Mercurial > repos > iuc > deseq2
annotate deseq2.R @ 13:3660c9088494 draft
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit c2e762b248fa86bb844c8e2d136e127d701d7b5a
author | iuc |
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date | Wed, 21 Feb 2018 00:06:27 -0500 |
parents | bd06df00180a |
children | d0c39b5e78cf |
rev | line source |
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4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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1 #!/usr/bin/env Rscript |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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2 |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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3 # A command-line interface to DESeq2 for use with Galaxy |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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4 # written by Bjoern Gruening and modified by Michael Love 2016.03.30 |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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5 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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6 # one of these arguments is required: |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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7 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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8 # 'factors' a JSON list object from Galaxy |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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9 # |
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6f91b8ce6e07
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 31371b7f8c564c4b4edc8914e83492ee7f04ea31
iuc
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10 # 'sample_table' is a sample table as described in ?DESeqDataSetFromHTSeqCount |
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4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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11 # with columns: sample name, filename, then factors (variables) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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12 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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13 # the output file has columns: |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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14 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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15 # baseMean (mean normalized count) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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16 # log2FoldChange (by default a moderated LFC estimate) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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17 # lfcSE (the standard error) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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18 # stat (the Wald statistic) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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19 # pvalue (p-value from comparison of Wald statistic to a standard Normal) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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20 # padj (adjusted p-value, Benjamini Hochberg correction on genes which pass the mean count filter) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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21 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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22 # the first variable in 'factors' and first column in 'sample_table' will be the primary factor. |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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23 # the levels of the primary factor are used in the order of appearance in factors or in sample_table. |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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24 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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25 # by default, levels in the order A,B,C produces a single comparison of B vs A, to a single file 'outfile' |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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26 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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27 # for the 'many_contrasts' flag, levels in the order A,B,C produces comparisons C vs A, B vs A, C vs B, |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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28 # to a number of files using the 'outfile' prefix: 'outfile.condition_C_vs_A' etc. |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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29 # all plots will still be sent to a single PDF, named by the arg 'plots', with extra pages. |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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30 # |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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31 # fit_type is an integer valued argument, with the options from ?estimateDisperions |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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32 # 1 "parametric" |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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33 # 2 "local" |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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34 # 3 "mean" |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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35 |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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36 # setup R error handling to go to stderr |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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37 options( show.error.messages=F, error = function () { cat( geterrmessage(), file=stderr() ); q( "no", 1, F ) } ) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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38 |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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39 # we need that to not crash galaxy with an UTF8 error on German LC settings. |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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40 loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8") |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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41 |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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42 library("getopt") |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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43 library("tools") |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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44 options(stringAsFactors = FALSE, useFancyQuotes = FALSE) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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45 args <- commandArgs(trailingOnly = TRUE) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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46 |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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47 # get options, using the spec as defined by the enclosed list. |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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48 # we read the options from the default: commandArgs(TRUE). |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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49 spec <- matrix(c( |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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50 "quiet", "q", 0, "logical", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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51 "help", "h", 0, "logical", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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52 "outfile", "o", 1, "character", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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53 "countsfile", "n", 1, "character", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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54 "factors", "f", 1, "character", |
