Mercurial > repos > shians > shrnaseq
annotate hairpinTool.xml @ 8:548802b3492f
Version 1.0.9
- Added R session info to output
- Added email for bug reports
- Changed edgeR requirement to be stopping condition
- Changed names of gene-wise comparison tables to "gene_level" from "roast"
- Fixed selection of multiple genes for barcode plotting
| author | shian_su <registertonysu@gmail.com> |
|---|---|
| date | Fri, 02 May 2014 17:22:24 +1000 |
| parents | 91e411fcdecc |
| children | f1076bfb0ed1 |
| rev | line source |
|---|---|
| 8 | 1 <tool id="shRNAseq" name="shRNAseq Tool" version="1.0.9"> |
| 2 | 2 <description> |
| 3 Analyse hairpin differential representation using edgeR | |
| 4 </description> | |
| 5 | |
| 6 <requirements> | |
|
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7 <requirement type="R-module" version="3.5.27">edgeR</requirement> |
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8 <requirement type="R-module" version="3.18.13">limma</requirement> |
| 2 | 9 </requirements> |
| 10 | |
| 11 <stdio> | |
| 12 <exit_code range="1:" level="fatal" description="Tool exception" /> | |
| 13 </stdio> | |
| 14 | |
| 15 <command interpreter="Rscript"> | |
|
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16 hairpinTool.R $inputOpt.inputType |
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17 #if $inputOpt.inputType=="fastq": |
| 2 | 18 #for $i, $fas in enumerate($inputOpt.fastq): |
| 19 fastq::$fas.file | |
| 20 #end for | |
| 21 | |
| 22 $inputOpt.hairpin | |
| 23 $inputOpt.samples | |
| 24 | |
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25 #if $inputOpt.positions.posOption=="yes": |
| 2 | 26 $inputOpt.positions.barstart |
| 27 $inputOpt.positions.barend | |
| 28 $inputOpt.positions.hpstart | |
| 29 $inputOpt.positions.hpend | |
| 30 #else: | |
| 31 1 | |
| 32 5 | |
| 33 37 | |
| 34 57 | |
| 35 #end if | |
| 36 #else: | |
| 37 $inputOpt.counts | |
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38 $inputOpt.hairpin |
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39 $inputOpt.samples |
| 2 | 40 0 0 0 |
| 41 #end if | |
| 42 | |
|
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43 #if $filterCPM.filtOption=="yes": |
| 2 | 44 $filterCPM.cpmReq |
| 45 $filterCPM.sampleReq | |
| 46 #else: | |
| 47 -Inf | |
| 48 -Inf | |
| 49 #end if | |
| 50 | |
| 51 $fdr | |
| 52 $lfc | |
| 53 $workMode.mode | |
| 54 $outFile | |
| 55 $outFile.files_path | |
| 56 | |
| 57 #if $workMode.mode=="classic": | |
| 58 "$workMode.pair1" | |
| 59 "$workMode.pair2" | |
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60 #elif $workMode.mode=="glm": |
| 2 | 61 "$workMode.contrast" |
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62 $workMode.roast.roastOption |
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63 #if $workMode.roast.roastOption=="yes": |
| 2 | 64 $workMode.roast.hairpinReq |
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65 $workMode.roast.select.selOption |
| 2 | 66 "$workMode.roast.select.selection" |
| 67 #else: | |
| 68 0 | |
| 69 0 | |
| 70 0 | |
| 71 #end if | |
| 72 #end if | |
| 73 </command> | |
| 74 | |
| 75 <inputs> | |
| 76 <conditional name="inputOpt"> | |
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77 <param name="inputType" type="select" label="Input File Type"> |
| 2 | 78 <option value="fastq">FastQ File</option> |
| 79 <option value="counts">Table of Counts</option> | |
| 80 </param> | |
| 81 | |
| 82 <when value="fastq"> | |
| 83 <param name="hairpin" type="data" format="tabular" | |
