comparison limma_voom.xml @ 1:76d01fe0ec36 draft

planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/limma_voom commit 58c05c0ce9334f8b9c800283cfd1f40573546edd
author iuc
date Wed, 05 Jul 2017 04:39:42 -0400
parents bdebdea5f6a7
children a330ddf43861
comparison
equal deleted inserted replaced
0:bdebdea5f6a7 1:76d01fe0ec36
1 <tool id="limma_voom" name="limma-voom" version="1.1.1"> 1 <tool id="limma_voom" name="limma-voom" version="1.2.0">
2 <description> 2 <description>
3 Differential expression with optional sample weights 3 Differential expression with optional sample weights
4 </description> 4 </description>
5 5
6 <requirements> 6 <requirements>
7 <requirement type="package" version="3.16.5">bioconductor-edger</requirement> 7 <requirement type="package" version="3.16.5">bioconductor-edger</requirement>
8 <requirement type="package" version="3.30.13">bioconductor-limma</requirement> 8 <requirement type="package" version="3.30.13">bioconductor-limma</requirement>
9 <requirement type="package" version="1.4.29">r-statmod</requirement> 9 <requirement type="package" version="1.4.29">r-statmod</requirement>
10 <requirement type="package" version="0.4.1">r-scales</requirement> 10 <requirement type="package" version="0.4.1">r-scales</requirement>
11 </requirements> 11 </requirements>
12 12
13 <version_command> 13 <version_command><![CDATA[
14 <![CDATA[
15 echo $(R --version | grep version | grep -v GNU)", limma version" $(R --vanilla --slave -e "library(limma); cat(sessionInfo()\$otherPkgs\$limma\$Version)" 2> /dev/null | grep -v -i "WARNING: ")", edgeR version" $(R --vanilla --slave -e "library(edgeR); cat(sessionInfo()\$otherPkgs\$edgeR\$Version)" 2> /dev/null | grep -v -i "WARNING: ") 14 echo $(R --version | grep version | grep -v GNU)", limma version" $(R --vanilla --slave -e "library(limma); cat(sessionInfo()\$otherPkgs\$limma\$Version)" 2> /dev/null | grep -v -i "WARNING: ")", edgeR version" $(R --vanilla --slave -e "library(edgeR); cat(sessionInfo()\$otherPkgs\$edgeR\$Version)" 2> /dev/null | grep -v -i "WARNING: ")
16 ]]> 15 ]]></version_command>
17 </version_command> 16
18 17 <command detect_errors="exit_code"><![CDATA[
19 <command detect_errors="exit_code"> 18 Rscript '$__tool_directory__/limma_voom.R'
20 <![CDATA[ 19 '$counts'
21 Rscript '$__tool_directory__/limma_voom.R' 20
22 '$counts' 21 #if $anno.annoOpt=='yes':
23 22 '$geneanno'
24 #if $anno.annoOpt=='yes': 23 #else:
25 '$geneanno' 24 None
26 #else: 25 #end if
27 None 26
28 #end if 27 '$outReport'
29 28 '$outReport.files_path'
30 '$outReport' 29 $rdaOption
31 '$outReport.files_path' 30 $normalisationOption
32 $rdaOption 31 $weightOption
33 $normalisationOption 32 '$contrast'
34 $weightOption 33
35 '$contrast' 34 #if $filterCPM.filterLowCPM=='yes':
36 35 '$filterCPM.cpmReq'
37 #if $filterCPM.filterLowCPM=='yes': 36 '$filterCPM.sampleReq'
38 '$filterCPM.cpmReq' 37 #else:
39 '$filterCPM.sampleReq' 38 0
40 #else: 39 0
41 0 40 #end if
42 0 41
43 #end if 42 #if $testOpt.wantOpt=='yes':
44 43 '$testOpt.pAdjust'
45 #if $testOpt.wantOpt=='yes': 44 '$testOpt.pVal'
46 '$testOpt.pAdjust' 45 '$testOpt.lfc'
47 '$testOpt.pVal' 46 #else:
48 '$testOpt.lfc' 47 "BH"
49 #else: 48 0.05
50 "BH" 49 0
51 0.05 50 #end if
52 0 51
53 #end if 52 $normCounts
54 53
55 '$factName::$factLevel' 54 #if $fact.ffile=='yes':
56 55 '$finfo'
57 && 56 'None'
58 mkdir ./output_dir 57 #else:
59 58 'None'
60 && 59 '$fact.pfactName::$fact.pfactLevel'
61 mv '$outReport.files_path'/*.tsv output_dir/ 60 #for $sfact in $fact.sfactors:
62 61 '$sfact.sfactName::$sfact.sfactLevel'
63 ]]> 62 #end for
64 </command> 63 #end if
65 64
65 &&
66 mkdir ./output_dir
67
68 &&
69 mv '$outReport.files_path'/*.tsv output_dir/
70 ]]></command>
71
66 <inputs> 72 <inputs>
67 <param name="counts" type="data" format="tabular" label="Counts Data"/> 73 <param name="counts" type="data" format="tabular" label="Counts Data"/>
68 74
69 <conditional name="anno"> 75 <conditional name="anno">
70 <param name="annoOpt" type="select" 76 <param name="annoOpt" type="select" label="Use Gene Annotations?"