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55 "files_to_labels", "l", 1, "character", |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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56 "plots" , "p", 1, "character", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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57 "sample_table", "s", 1, "character", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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58 "tximport", "i", 0, "logical", |
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25204a860b79
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 0a56599c36b4968095ec5a3cb589f94fb139466c
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59 "txtype", "y", 1, "character", |
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4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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60 "tx2gene", "x", 1, "character", # a space-sep tx-to-gene map or GTF file (auto detect .gtf/.GTF) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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61 "fit_type", "t", 1, "integer", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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62 "many_contrasts", "m", 0, "logical", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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63 "outlier_replace_off" , "a", 0, "logical", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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64 "outlier_filter_off" , "b", 0, "logical", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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65 "auto_mean_filter_off", "c", 0, "logical", |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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66 "beta_prior_off", "d", 0, "logical"), |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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67 byrow=TRUE, ncol=4) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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68 opt <- getopt(spec) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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69 |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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70 # if help was asked for print a friendly message |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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71 # and exit with a non-zero error code |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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72 if (!is.null(opt$help)) { |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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73 cat(getopt(spec, usage=TRUE)) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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74 q(status=1) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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75 } |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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76 |
4939397c4706
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77 # enforce the following required arguments |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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78 if (is.null(opt$outfile)) { |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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79 cat("'outfile' is required\n") |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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80 q(status=1) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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81 } |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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82 if (is.null(opt$sample_table) & is.null(opt$factors)) { |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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83 cat("'factors' or 'sample_table' is required\n") |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
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84 q(status=1) |
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parents:
diff
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85 } |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
parents:
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changeset
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86 |
4939397c4706
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87 verbose <- if (is.null(opt$quiet)) { |
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iuc
parents:
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88 TRUE |
4939397c4706
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89 } else { |
4939397c4706
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iuc
parents:
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90 FALSE |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
parents:
diff
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91 } |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
iuc
parents:
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92 |
4939397c4706
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93 if (!is.null(opt$tximport)) { |
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94 if (is.null(opt$tx2gene)) stop("A transcript-to-gene map or a GTF file is required for tximport") |
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95 if (tolower(file_ext(opt$tx2gene)) == "gtf") { |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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96 gtfFile <- opt$tx2gene |
4939397c4706
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97 } else { |
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98 gtfFile <- NULL |
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99 tx2gene <- read.table(opt$tx2gene, header=FALSE) |
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parents:
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100 } |
4939397c4706
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101 useTXI <- TRUE |
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102 } else { |
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103 useTXI <- FALSE |
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104 } |
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105 |
4939397c4706
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106 suppressPackageStartupMessages({ |
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107 library("DESeq2") |
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108 library("RColorBrewer") |
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109 library("gplots") |
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110 }) |
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111 |
4939397c4706
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112 # build or read sample table |
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113 |
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114 trim <- function (x) gsub("^\\s+|\\s+$", "", x) |
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115 |
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116 # switch on if 'factors' was provided: |
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117 if (!is.null(opt$factors)) { |
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118 library("rjson") |
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119 parser <- newJSONParser() |
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120 parser$addData(opt$factors) |
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121 factorList <- parser$getObject() |
12