| 84 label="Hairpin Annotation"/> | |
| 85 | |
| 86 | |
| 87 <param name="samples" type="data" format="tabular" | |
| 88 label="Sample Annotation"/> | |
| 89 | |
| 90 <repeat name="fastq" title="FastQ Files"> | |
| 91 <param name="file" type="data" format="fastq"/> | |
| 92 </repeat> | |
| 93 | |
| 94 <conditional name="positions"> | |
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95 <param name="posOption" type="select" |
| 2 | 96 label="Specify Barcode and Hairpin Locations?" |
| 97 help="Default Positions: Barcode: 1 to 5, Hairpin: 37 to 57."> | |
| 98 <option value="no" selected="True">No</option> | |
| 99 <option value="yes">Yes</option> | |
| 100 </param> | |
| 101 | |
| 102 <when value="yes"> | |
| 103 <param name="barstart" type="integer" value="1" | |
| 104 label="Barcode Starting Position"/> | |
| 105 <param name="barend" type="integer" value="5" | |
| 106 label="Barcode Ending Position"/> | |
| 107 | |
| 108 <param name="hpstart" type="integer" value="37" | |
| 109 label="Hairpin Starting Position"/> | |
| 110 | |
| 111 <param name="hpend" type="integer" value="57" | |
| 112 label="Hairpin Ending Position"/> | |
| 113 </when> | |
| 114 | |
| 115 <when value="no"/> | |
| 116 </conditional> | |
| 117 </when> | |
| 118 | |
| 119 <when value="counts"> | |
| 120 <param name="counts" type="data" format="tabular" label="Counts Table"/> | |
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121 <param name="hairpin" type="data" format="tabular" |
| 2 | 122 label="Hairpin Annotation"/> |
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123 <param name="samples" type="data" format="tabular" |
| 2 | 124 label="Sample Annotation"/> |
| 125 </when> | |
| 126 </conditional> | |
| 127 | |
| 128 <conditional name="filterCPM"> | |
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129 <param name="filtOption" type="select" label="Filter Low CPM?" |
| 2 | 130 help="Ignore hairpins with very low representation when performing |
| 131 analysis."> | |
| 132 <option value="yes">Yes</option> | |
| 133 <option value="no">No</option> | |
| 134 </param> | |
| 135 | |
| 136 <when value="yes"> | |
| 137 <param name="cpmReq" type="float" value="0.5" min="0" max="1" | |
| 138 label="Minimum CPM"/> | |
| 139 | |
| 140 <param name="sampleReq" type="integer" value="1" min="0" | |
| 141 label="Minimum Samples" | |
| 142 help="Filter out all the genes that do not meet the minimum | |
| 143 CPM in at least this many samples."/> | |
| 144 </when> | |
| 145 | |
| 146 <when value="no"/> | |
| 147 | |
| 148 </conditional> | |
| 149 | |
| 150 <conditional name="workMode"> | |
| 151 <param name="mode" type="select" label="Analysis Type" | |
| 152 help="Classic Exact Tests are useful for simple comparisons across | |
| 153 two sampling groups. Generalised linear models allow for more | |
| 154 complex contrasts and gene level analysis to be made."> | |
| 155 <option value="classic">Classic Exact Test</option> | |
| 156 <option value="glm">Generalised Linear Model</option> | |
| 157 </param> | |
| 158 | |
| 159 <when value="classic"> | |
| 160 <param name="pair1" type="text" label="Compare" size="40"/> | |
| 161 <param name="pair2" type="text" label="To" size="40" | |
| 162 help="The analysis will subtract values of this group from those | |
| 163 in the group above to establish the difference."/> | |
| 164 </when> | |
| 165 | |
| 166 <when value="glm"> | |
| 167 <param name="contrast" type="text" size="60" | |
| 168 label="Contrasts of interest" | |
| 169 help="Specify equations defining contrasts to be made. Eg. | |