71 label="Use Gene Annotations?" 77 help="If an annotation file is provided, annotations will be added to the table of differential expression results to provide descriptions for each gene.">
72 help="If an annotation file is provided, annotations will be added to the table of differential expression results to provide descriptions for each gene.">
73 <option value="no">No</option> 78 <option value="no">No</option>
74 <option value="yes">Yes</option> 79 <option value="yes">Yes</option>
75 </param> 80 </param>
76 <when value="yes"> 81 <when value="yes">
77 <param name="geneanno" type="data" format="tabular" label="Gene Annotations"/> 82 <param name="geneanno" type="data" format="tabular" label="Gene Annotations"/>
78 </when> 83 </when>
79 <when value="no" /> 84 <when value="no" />
80 </conditional> 85 </conditional>
81 86
82 <!--*Code commented until solution for multiple factors is found* 87 <conditional name="fact">
83 <repeat name="factors" title="Factors" min="1" max="5" default="1"> 88 <param name="ffile" type="select" label="Input Factor Information from file?"
84 <param name="factName" type="text" label="Factor Name (No spaces)" 89 help="You can choose to input the factor information from a file or manually enter below.">
85 help="Eg. Genotype"/> 90 <option value="no">No</option>
86 <param name="factLevel" type="text" size="100" 91 <option value="yes">Yes</option>
87 label="Factor Levels (No spaces)" 92 </param>
88 help="Eg. WT,WT,Mut,Mut,WT"/> 93 <when value="yes">
89 </repeat> 94 <param name="finfo" type="data" format="tabular" label="Factor Information"/>
90 --> 95 </when>
91 96 <when value="no" >
92 <param name="factName" type="text" label="Factor Name" help="Eg. Genotype."/> 97 <param name="pfactName" type="text" label="Primary Factor Name"
93 <param name="factLevel" type="text" label="Factor Values" 98 help="Eg. Genotype NOTE: Please only use letters, numbers or underscores.">
94 help="Eg. WT,WT,WT,Mut,Mut,Mut 99 <validator type="empty_field" />
95 NOTE: Please ensure that the same levels are typed identically with cases matching."/> 100 <validator type="regex" message="Please only use letters, numbers or underscores">^[\w]+$</validator>
96 <param name="contrast" type="text" label="Contrasts of interest" help="Eg. Mut-WT,KD-Control"/> 101 </param>
97 102 <param name="pfactLevel" type="text" label="Primary Factor Levels"
103 help="Eg. WT,WT,WT,Mut,Mut,Mut NOTE: Please only use letters, numbers or underscores and ensure that the same levels are typed identically with cases matching.">
104 <validator type="empty_field" />
105 <validator type="regex" message="Please only use letters, numbers or underscores, and separate levels by commas">^[\w,]+$</validator>
106 </param>
107 <repeat name="sfactors" title="Secondary Factor" >
108 <param name="sfactName" type="text" label="Secondary Factor Name" help="Eg. Batch">
109 <validator type="empty_field" />
110 <validator type="regex" message="Please only use letters, numbers or underscores">^[\w]+$</validator>
111 </param>
112 <param name="sfactLevel" type="text" label="Secondary Factor Levels"
113 help="Eg. b1,b2,b3,b1,b2,b3 NOTE: Please only use letters, numbers or underscores and ensure that the same levels are typed identically with cases matching.">
114 <validator type="empty_field" />
115 <validator type="regex" message="Please only use letters, numbers or underscores">^[\w,]+$</validator>
116 </param>
117 </repeat>
118 </when>
119 </conditional>
120
121 <param name="contrast" type="text" label="Contrasts of interest" help="Eg. Mut-WT,KD-Control">
122 <validator type="empty_field" />
123 <validator type="regex" message="Please only use letters, numbers or underscores">^[\w,-]+$</validator>
124 </param>
125
98 <conditional name="filterCPM"> 126 <conditional name="filterCPM">
99 <param name="filterLowCPM" type="select" label="Filter Low CPM?" 127 <param name="filterLowCPM" type="select" label="Filter Low CPM?"