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122 filenames_to_labels <- fromJSON(opt$files_to_labels) |
6
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123 factors <- sapply(factorList, function(x) x[[1]]) |
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124 primaryFactor <- factors[1] |
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125 filenamesIn <- unname(unlist(factorList[[1]][[2]])) |
12
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126 labs = unname(unlist(filenames_to_labels[basename(filenamesIn)])) |
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127 sampleTable <- data.frame(sample=basename(filenamesIn), |
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128 filename=filenamesIn, |
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129 row.names=filenamesIn, |
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130 stringsAsFactors=FALSE) |
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131 for (factor in factorList) { |
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132 factorName <- trim(factor[[1]]) |
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133 sampleTable[[factorName]] <- character(nrow(sampleTable)) |
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134 lvls <- sapply(factor[[2]], function(x) names(x)) |
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135 for (i in seq_along(factor[[2]])) { |
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136 files <- factor[[2]][[i]][[1]] |
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137 sampleTable[files,factorName] <- trim(lvls[i]) |
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138 } |
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139 sampleTable[[factorName]] <- factor(sampleTable[[factorName]], levels=lvls) |
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140 } |
12
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141 rownames(sampleTable) <- labs |
6
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142 } else { |
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143 # read the sample_table argument |
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144 # this table is described in ?DESeqDataSet |
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145 # one column for the sample name, one for the filename, and |
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146 # the remaining columns for factors in the analysis |
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147 sampleTable <- read.delim(opt$sample_table, stringsAsFactors=FALSE) |
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148 factors <- colnames(sampleTable)[-c(1:2)] |
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149 for (factor in factors) { |
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150 lvls <- unique(as.character(sampleTable[[factor]])) |
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151 sampleTable[[factor]] <- factor(sampleTable[[factor]], levels=lvls) |
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152 } |
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153 } |
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154 |
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155 primaryFactor <- factors[1] |
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156 designFormula <- as.formula(paste("~", paste(rev(factors), collapse=" + "))) |
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157 |
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158 if (verbose) { |
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159 cat("DESeq2 run information\n\n") |
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160 cat("sample table:\n") |
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161 print(sampleTable[,-c(1:2),drop=FALSE]) |
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162 cat("\ndesign formula:\n") |
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163 print(designFormula) |
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164 cat("\n\n") |
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165 } |
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166 |
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167 # these are plots which are made once for each analysis |
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168 generateGenericPlots <- function(dds, factors) { |
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169 library("ggplot2") |
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170 library("ggrepel") |
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171 library("pheatmap") |
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172 |
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173 rld <- rlog(dds) |
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174 p <- plotPCA(rld, intgroup=rev(factors)) |
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175 print(p + geom_text_repel(aes_string(x = "PC1", y = "PC2", label = factor(colnames(dds))), size=3) + geom_point()) |
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176 dat <- assay(rld) |
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177 distsRL <- dist(t(dat)) |
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178 mat <- as.matrix(distsRL) |
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179 colors <- colorRampPalette( rev(brewer.pal(9, "Blues")) )(255) |
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180 pheatmap(mat, |
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181 clustering_distance_rows=distsRL, |
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182 clustering_distance_cols=distsRL, |
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183 col=colors, |
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184 main="Sample-to-sample distances") |
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185 plotDispEsts(dds, main="Dispersion estimates") |
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186 } |
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187 |
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188 # these are plots which can be made for each comparison, e.g. |
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189 # once for C vs A and once for B vs A |
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190 generateSpecificPlots <- function(res, threshold, title_suffix) { |
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191 use <- res$baseMean > threshold |
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192 if (sum(!use) == 0) { |
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193 h <- hist(res$pvalue, breaks=0:50/50, plot=FALSE) |
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194 barplot(height = h$counts, |
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195 col = "powderblue", space = 0, xlab="p-values", ylab="frequency", |
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196 main=paste("Histogram of p-values for",title_suffix)) |
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197 text(x = c(0, length(h$counts)), y = 0, label=paste(c(0,1)), adj=c(0.5,1.7), xpd=NA) |
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198 } else { |
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199 h1 <- hist(res$pvalue[!use], breaks=0:50/50, plot=FALSE) |
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200 h2 <- hist(res$pvalue[use], breaks=0:50/50, plot=FALSE) |
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201 colori <- c("filtered (low count)"="khaki", "not filtered"="powderblue") |
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202 barplot(height = rbind(h1$counts, h2$counts), beside = FALSE, |
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203 col = colori, space = 0, xlab="p-values", ylab="frequency", |
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204 main=paste("Histogram of p-values for",title_suffix)) |
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205 text(x = c(0, length(h1$counts)), y = 0, label=paste(c(0,1)), adj=c(0.5,1.7), xpd=NA) |
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206 legend("topright", fill=rev(colori), legend=rev(names(colori)), bg="white") |
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207 } |