| 170 KD-Control will result in positive fold change if KD has | |
| 171 greater expression and negative if Control has greater | |
| 172 expression."/> | |
| 173 | |
| 174 <conditional name="roast"> | |
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175 <param name="roastOption" type="select" |
| 2 | 176 label="Perform Gene Level Analysis?" |
| 177 help="Analyse LogFC tendencies for hairpins belonging | |
| 178 to the same gene."> | |
| 179 <option value="no">No</option> | |
| 180 <option value="yes">Yes</option> | |
| 181 </param> | |
| 182 | |
| 183 <when value="yes"> | |
| 184 <param name="hairpinReq" type="integer" value="2" min="2" | |
| 185 label="Minimum Hairpins" | |
| 186 help="Only genes with at least this many hairpins will | |
| 187 be analysed."/> | |
| 188 | |
| 189 <conditional name="select"> | |
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190 <param name="selOption" type="select" |
| 2 | 191 label="Gene Selection Method"> |
| 192 <option value="rank">By p-value Rank</option> | |
| 193 <option value="geneID">By Gene Identifier</option> | |
| 194 </param> | |
| 195 <when value="rank"> | |
| 196 <param name="selection" type="text" size="40" value="1:5" | |
| 197 label="Ranks of Top Genes to Plot" | |
| 198 help="Genes are ranked in ascending p-value for | |
| 199 differential representation, individual ranks can | |
| 200 be entered seperated by comma or a range seperated | |
| 201 by colon."/> | |
| 202 </when> | |
| 203 <when value="geneID"> | |
| 204 <param name="selection" type="text" size="80" value="" | |
| 205 label="Symbols of Genes to Plot" | |
| 206 help="Select genes based on their identifier in the | |
| 207 'Gene' column of the sample information file. | |
| 208 Please ensure exact match with the values in input | |
| 209 file and separate selections with commas."/> | |
| 210 </when> | |
| 211 </conditional> | |
| 212 | |
| 213 | |
| 214 </when> | |
| 215 | |
| 216 <when value="no"/> | |
| 217 </conditional> | |
| 218 </when> | |
| 219 </conditional> | |
| 220 | |
| 221 <param name="fdr" type="float" value="0.05" min="0" max="1" | |
| 222 label="FDR Threshold" | |
| 223 help="All observations below this threshold will be highlighted | |
| 224 in the smear plot."/> | |
| 225 <param name="lfc" type="float" value="0" min="0" | |
| 226 label="Absolute LogFC Threshold" | |
| 227 help="In additional to meeting the FDR requirement, the absolute | |
| 228 value of the log-fold-change of the observation must be above | |
| 229 this threshold to be highlighted."/> | |
| 230 </inputs> | |
| 231 | |
| 232 <outputs> | |
| 233 <data format="html" name="outFile" label="shRNAseq Analysis"/> | |
| 234 </outputs> | |
| 235 <help> | |
| 236 .. class:: infomark | |
| 237 | |
| 238 **What it does** | |
| 239 | |
| 240 Given tables containing information about the hairpins and their associated | |
| 241 barcodes, information about the samples and fastq file containing the hairpin | |
| 242 reads. This tool will generate plots and tables for the analysis of differential | |
| 243 representation. | |
| 244 | |
| 7 | 245 .. class:: infomark |
| 246 | |
| 247 A tutorial of how to use this tool is available at: | |
| 248 http://bioinf.wehi.edu.au/shRNAseq/galaxy.html | |
| 249 | |
| 2 | 250 ----- |
| 251 | |
| 252 .. class:: infomark | |
| 253 | |
| 254 **INPUTS** | |
| 255 | |
| 256 **Input File Type:** | |
| 257 | |
| 258 This tool is able to either generate counts from a raw FastQ file given the | |
| 259 information regarding the samples and hairpins. Alternatively if a table of | |