100 help="Treat genes with very low expression as unexpressed and filter out to speed up computation."> 128 help="Treat genes with very low expression as unexpressed and filter out to speed up computation.">
101 <option value="yes" selected="True">Yes</option> 129 <option value="yes" selected="True">Yes</option>
102 <option value="no">No</option> 130 <option value="no">No</option>
103 </param> 131 </param>
104 <when value="yes"> 132 <when value="yes">
105 <param name="cpmReq" type="float" value="0.5" min="0" label="Minimum CPM"/> 133 <param name="cpmReq" type="float" value="0.5" min="0" label="Minimum CPM"/>
106
107 <param name="sampleReq" type="integer" value="1" min="0" label="Minimum Samples" 134 <param name="sampleReq" type="integer" value="1" min="0" label="Minimum Samples"
108 help="Filter out all the genes that do not meet the minimum CPM in at least this many samples."/> 135 help="Filter out all the genes that do not meet the minimum CPM in at least this many samples."/>
109 </when> 136 </when>
110 <when value="no"/> 137 <when value="no"/>
111 </conditional> 138 </conditional>
112 139
113 <param name="weightOption" type="boolean" truevalue="yes" falsevalue="no" checked="false" label="Apply sample weights?" 140 <param name="weightOption" type="boolean" truevalue="yes" falsevalue="no" checked="false" label="Apply sample weights?"
114 help="Apply weights if outliers are present."> 141 help="Apply weights if outliers are present.">
115 </param> 142 </param>
116 143
117 <param name="normalisationOption" type="select" label="Normalisation Method"> 144 <param name="normalisationOption" type="select" label="Normalisation Method">
118 <option value="TMM">TMM</option> 145 <option value="TMM">TMM</option>
119 <option value="RLE">RLE</option> 146 <option value="RLE">RLE</option>
120 <option value="upperquartile">Upperquartile</option> 147 <option value="upperquartile">Upperquartile</option>
121 <option value="none">None (Don't normalise)</option> 148 <option value="none">None (Don't normalise)</option>
122 </param> 149 </param>
123 150
124 <param name="rdaOption" type="boolean" truevalue="yes" falsevalue="no" checked="false" 151 <param name="normCounts" type="boolean" truevalue="yes" falsevalue="no" checked="false"
152 label="Output normalised counts table?"
153 help="Output a file containing the normalised counts, these are in log2 counts per million (logCPM).">
154 </param>
155
156 <param name="rdaOption" type="boolean" truevalue="yes" falsevalue="no" checked="false"
125 label="Output RData?" 157 label="Output RData?"
126 help="Output all the data used by R to construct the plots and tables, can be loaded into R. A link to the RData file will be provided in the HTML report."> 158 help="Output all the data used by R to construct the plots and tables, can be loaded into R. A link to the RData file will be provided in the HTML report.">
127 </param> 159 </param>
128 160
129 <conditional name="testOpt"> 161 <conditional name="testOpt">
130 <param name="wantOpt" type="select" label="Use Advanced Testing Options?" 162 <param name="wantOpt" type="select" label="Use Advanced Testing Options?"