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208 plotMA(res, main= paste("MA-plot for",title_suffix), ylim=range(res$log2FoldChange, na.rm=TRUE)) |
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209 } |
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210 |
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211 if (verbose) { |
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212 cat(paste("primary factor:",primaryFactor,"\n")) |
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213 if (length(factors) > 1) { |
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214 cat(paste("other factors in design:",paste(factors[-length(factors)],collapse=","),"\n")) |
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215 } |
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216 cat("\n---------------------\n") |
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217 } |
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218 |
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219 # if JSON input from Galaxy, path is absolute |
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220 # otherwise, from sample_table, assume it is relative |
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221 dir <- if (is.null(opt$factors)) { |
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222 "." |
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223 } else { |
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224 "" |
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225 } |
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226 |
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227 if (!useTXI) { |
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228 # construct the object from HTSeq files |
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229 dds <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, |
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230 directory = dir, |
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231 design = designFormula) |
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232 labs <- unname(unlist(filenames_to_labels[colnames(dds)])) |
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233 colnames(dds) <- labs |
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234 } else { |
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235 # construct the object using tximport |
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236 # first need to make the tx2gene table |
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237 # this takes ~2-3 minutes using Bioconductor functions |
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238 if (!is.null(gtfFile)) { |
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239 suppressPackageStartupMessages({ |
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240 library("GenomicFeatures") |
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241 }) |
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242 txdb <- makeTxDbFromGFF(gtfFile, format="gtf") |
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243 k <- keys(txdb, keytype = "GENEID") |
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244 df <- select(txdb, keys = k, keytype = "GENEID", columns = "TXNAME") |
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245 tx2gene <- df[, 2:1] # tx ID, then gene ID |
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246 } |
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247 library("tximport") |
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248 txiFiles <- as.character(sampleTable[,2]) |
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249 labs <- unname(unlist(filenames_to_labels[sampleTable[,1]])) |
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250 names(txiFiles) <- labs |
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251 txi <- tximport(txiFiles, type=opt$txtype, tx2gene=tx2gene) |
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252 dds <- DESeqDataSetFromTximport(txi, |
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253 sampleTable[,3:ncol(sampleTable),drop=FALSE], |
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254 designFormula) |
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255 } |
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256 |
4939397c4706
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257 if (verbose) cat(paste(ncol(dds), "samples with counts over", nrow(dds), "genes\n")) |
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258 # optional outlier behavior |
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259 if (is.null(opt$outlier_replace_off)) { |
4939397c4706
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260 minRep <- 7 |
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261 } else { |
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262 minRep <- Inf |
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263 if (verbose) cat("outlier replacement off\n") |
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264 } |
4939397c4706
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265 if (is.null(opt$outlier_filter_off)) { |
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266 cooksCutoff <- TRUE |
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267 } else { |
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268 cooksCutoff <- FALSE |
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269 if (verbose) cat("outlier filtering off\n") |
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270 } |
4939397c4706
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271 |
4939397c4706
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272 # optional automatic mean filtering |
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273 if (is.null(opt$auto_mean_filter_off)) { |
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274 independentFiltering <- TRUE |
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275 } else { |
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276 independentFiltering <- FALSE |
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277 if (verbose) cat("automatic filtering on the mean off\n") |
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278 } |
4939397c4706
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279 |
4939397c4706
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280 # shrinkage of LFCs |
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281 if (is.null(opt$beta_prior_off)) { |
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282 betaPrior <- TRUE |
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283 } else { |
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284 betaPrior <- FALSE |
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285 if (verbose) cat("beta prior off\n") |
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286 } |
4939397c4706
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287 |
4939397c4706
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288 # dispersion fit type |
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289 if (is.null(opt$fit_type)) { |
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290 fitType <- "parametric" |
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291 } else { |
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292 fitType <- c("parametric","local","mean")[opt$fit_type] |
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293 } |
4939397c4706
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294 |
4939397c4706
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295 if (verbose) cat(paste("using disperion fit type:",fitType,"\n")) |
4939397c4706
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296 |
4939397c4706
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297 # run the analysis |
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298 dds <- DESeq(dds, fitType=fitType, betaPrior=betaPrior, minReplicatesForReplace=minRep) |
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299 |
4939397c4706
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300 # create the generic plots and leave the device open |
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301 if (!is.null(opt$plots)) { |