| 260 counts has already been generated it can also be used. | |
| 261 | |
| 262 **Counts Table (Counts Input):** | |
| 263 | |
| 264 A tab delimited text table of information regarding the counts of hairpins. | |
| 265 Should have a column 'ID' to denote the hairpins that counts correspond to. Each | |
| 266 additional column should have titles corresponding to the label for the sample. | |
| 267 | |
| 268 Example:: | |
| 269 | |
| 270 ID Sample1 Sample2 Sample3 | |
| 271 Control1 49802 48014 40148 | |
| 272 Control2 12441 16352 14232 | |
| 273 Control3 9842 9148 9111 | |
| 274 Hairpin1 3300 3418 2914 | |
| 275 Hairpin2 91418 95812 93174 | |
| 276 Hairpin3 32985 31975 35104 | |
| 277 Hairpin4 12082 14081 14981 | |
| 278 Hairpin5 2491 2769 2691 | |
| 279 Hairpin6 1294 1486 1642 | |
| 280 Hairpin7 49501 49076 47611 | |
| 281 ... | |
| 282 | |
| 283 **Hairpin Annotation:** | |
| 284 | |
| 285 A tab delimited text table of information regarding the hairpins. Should have | |
| 286 columns 'ID', 'Sequences' and 'Gene' to uniquely identify the hairpin, align it | |
| 287 with the reads to produce counts and identify which gene the hairpin acts on. | |
| 288 | |
| 289 NOTE: the column names are case sensitive and should be input exactly as they | |
| 290 are shown here. | |
| 291 | |
| 292 Example:: | |
| 293 | |
| 294 ID Sequences Gene | |
| 295 Control1 TCTCGCTTGGGCGAGAGTAAG 2 | |
| 296 Control2 CCGCCTGAAGTCTCTGATTAA 2 | |
| 297 Control3 AGGAATTATAATGCTTATCTA 2 | |
| 298 Hairpin1 AAGGCAGAGACTGACCACCTA 4 | |
| 299 Hairpin2 GAGCGACCTGGTGTTACTCTA 4 | |
| 300 Hairpin3 ATGGTGTAAATAGAGCTGTTA 4 | |
| 301 Hairpin4 CAGCTCATCTTCTGTGAAGAA 4 | |
| 302 Hairpin5 CAGCTCTGTGGGTCAGAAGAA 4 | |
| 303 Hairpin6 CCAGGCACAGATCTCAAGATA 4 | |
| 304 Hairpin7 ATGACAAGAAAGACATCTCAA 7 | |
| 305 ... | |
| 306 | |
| 307 **Sample Annotation (FastQ Input):** | |
| 308 | |
| 309 A tab delimited text table of information regarding the samples. Should have | |
| 310 columns 'ID', 'Sequences' and 'group' to uniquely identify each sample, identify | |
| 311 the sample in the reads by its barcode sequence and correctly group replicates | |
| 312 for analysis. Additional columns may inserted for annotation purposes and will | |
| 313 not interfere with analysis as long as the necessary columns are present. | |
| 314 | |
| 315 NOTE: the column names are case sensitive and should be input exactly as they | |
| 316 are shown here. | |
| 317 | |
| 318 Example:: | |
| 319 | |
| 320 ID Sequences group Replicate | |
| 321 3 GAAAG Day 2 1 | |
| 322 6 GAACC Day 10 1 | |
| 323 9 GAAGA Day 5 GFP neg 1 | |
| 324 16 GAATT Day 5 GFP pos 1 | |
| 325 18 GACAC Day 2 2 | |
| 326 21 GACCA Day 10 2 | |
| 327 28 GACGT Day 5 GFP neg 2 | |
| 328 31 GACTG Day 5 GFP pos 2 | |
| 329 33 GAGAA Day 2 3 | |
| 330 40 GAGCT Day 10 3 | |
| 331 ... | |
| 332 | |
| 333 **Specify Barcode and Hairpin Locations (FastQ Input):** | |
| 334 | |
| 335 It is assumed that in the sequencing reads that the first 5 bases are the | |
| 336 barcodes and that bases 37-57 are the hairpins. If this is not the case then the | |
| 337 values of the positions can be changed, however it still requires the barcodes | |
| 338 and hairpins to be in a consistent location an in a continuous sequence. | |
| 339 | |
| 340 **Filter Low CPM?:** | |
| 341 | |
| 342 Often in a large screen there may members with very low counts which are of no | |
| 343 interest in the experiment, these may be filtered out to speed up computations. | |