131 help="Enable choices for p-value adjustment method, p-value threshold and log2-fold-change threshold."> 163 help="Enable choices for p-value adjustment method, p-value threshold and log2-fold-change threshold.">
132 <option value="no" selected="True">No</option> 164 <option value="no" selected="True">No</option>
133 <option value="yes">Yes</option> 165 <option value="yes">Yes</option>
134 </param> 166 </param>
135 <when value="yes"> 167 <when value="yes">
136 <param name="pAdjust" type="select" label="P-Value Adjustment Method."> 168 <param name="pAdjust" type="select" label="P-Value Adjustment Method.">
137 <option value="BH">Benjamini and Hochberg (1995)</option> 169 <option value="BH">Benjamini and Hochberg (1995)</option>
138 <option value="BY">Benjamini and Yekutieli (2001)</option> 170 <option value="BY">Benjamini and Yekutieli (2001)</option>
139 <option value="holm">Holm (1979)</option> 171 <option value="holm">Holm (1979)</option>
140 <option value="none">None</option> 172 <option value="none">None</option>
141 </param> 173 </param>
142 <param name="pVal" type="float" value="0.05" min="0" max="1" 174 <param name="pVal" type="float" value="0.05" min="0" max="1"
143 label="Adjusted Threshold" 175 label="Adjusted Threshold"
144 help="Genes below this threshold are considered significant and highlighted in the MA plot. If either BH(1995) or BY(2001) were selected then this value is a false-discovery-rate control. If Holm(1979) was selected then this is an adjusted p-value for family-wise error rate."/> 176 help="Genes below this threshold are considered significant and highlighted in the MA plot. If either BH(1995) or BY(2001) were selected then this value is a false-discovery-rate control. If Holm(1979) was selected then this is an adjusted p-value for family-wise error rate."/>
145 <param name="lfc" type="float" value="0" min="0" 177 <param name="lfc" type="float" value="0" min="0"
146 label="Minimum log2-fold-change Required" 178 label="Minimum log2-fold-change Required"
147 help="Genes above this threshold and below the p-value threshold are considered significant and highlighted in the MA plot."/> 179 help="Genes above this threshold and below the p-value threshold are considered significant and highlighted in the MA plot."/>
148 </when> 180 </when>
149 <when value="no"/> 181 <when value="no"/>
150 </conditional> 182 </conditional>
151 183
152 </inputs> 184 </inputs>
153 185
154 <outputs> 186 <outputs>
155 <data format="html" name="outReport" label="${tool.name} on ${on_string}: Report" /> 187 <data name="outReport" format="html" label="${tool.name} on ${on_string}: Report" />
156 <collection name="voom_results" type="list" label="${tool.name} on ${on_string}: DE genes"> 188 <collection name="outTables" type="list" label="${tool.name} on ${on_string}: Tables">
157 <discover_datasets pattern="(?P&lt;name&gt;.+)\.tsv$" format="tabular" directory="output_dir" visible="false" /> 189 <discover_datasets pattern="(?P&lt;name&gt;.+)\.tsv$" format="tabular" directory="output_dir" visible="false" />
158 </collection> 190 </collection>
159 </outputs> 191 </outputs>
160 192
161 <tests> 193 <tests>
162 <test> 194 <test>
163 <param name="counts" value="matrix.txt" /> 195 <param name="counts" value="matrix.txt" />
164 <param name="factName" value="Genotype" /> 196 <param name="pfactName" value="Genotype" />
165 <param name="factLevel" value="WT,WT,WT,Mut,Mut,Mut" /> 197 <param name="pfactLevel" value="WT,WT,WT,Mut,Mut,Mut" />
166 <param name="contrast" value="Mut-WT,WT-Mut" /> 198 <param name="contrast" value="Mut-WT,WT-Mut" />
167 <param name="normalisationOption" value="TMM" /> 199 <param name="normalisationOption" value="TMM" />
168 <output_collection name="voom_results" count="2"> 200 <output_collection name="outTables" count="2">
169 <element name="limma-voom_Mut-WT" ftype="tabular" file="limma-voom_Mut-WT.tsv" /> 201 <element name="limma-voom_Mut-WT" ftype="tabular" file="limma-voom_Mut-WT.tsv" />
170 <element name="limma-voom_WT-Mut" ftype="tabular" file="limma-voom_WT-Mut.tsv" /> 202 <element name="limma-voom_WT-Mut" ftype="tabular" file="limma-voom_WT-Mut.tsv" />
171 </output_collection> 203 </output_collection>
172 <output name="outReport" > 204 <output name="outReport" >
173 <assert_contents> 205 <assert_contents>
