51E-06 0.00303
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417 chr18 399013 400382 DiffBind 0 \. 7.62 7 8.05 -1.04 1.04E-05 0.00364
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418 chr18 371109 372102 DiffBind 0 \. 4.63 3.07 5.36 -2.3 8.10E-05 0.0226
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419 ===== ====== ====== ======== ===== ====== ==== =============== ============== ===== ======== ========
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420
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421
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422 **Binding Affinity Matrix**
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423
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424 The final result of counting is a binding affinity matrix containing a (normalized) read count for each sample at every potential binding site. With this matrix, the samples can be re-clustered using affinity, rather than occupancy, data. The binding affinity matrix can be used for QC plotting as well as for subsequent
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425 differential analysis. Note that this output is a tabular 0-based format.
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426
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427 Example:
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428
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429 ===== ====== ====== ========= ========= ========== ==========
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430 Chrom Start End MCF7_ER_1 MCF7_ER_2 BT474_ER_1 BT474_ER_2
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431 ===== ====== ====== ========= ========= ========== ==========
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432 chr18 111567 112005 137.6152 59.87837 29.41393 19.95945
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433 chr18 189223 189652 19.95945 12.60597 11.55547 23.11095
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
iuc
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434 chr18 215232 216063 11.55547 15.75746 31.51493 72.48434
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
iuc
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435 chr18 311530 312172 17.85846 11.55547 54.62588 43.07040
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
iuc
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436 chr18 346464 347342 75.63583 40.96941 21.00995 16.80796
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
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437 chr18 356560 357362 11.55547 14.70696 57.77737 53.57538
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
iuc
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438 chr18 371110 372102 8.403982 9.454479 81.93882 82.98932
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
iuc
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439 chr18 394600 396513 56.72687 43.07040 510.5419 438.0575
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
iuc
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440 chr18 399014 400382 156.5241 117.6557 558.8648 496.8854
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
iuc
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441 chr18 498906 500200 767.9138 278.3819 196.4430 181.7361
fa56d93f7980 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 11f68fe2b872f5abc5b660adb10336b0955fa0ee
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442 ===== ====== ====== ========= ========= ========== ==========
9
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443
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444 -----
9
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445
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446 **More Information**
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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447
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448 Generally, processing data with DiffBind involves five phases:
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449
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450 #. Reading in peaksets
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451 #. Occupancy analysis
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452 #. Counting reads
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453 #. Differential binding affinity analysis
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454 #. Plotting and reporting
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455
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456
10
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457 **Reading in peaksets**:
9
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458
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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459 The first step is to read in a set of peaksets and associated
10
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460 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
d7725c5596ab planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit f970dcbe9d0e4c3714b1db74c404ea34223cf8ed
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461 in a genome). A single experiment can have more than
9
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462 one associated peakset; e.g. if multiple peak callers are used for comparison purposes
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463 each sample would have more than one line in the sample sheet. Once the peaksets
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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464 are read in, a merging function finds all overlapping peaks and derives a single set of
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465 unique genomic intervals covering all the supplied peaks (a consensus peakset for the
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466 experiment).
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467
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468 **Occupancy analysis**:
9
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469
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470 Peaksets, especially those generated by peak callers, provide
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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471 an insight into the potential occupancy of the protein being ChIPed for at specific
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472 genomic loci. After the peaksets have been loaded, it can be useful to perform some
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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473 exploratory plotting to determine how these occupancy maps agree with each other,
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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474 e.g. between experimental replicates (re-doing the ChIP under the same conditions),
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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475 between different peak callers on the same experiment, and within groups of samples
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476 representing a common experimental condition. DiffBind provides functions to enable
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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477 overlaps to be examined, as well as functions to determine how well similar samples
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478 cluster together. Beyond quality control, the product of an occupancy analysis may be
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479 a consensus peakset, representing an overall set of candidate binding sites to be used
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480 in further analysis.
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481
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482 **Counting reads**:
9
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483
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484 Once a consensus peakset has been derived, DiffBind can use the
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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485 supplied sequence read files to count how many reads overlap each interval for each
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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486 unique sample. The peaks in the consensus peakset may be re-centered and trimmed
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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487 based on calculating their summits (point of greatest read overlap) in order to provide
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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488 more standardized peak intervals. The final result of counting is a binding affinity matrix
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489 containing a (normalized) read count for each sample at every potential binding site.
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490 With this matrix, the samples can be re-clustered using affinity, rather than occupancy,
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491 data. The binding affinity matrix is used for QC plotting as well as for subsequent
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492 differential analysis.
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493
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494 **Differential binding affinity analysis**:
9
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495
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496 The core functionality of DiffBind is the
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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497 differential binding affinity analysis, which enables binding sites to be identified that
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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498 are statistically significantly differentially bound between sample groups. To accomplish
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499 this, first a contrast (or contrasts) is established, dividing the samples into groups to
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500 be compared. Next the core analysis routines are executed, by default using DESeq2.
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501 This will assign a p-value and FDR to each candidate binding site indicating confidence
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502 that they are differentially bound.
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503
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504 **Plotting and reporting**:
9
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505
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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506 Once one or more contrasts have been run, DiffBind provides
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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507 a number of functions for reporting and plotting the results. MA plots give an
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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508 overview of the results of the analysis, while correlation heatmaps and PCA plots show
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509 how the groups cluster based on differentially bound sites. Boxplots show the distribution
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510 of reads within differentially bound sites corresponding to whether they gain or
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511 lose affinity between the two sample groups. A reporting mechanism enables differentially
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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512 bound sites to be extracted for further processing, such as annotation, motif, and
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513 pathway analyses.
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514
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515 -----
9
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516
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517 **References**
6171163112de planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/diffbind commit 9de99de5fb4c62f889814ea43b8800ce8d28eb83
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518
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519 DiffBind Authors: Rory Stark, Gordon Brown (2011)
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520 Wrapper authors: Bjoern Gruening, Pavankumar Videm
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521
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522 .. _DiffBind: https://bioconductor.org/packages/release/bioc/html/DiffBind.html
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523 .. _`Bioconductor package`: https://bioconductor.org/packages/release/bioc/html/DiffBind.html
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524 .. _`DiffBind User Guide`: https://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf
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525 .. _`Bioconductor post`: https://support.bioconductor.org/p/69924/
13
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526 .. _here: https://galaxyproject.org/learn/datatypes/#interval
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527 .. _`Biostars post`: https://www.biostars.org/p/84686/
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528
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529 ]]>
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530 </help>
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531 <citations>
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532 <citation type="doi">doi:10.1038/nature10730</citation>
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533 </citations>
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534 </tool>