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302 if (verbose) cat("creating plots\n") |
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303 pdf(opt$plots) |
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304 generateGenericPlots(dds, factors) |
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305 } |
4939397c4706
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306 |
4939397c4706
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307 n <- nlevels(colData(dds)[[primaryFactor]]) |
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308 allLevels <- levels(colData(dds)[[primaryFactor]]) |
4939397c4706
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309 |
4939397c4706
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310 if (!is.null(opt$countsfile)) { |
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311 normalizedCounts<-counts(dds,normalized=TRUE) |
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312 write.table(normalizedCounts, file=opt$countsfile, sep="\t", col.names=NA, quote=FALSE) |
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313 } |
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314 |
4939397c4706
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315 if (is.null(opt$many_contrasts)) { |
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316 # only contrast the first and second level of the primary factor |
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317 ref <- allLevels[1] |
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318 lvl <- allLevels[2] |
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319 res <- results(dds, contrast=c(primaryFactor, lvl, ref), |
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320 cooksCutoff=cooksCutoff, |
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321 independentFiltering=independentFiltering) |
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322 if (verbose) { |
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323 cat("summary of results\n") |
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324 cat(paste0(primaryFactor,": ",lvl," vs ",ref,"\n")) |
4939397c4706
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325 print(summary(res)) |
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326 } |
4939397c4706
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327 resSorted <- res[order(res$padj),] |
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328 outDF <- as.data.frame(resSorted) |
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329 outDF$geneID <- rownames(outDF) |
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330 outDF <- outDF[,c("geneID", "baseMean", "log2FoldChange", "lfcSE", "stat", "pvalue", "padj")] |
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331 filename <- opt$outfile |
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332 write.table(outDF, file=filename, sep="\t", quote=FALSE, row.names=FALSE, col.names=FALSE) |
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333 if (independentFiltering) { |
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334 threshold <- unname(attr(res, "filterThreshold")) |
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335 } else { |
4939397c4706
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336 threshold <- 0 |
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337 } |
4939397c4706
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338 title_suffix <- paste0(primaryFactor,": ",lvl," vs ",ref) |
4939397c4706
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339 if (!is.null(opt$plots)) { |
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340 generateSpecificPlots(res, threshold, title_suffix) |
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341 } |
4939397c4706
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parents:
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342 } else { |
4939397c4706
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iuc
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343 # rotate through the possible contrasts of the primary factor |
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344 # write out a sorted table of results with the contrast as a suffix |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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345 # add contrast specific plots to the device |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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346 for (i in seq_len(n-1)) { |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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347 ref <- allLevels[i] |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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348 contrastLevels <- allLevels[(i+1):n] |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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349 for (lvl in contrastLevels) { |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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350 res <- results(dds, contrast=c(primaryFactor, lvl, ref), |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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351 cooksCutoff=cooksCutoff, |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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352 independentFiltering=independentFiltering) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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353 resSorted <- res[order(res$padj),] |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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354 outDF <- as.data.frame(resSorted) |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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355 outDF$geneID <- rownames(outDF) |
4939397c4706
planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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356 outDF <- outDF[,c("geneID", "baseMean", "log2FoldChange", "lfcSE", "stat", "pvalue", "padj")] |
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357 filename <- paste0(opt$outfile,".",primaryFactor,"_",lvl,"_vs_",ref) |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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358 write.table(outDF, file=filename, sep="\t", quote=FALSE, row.names=FALSE, col.names=FALSE) |
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359 if (independentFiltering) { |
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360 threshold <- unname(attr(res, "filterThreshold")) |
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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 3bc8d91ee546682ef8e9303bd1044bb14cf21b07
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361 } else { |
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362 threshold <- 0 |
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363 } |
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364 title_suffix <- paste0(primaryFactor,": ",lvl," vs ",ref) |
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365 if (!is.null(opt$plots)) { |
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366 generateSpecificPlots(res, threshold, title_suffix) |
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367 } |
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368 } |
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369 } |
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370 } |
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371 |
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372 # close the plot device |
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373 if (!is.null(opt$plots)) { |
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374 cat("closing plot device\n") |
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375 dev.off() |
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376 } |
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377 |
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378 cat("Session information:\n\n") |
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379 |
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380 sessionInfo() |
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381 |