174 <has_text text="Limma-voom Analysis Output" /> 206 <has_text text="Limma-voom Analysis Output" />
175 <not_has_text text="RData" /> 207 <not_has_text text="RData" />
176 </assert_contents> 208 </assert_contents>
177 </output> 209 </output>
178 </test> 210 </test>
179 <test> 211 <test>
180 <param name="annoOpt" value="yes" /> 212 <param name="annoOpt" value="yes" />
181 <param name="geneanno" value="anno.txt" /> 213 <param name="geneanno" value="anno.txt" />
182 <param name="counts" value="matrix.txt" /> 214 <param name="counts" value="matrix.txt" />
183 <param name="factName" value="Genotype" /> 215 <param name="pfactName" value="Genotype" />
184 <param name="factLevel" value="WT,WT,WT,Mut,Mut,Mut" /> 216 <param name="pfactLevel" value="WT,WT,WT,Mut,Mut,Mut" />
185 <param name="contrast" value="Mut-WT" /> 217 <param name="contrast" value="Mut-WT" />
186 <param name="normalisationOption" value="TMM" /> 218 <param name="normalisationOption" value="TMM" />
187 <output_collection name="voom_results" > 219 <output_collection name="outTables" >
188 <element name="limma-voom_Mut-WT" ftype="tabular" file="limma-voom_Mut-WTanno.tsv" /> 220 <element name="limma-voom_Mut-WT" ftype="tabular" file="limma-voom_Mut-WTanno.tsv" />
189 </output_collection> 221 </output_collection>
190 </test> 222 </test>
191 <test> 223 <test>
192 <param name="rdaOption" value="yes" /> 224 <param name="rdaOption" value="yes" />
193 <param name="counts" value="matrix.txt" /> 225 <param name="counts" value="matrix.txt" />
194 <param name="factName" value="Genotype" /> 226 <param name="pfactName" value="Genotype" />
195 <param name="factLevel" value="WT,WT,WT,Mut,Mut,Mut" /> 227 <param name="pfactLevel" value="WT,WT,WT,Mut,Mut,Mut" />
196 <param name="contrast" value="Mut-WT" /> 228 <param name="contrast" value="Mut-WT" />
197 <param name="normalisationOption" value="TMM" /> 229 <param name="normalisationOption" value="TMM" />
198 <output name="outReport" > 230 <output name="outReport" >
199 <assert_contents> 231 <assert_contents>
200 <has_text text="RData" /> 232 <has_text text="RData" />
201 </assert_contents> 233 </assert_contents>
202 </output> 234 </output>
203 </test> 235 </test>
236 <test>
237 <param name="counts" value="matrix.txt" />
238 <param name="pfactName" value="Genotype"/>
239 <param name="pfactLevel" value="WT,WT,WT,Mut,Mut,Mut"/>
240 <repeat name="sfactors">
241 <param name="sfactName" value="Batch"/>
242 <param name="sfactLevel" value="b1,b2,b3,b1,b2,b3"/>
243 </repeat>
244 <param name="contrast" value="Mut-WT" />
245 <param name="normalisationOption" value="TMM" />
246 <output_collection name="outTables" >
247 <element name="limma-voom_Mut-WT" ftype="tabular" file="limma-voom_Mut-WTmultifact.tsv" />
248 </output_collection>
249 </test>
250 <test>
251 <param name="ffile" value="yes" />
252 <param name="finfo" value="factorinfo.txt" />
253 <param name="counts" value="matrix.txt" />
254 <param name="contrast" value="Mut-WT" />
255 <param name="normalisationOption" value="TMM" />
256 <output_collection name="outTables" >
257 <element name="limma-voom_Mut-WT" ftype="tabular" file="limma-voom_Mut-WTmultifact.tsv" />
258 </output_collection>
259 </test>
260 <test>
261 <param name="normCounts" value="yes" />
262 <param name="counts" value="matrix.txt" />
263 <param name="pfactName" value="Genotype" />
264 <param name="pfactLevel" value="WT,WT,WT,Mut,Mut,Mut" />
265 <param name="contrast" value="Mut-WT" />
266 <param name="normalisationOption" value="TMM" />
267 <output_collection name="outTables" count="2">
268 <element name="limma-voom_Mut-WT" ftype="tabular" file="limma-voom_Mut-WT.tsv" />
269 <element name="limma-voom_normcounts" ftype="tabular" file="limma-voom_normcounts.tsv" />
270 </output_collection>
271 </test>
204 </tests> 272 </tests>
205 273
206 <help> 274 <help><![CDATA[
207 <![CDATA[
208 .. class:: infomark 275 .. class:: infomark
209 276
210 **What it does** 277 **What it does**
211 278
212 Given a matrix of counts (e.g. from featureCounts) and optional information about the genes, this tool 279 Given a matrix of counts (e.g. from featureCounts) and optional information about the genes, this tool
213 produces plots and tables useful in the analysis of differential gene 280 produces plots and tables useful in the analysis of differential gene
214 expression. 281 expression.