| 344 Filtering will be based on counts per million in a required number of samples. | |
| 345 | |
| 346 **Analysis Type:** | |
| 347 | |
| 348 * **Classic Exact Test:** This allows two experimental groups to be compared and | |
| 349 p-values for differential representation derivec for each hairpin. Simple and | |
| 350 fast for straightforward comparisons. In this option you will have the option of | |
| 351 "*Compare* x *To* y" which implicitly subtracts the data from y from that of x | |
| 352 to produce the comparison. | |
| 353 | |
| 354 * **Generalised Linear Model:** This allow for complex contrasts to be specified | |
| 355 and also gene level analysis to be performed. If this option is chosen then | |
| 356 contrasts must be explicitly stated in equations and multiple contrasts can be | |
| 357 made. In addition there will be the option to analyse hairpins on a per-gene | |
| 358 basis to see if hairpins belonging to a particular gene have any overall | |
| 359 tendencies for the direction of their log-fold-change. | |
| 360 | |
| 361 **FDR Threshold:** | |
| 362 The smear plot in the output will have hairpins highlighted to signify | |
| 363 significant differential representation. The significance is determined by | |
| 364 contorlling the false discovery rate, only those with a FDR lower than the | |
| 365 threshold will be highlighted in the plot. | |
| 366 | |
| 367 ----- | |
| 368 | |
| 369 **Citations:** | |
| 370 | |
| 371 .. class:: infomark | |
| 372 | |
| 373 limma | |
| 374 | |
| 375 Please cite the paper below for the limma software itself. Please also try | |
| 376 to cite the appropriate methodology articles that describe the statistical | |
| 377 methods implemented in limma, depending on which limma functions you are | |
| 378 using. The methodology articles are listed in Section 2.1 of the limma | |
| 379 User's Guide. | |
| 380 | |
| 381 * Smyth, GK (2005). Limma: linear models for microarray data. In: | |
| 382 'Bioinformatics and Computational Biology Solutions using R and | |
| 383 Bioconductor'. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, | |
| 384 W. Huber (eds), Springer, New York, pages 397-420. | |
| 385 | |
| 386 .. class:: infomark | |
| 387 | |
| 388 edgeR | |
| 389 | |
| 390 Please cite the first paper for the software itself and the other papers for | |
| 391 the various original statistical methods implemented in edgeR. See | |
| 392 Section 1.2 in the User's Guide for more detail. | |
| 393 | |
| 394 * Robinson MD, McCarthy DJ and Smyth GK (2010). edgeR: a Bioconductor | |
| 395 package for differential expression analysis of digital gene expression | |
| 396 data. Bioinformatics 26, 139-140 | |
| 397 | |
| 398 * Robinson MD and Smyth GK (2007). Moderated statistical tests for assessing | |
| 399 differences in tag abundance. Bioinformatics 23, 2881-2887 | |
| 400 | |
| 401 * Robinson MD and Smyth GK (2008). Small-sample estimation of negative | |
| 402 binomial dispersion, with applications to SAGE data. | |
| 403 Biostatistics, 9, 321-332 | |
| 404 | |
| 405 * McCarthy DJ, Chen Y and Smyth GK (2012). Differential expression analysis | |
| 406 of multifactor RNA-Seq experiments with respect to biological variation. | |
| 407 Nucleic Acids Research 40, 4288-4297 | |
| 8 | 408 |
| 409 Report problems to: su.s@wehi.edu.au | |
| 2 | 410 |
| 411 .. _edgeR: http://www.bioconductor.org/packages/release/bioc/html/edgeR.html | |
| 412 .. _limma: http://www.bioconductor.org/packages/release/bioc/html/limma.html | |
| 413 </help> | |
| 414 </tool> | |
| 415 |