215 282
216 ----- 283 -----
217 284
218 **Inputs** 285 **Inputs**
226 Example: 293 Example:
227 294
228 ========== ======= ======= ======= ======== ======== ======== 295 ========== ======= ======= ======= ======== ======== ========
229 **GeneID** **WT1** **WT2** **WT3** **Mut1** **Mut2** **Mut3** 296 **GeneID** **WT1** **WT2** **WT3** **Mut1** **Mut2** **Mut3**
230 ---------- ------- ------- ------- -------- -------- -------- 297 ---------- ------- ------- ------- -------- -------- --------
231 11287 1699 1528 1601 1463 1441 1495 298 11287 1699 1528 1601 1463 1441 1495
232 11298 1905 1744 1834 1345 1291 1346 299 11298 1905 1744 1834 1345 1291 1346
233 11302 6 8 7 5 6 5 300 11302 6 8 7 5 6 5
234 11303 2099 1974 2100 1574 1519 1654 301 11303 2099 1974 2100 1574 1519 1654
235 11304 356 312 337 361 397 346 302 11304 356 312 337 361 397 346
236 11305 2528 2438 2493 1762 1942 2027 303 11305 2528 2438 2493 1762 1942 2027
237 ========== ======= ======= ======= ======== ======== ======== 304 ========== ======= ======= ======= ======== ======== ========
238 305
239 **Gene Annotations:** 306 **Gene Annotations:**
240 Optional input for gene annotations, this can contain more 307 Optional input for gene annotations, this can contain more
241 information about the genes than just an ID number. The annotations will 308 information about the genes than just an ID number. The annotations will
242 be avaiable in the differential expression results table. 309 be available in the differential expression results table and the optional normalised counts table.
243 310
244 Example: 311 Example:
245 312
246 ========== ========== =================================================== 313 ========== ========== ===================================================
247 **GeneID** **Symbol** **GeneName** 314 **GeneID** **Symbol** **GeneName**
252 1303 Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 319 1303 Abca1 ATP-binding cassette, sub-family A (ABC1), member 1
253 1304 Abca4 ATP-binding cassette, sub-family A (ABC1), member 4 320 1304 Abca4 ATP-binding cassette, sub-family A (ABC1), member 4
254 1305 Abca2 ATP-binding cassette, sub-family A (ABC1), member 2 321 1305 Abca2 ATP-binding cassette, sub-family A (ABC1), member 2
255 ========== ========== =================================================== 322 ========== ========== ===================================================
256 323
257 **Factor Name:** 324 **Factor Information:**
258 The name of the factor being investigated. This tool currently assumes 325 Enter Factor Names and Levels in the tool form or provide a tab-separated file that has the samples in the same order as listed in the columns of the counts matrix. The second column should contain the Primary Factor levels (e.g. Genotype) with optional additional columns for any Secondary Factors (e.g. Batch).
259 that only one factor is of interest. 326
260 327 Example:
261 **Factor Levels:** 328
262 The levels of the factor of interest, this must be entered in the same 329 ========== ============ =========
263 order as the samples to which the levels correspond as listed in the 330 **Sample** **Genotype** **Batch**
264 columns of the counts matrix. 331 ---------- ------------ ---------
265 332 WT1 WT b1
266 The values should be seperated by commas, and spaces must not be used. 333 WT2 WT b2
334 WT3 WT b3
335 Mut1 Mut b1
336 Mut2 Mut b2
337 Mut3 Mut b3
338 ========== ============ =========
339
340 **Primary Factor Name:** The name of the primary factor being investigated e.g. Genotype. One primary factor must be entered and spaces must not be used.
341
342 **Primary Factor Levels:** The levels of the primary factor of interest, these must be entered in the same order as the samples to which the levels correspond, as listed in the columns of the counts matrix. Spaces must not be used and if entered in the tool form the values should be separated by commas.
343
344 **Secondary Factor Name:** Optionally, one or more secondary factors can be included. These are variables that might influence your experiment e.g. Batch, Gender. Spaces must not be used.
345
346 **Secondary Factor Levels:** The levels of the secondary factor of interest, these must be entered in the same order as the samples to which the levels correspond, as listed in the columns of the counts matrix. Spaces must not be used and if entered in the tool form the values should be separated by commas.
347
267 348
268 **Contrasts of Interest:** 349 **Contrasts of Interest:**
269 The contrasts you wish to make between levels. 350 The contrasts you wish to make between levels.
270 351 A common contrast would be a simple difference between two levels: "Mut-WT"
271 A common contrast would be a simple difference between two levels: "Mut-WT"
272 represents the difference between the mutant and wild type genotypes. 352 represents the difference between the mutant and wild type genotypes.
273 353 Multiple contrasts should be separated by commas and spaces must not be used.
274 The values should be seperated by commas and spaces must not be used.
275 354
276 **Filter Low CPM:** 355 **Filter Low CPM:**
277 Option to ignore the genes that do not show significant levels of 356 Option to ignore the genes that do not show significant levels of
278 expression, this filtering is dependent on two criteria: 357 expression, this filtering is dependent on two criteria:
279 358
289 with two experimental groups each with two members, if there is a change from 368 with two experimental groups each with two members, if there is a change from
290 insignificant cpm to significant cpm but the sample requirement is set to 3, 369 insignificant cpm to significant cpm but the sample requirement is set to 3,
291 then this will cause that gene to fail the criteria. When in doubt simply do not 370 then this will cause that gene to fail the criteria. When in doubt simply do not
292 filter. 371 filter.
293 372
294
295 **Normalisation Method:** 373 **Normalisation Method:**
296 Option for using different methods to rescale the raw library 374 Option for using different methods to rescale the raw library
297 size. For more information, see calcNormFactor section in the edgeR_ user's 375 size. For more information, see calcNormFactor section in the edgeR_ user's
298 manual. 376 manual.
299 377
307 By default error rate for multiple testing is controlled using Benjamini and 385 By default error rate for multiple testing is controlled using Benjamini and
308 Hochberg's false discovery rate control at a threshold value of 0.05. However 386 Hochberg's false discovery rate control at a threshold value of 0.05. However
309 there are options to change this to custom values. 387 there are options to change this to custom values.
310 388
311 * **P-Value Adjustment Method:** 389 * **P-Value Adjustment Method:**
312 Change the multiple testing control method, the options are BH(1995) and 390 Change the multiple testing control method, the options are BH(1995) and
313 BY(2001) which are both false discovery rate controls. There is also 391 BY(2001) which are both false discovery rate controls. There is also
314 Holm(1979) which is a method for family-wise error rate control. 392 Holm(1979) which is a method for family-wise error rate control.
315 393
316 * **Adjusted Threshold:** 394 * **Adjusted Threshold:**
317 Set the threshold for the resulting value of the multiple testing control 395 Set the threshold for the resulting value of the multiple testing control
318 method. Only observations whose statistic falls below this value is 396 method. Only observations whose statistic falls below this value is
319 considered significant, thus highlighted in the MA plot. 397 considered significant, thus highlighted in the MA plot.
320 398
321 * **Minimum log2-fold-change Required:** 399 * **Minimum log2-fold-change Required:**
322 In addition to meeting the requirement for the adjusted statistic for 400 In addition to meeting the requirement for the adjusted statistic for
323 multiple testing, the observation must have an absolute log2-fold-change 401 multiple testing, the observation must have an absolute log2-fold-change
324 greater than this threshold to be considered significant, thus highlighted 402 greater than this threshold to be considered significant, thus highlighted
325 in the MA plot. 403 in the MA plot.
326 404
405 **Outputs**
406
407 This tool outputs a table of differentially expressed genes for each contrast of interest and a HTML report with plots and additional information. Optionally you can choose to output the normalised counts table and the RData file.
408
327 ----- 409 -----
328 410
329 **Citations:** 411 **Citations:**
330 412
331 .. class:: infomark 413 .. class:: infomark
332 414
333 limma 415 limma
334 416
335 Please cite the paper below for the limma software itself. Please also try 417 Please cite the paper below for the limma software itself. Please also try
336 to cite the appropriate methodology articles that describe the statistical 418 to cite the appropriate methodology articles that describe the statistical
337 methods implemented in limma, depending on which limma functions you are 419 methods implemented in limma, depending on which limma functions you are
338 using. The methodology articles are listed in Section 2.1 of the limma 420 using. The methodology articles are listed in Section 2.1 of the limma
339 User's Guide. 421 User's Guide.
340 422
341 * Smyth GK (2005). Limma: linear models for microarray data. In: 423 * Smyth GK (2005). Limma: linear models for microarray data. In:
342 'Bioinformatics and Computational Biology Solutions using R and 424 'Bioinformatics and Computational Biology Solutions using R and
343 Bioconductor'. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, 425 Bioconductor'. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry,
344 W. Huber (eds), Springer, New York, pages 397-420. 426 W. Huber (eds), Springer, New York, pages 397-420.
345 427
346 * Law CW, Chen Y, Shi W, and Smyth GK (2014). Voom: 428 * Law CW, Chen Y, Shi W, and Smyth GK (2014). Voom:
347 precision weights unlock linear model analysis tools for 429 precision weights unlock linear model analysis tools for
348 RNA-seq read counts. Genome Biology 15, R29. 430 RNA-seq read counts. Genome Biology 15, R29.
349 431
350 * Liu R, Holik AZ, Su S, Jansz N, Chen K, Leong HS, Blewitt ME, Asselin-Labat ML, Smyth GK, Ritchie ME (2015). Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses. Nucleic Acids Research, 43(15), e97. 432 * Liu R, Holik AZ, Su S, Jansz N, Chen K, Leong HS, Blewitt ME, Asselin-Labat ML, Smyth GK, Ritchie ME (2015). Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses. Nucleic Acids Research, 43(15), e97.
351 433
352 * Ritchie, M. E., Diyagama, D., Neilson, J., van Laar, R., Dobrovic, 434 * Ritchie, M. E., Diyagama, D., Neilson, J., van Laar, R., Dobrovic,
353 A., Holloway, A., and Smyth, G. K. (2006). Empirical array quality weights 435 A., Holloway, A., and Smyth, G. K. (2006). Empirical array quality weights
354 for microarray data. BMC Bioinformatics 7, Article 261. 436 for microarray data. BMC Bioinformatics 7, Article 261.
355 437
356 .. class:: infomark 438 .. class:: infomark
357 439
358 edgeR 440 edgeR
359 441
360 Please cite the first paper for the software itself and the other papers for 442 Please cite the first paper for the software itself and the other papers for
361 the various original statistical methods implemented in edgeR. See 443 the various original statistical methods implemented in edgeR. See
362 Section 1.2 in the User's Guide for more detail. 444 Section 1.2 in the User's Guide for more detail.
363 445
364 * Robinson MD, McCarthy DJ and Smyth GK (2010). edgeR: a Bioconductor 446 * Robinson MD, McCarthy DJ and Smyth GK (2010). edgeR: a Bioconductor
365 package for differential expression analysis of digital gene expression 447 package for differential expression analysis of digital gene expression
366 data. Bioinformatics 26, 139-140 448 data. Bioinformatics 26, 139-140
367 449
368 * Robinson MD and Smyth GK (2007). Moderated statistical tests for assessing 450 * Robinson MD and Smyth GK (2007). Moderated statistical tests for assessing
369 differences in tag abundance. Bioinformatics 23, 2881-2887 451 differences in tag abundance. Bioinformatics 23, 2881-2887
370 452
371 * Robinson MD and Smyth GK (2008). Small-sample estimation of negative 453 * Robinson MD and Smyth GK (2008). Small-sample estimation of negative
372 binomial dispersion, with applications to SAGE data. 454 binomial dispersion, with applications to SAGE data.
373 Biostatistics, 9, 321-332 455 Biostatistics, 9, 321-332
374 456
375 * McCarthy DJ, Chen Y and Smyth GK (2012). Differential expression analysis 457 * McCarthy DJ, Chen Y and Smyth GK (2012). Differential expression analysis
376 of multifactor RNA-Seq experiments with respect to biological variation. 458 of multifactor RNA-Seq experiments with respect to biological variation.
377 Nucleic Acids Research 40, 4288-4297 459 Nucleic Acids Research 40, 4288-4297
378 460
379 Please report problems or suggestions to: su.s@wehi.edu.au 461 Please report problems or suggestions to: su.s@wehi.edu.au
380 462
381 .. _edgeR: http://www.bioconductor.org/packages/release/bioc/html/edgeR.html 463 .. _edgeR: http://www.bioconductor.org/packages/release/bioc/html/edgeR.html
382 .. _limma: http://www.bioconductor.org/packages/release/bioc/html/limma.html 464 .. _limma: http://www.bioconductor.org/packages/release/bioc/html/limma.html
383 ]]> 465 ]]></help>
384 </help>
385 <citations> 466 <citations>
386 <citation type="doi">10.1093/nar/gkv412</citation> 467 <citation type="doi">10.1093/nar/gkv412</citation>
387 </citations> 468 </citations>
388 </tool> 469 </tool>