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annotate deseq2.R @ 26:47267a044ef1 draft

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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit e811a7887db870f4f94f620f52bce656c8d5ba23
author iuc
date Thu, 12 Apr 2018 17:29:07 -0400
parents 1a34aec44d60
children dc6bc19f05ab
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1 #!/usr/bin/env Rscript
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3 # A command-line interface to DESeq2 for use with Galaxy
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4 # written by Bjoern Gruening and modified by Michael Love 2016.03.30
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5 #
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6 # This argument is required:
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7 #
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8 # 'factors' a JSON list object from Galaxy
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9 #
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10 # the output file has columns:
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11 #
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12 # baseMean (mean normalized count)
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13 # log2FoldChange (by default a moderated LFC estimate)
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14 # lfcSE (the standard error)
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15 # stat (the Wald statistic)
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16 # pvalue (p-value from comparison of Wald statistic to a standard Normal)
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17 # padj (adjusted p-value, Benjamini Hochberg correction on genes which pass the mean count filter)
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18 #
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19 # the first variable in 'factors' will be the primary factor.
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20 # the levels of the primary factor are used in the order of appearance in factors.
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21 #
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22 # by default, levels in the order A,B,C produces a single comparison of B vs A, to a single file 'outfile'
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23 #
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24 # for the 'many_contrasts' flag, levels in the order A,B,C produces comparisons C vs A, B vs A, C vs B,
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25 # to a number of files using the 'outfile' prefix: 'outfile.condition_C_vs_A' etc.
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26 # all plots will still be sent to a single PDF, named by the arg 'plots', with extra pages.
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27 #
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28 # fit_type is an integer valued argument, with the options from ?estimateDisperions
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29 # 1 "parametric"
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30 # 2 "local"
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31 # 3 "mean"
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33 # setup R error handling to go to stderr
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34 options( show.error.messages=F, error = function () { cat( geterrmessage(), file=stderr() ); q( "no", 1, F ) } )
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36 # we need that to not crash galaxy with an UTF8 error on German LC settings.
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37 loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8")
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39 library("getopt")
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40 library("tools")
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41 options(stringAsFactors = FALSE, useFancyQuotes = FALSE)
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42 args <- commandArgs(trailingOnly = TRUE)
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44 # get options, using the spec as defined by the enclosed list.
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45 # we read the options from the default: commandArgs(TRUE).
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46 spec <- matrix(c(
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47 "quiet", "q", 0, "logical",
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48 "help", "h", 0, "logical",
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49 "outfile", "o", 1, "character",
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50 "countsfile", "n", 1, "character",
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51 "factors", "f", 1, "character",
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52 "files_to_labels", "l", 1, "character",
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53 "plots" , "p", 1, "character",
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54 "tximport", "i", 0, "logical",
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55 "txtype", "y", 1, "character",
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56 "tx2gene", "x", 1, "character", # a space-sep tx-to-gene map or GTF file (auto detect .gtf/.GTF)
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57 "fit_type", "t", 1, "integer",
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58 "many_contrasts", "m", 0, "logical",
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59 "outlier_replace_off" , "a", 0, "logical",
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60 "outlier_filter_off" , "b", 0, "logical",
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61 "auto_mean_filter_off", "c", 0, "logical",
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62 "beta_prior_off", "d", 0, "logical"),
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63 byrow=TRUE, ncol=4)
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64 opt <- getopt(spec)
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66 # if help was asked for print a friendly message
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67 # and exit with a non-zero error code
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68 if (!is.null(opt$help)) {
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69 cat(getopt(spec, usage=TRUE))
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70 q(status=1)
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71 }
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73 # enforce the following required arguments
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74 if (is.null(opt$outfile)) {
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75 cat("'outfile' is required\n")
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76 q(status=1)
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77 }
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78 if (is.null(opt$factors)) {
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79 cat("'factors' is required\n")
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80 q(status=1)
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81 }
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82
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83 verbose <- if (is.null(opt$quiet)) {
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84 TRUE
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85 } else {
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86 FALSE
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87 }
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88
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89 if (!is.null(opt$tximport)) {
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90 if (is.null(opt$tx2gene)) stop("A transcript-to-gene map or a GTF file is required for tximport")
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91 if (tolower(file_ext(opt$tx2gene)) == "gtf") {
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92 gtfFile <- opt$tx2gene
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93 } else {
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94 gtfFile <- NULL
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95 tx2gene <- read.table(opt$tx2gene, header=FALSE)
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96 }
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97 useTXI <- TRUE
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98 } else {
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99 useTXI <- FALSE
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100 }
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101
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102 suppressPackageStartupMessages({
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103 library("DESeq2")
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104 library("RColorBrewer")
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105 library("gplots")
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106 })
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107
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108 # build or read sample table
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109
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110 trim <- function (x) gsub("^\\s+|\\s+$", "", x)
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111
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112 # switch on if 'factors' was provided:
26
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113 library("rjson")
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114 parser <- newJSONParser()
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115 parser$addData(opt$factors)
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116 factorList <- parser$getObject()
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117 filenames_to_labels <- fromJSON(opt$files_to_labels)
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118 factors <- sapply(factorList, function(x) x[[1]])
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119 primaryFactor <- factors[1]
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120 filenamesIn <- unname(unlist(factorList[[1]][[2]]))
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121 labs = unname(unlist(filenames_to_labels[basename(filenamesIn)]))
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122 sampleTable <- data.frame(sample=basename(filenamesIn),
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123 filename=filenamesIn,
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124 row.names=filenamesIn,
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125 stringsAsFactors=FALSE)
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126 for (factor in factorList) {
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127 factorName <- trim(factor[[1]])
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128 sampleTable[[factorName]] <- character(nrow(sampleTable))
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129 lvls <- sapply(factor[[2]], function(x) names(x))
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130 for (i in seq_along(factor[[2]])) {
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131 files <- factor[[2]][[i]][[1]]
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132 sampleTable[files,factorName] <- trim(lvls[i])
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133 }
26
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134 sampleTable[[factorName]] <- factor(sampleTable[[factorName]], levels=lvls)
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135 }
26
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136 rownames(sampleTable) <- labs
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137
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138 primaryFactor <- factors[1]
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139 designFormula <- as.formula(paste("~", paste(rev(factors), collapse=" + ")))
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140
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141 if (verbose) {
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142 cat("DESeq2 run information\n\n")
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143 cat("sample table:\n")
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144 print(sampleTable[,-c(1:2),drop=FALSE])
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145 cat("\ndesign formula:\n")
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146 print(designFormula)
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147 cat("\n\n")
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148 }
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149
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150 # these are plots which are made once for each analysis
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151 generateGenericPlots <- function(dds, factors) {
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152 library("ggplot2")
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153 library("ggrepel")
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154 library("pheatmap")
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155
17
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156 rld <- rlog(dds)
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157 p <- plotPCA(rld, intgroup=rev(factors))
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158 print(p + geom_text_repel(aes_string(x = "PC1", y = "PC2", label = factor(colnames(dds))), size=3) + geom_point())
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159 dat <- assay(rld)
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160 distsRL <- dist(t(dat))
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161 mat <- as.matrix(distsRL)
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162 colors <- colorRampPalette( rev(brewer.pal(9, "Blues")) )(255)
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163 pheatmap(mat,
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164 clustering_distance_rows=distsRL,
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165 clustering_distance_cols=distsRL,
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166 col=colors,
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167 main="Sample-to-sample distances")
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168 plotDispEsts(dds, main="Dispersion estimates")
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169 }
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170
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171 # these are plots which can be made for each comparison, e.g.
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172 # once for C vs A and once for B vs A
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173 generateSpecificPlots <- function(res, threshold, title_suffix) {
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174 use <- res$baseMean > threshold
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175 if (sum(!use) == 0) {
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176 h <- hist(res$pvalue, breaks=0:50/50, plot=FALSE)
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177 barplot(height = h$counts,
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178 col = "powderblue", space = 0, xlab="p-values", ylab="frequency",
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179 main=paste("Histogram of p-values for",title_suffix))
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180 text(x = c(0, length(h$counts)), y = 0, label=paste(c(0,1)), adj=c(0.5,1.7), xpd=NA)
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181 } else {
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182 h1 <- hist(res$pvalue[!use], breaks=0:50/50, plot=FALSE)
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183 h2 <- hist(res$pvalue[use], breaks=0:50/50, plot=FALSE)
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184 colori <- c("filtered (low count)"="khaki", "not filtered"="powderblue")
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185 barplot(height = rbind(h1$counts, h2$counts), beside = FALSE,
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186 col = colori, space = 0, xlab="p-values", ylab="frequency",
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187 main=paste("Histogram of p-values for",title_suffix))
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188 text(x = c(0, length(h1$counts)), y = 0, label=paste(c(0,1)), adj=c(0.5,1.7), xpd=NA)
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189 legend("topright", fill=rev(colori), legend=rev(names(colori)), bg="white")
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190 }
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191 plotMA(res, main= paste("MA-plot for",title_suffix), ylim=range(res$log2FoldChange, na.rm=TRUE))
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192 }
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193
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194 if (verbose) {
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195 cat(paste("primary factor:",primaryFactor,"\n"))
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196 if (length(factors) > 1) {
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197 cat(paste("other factors in design:",paste(factors[-length(factors)],collapse=","),"\n"))
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198 }
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199 cat("\n---------------------\n")
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200 }
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201
26
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202 # For JSON input from Galaxy, path is absolute
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203 dir <- ""
17
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204
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205 if (!useTXI) {
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206 # construct the object from HTSeq files
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207 dds <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable,
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208 directory = dir,
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209 design = designFormula)
24
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210 labs <- unname(unlist(filenames_to_labels[colnames(dds)]))
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211 colnames(dds) <- labs
17
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212 } else {
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213 # construct the object using tximport
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214 # first need to make the tx2gene table
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215 # this takes ~2-3 minutes using Bioconductor functions
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216 if (!is.null(gtfFile)) {
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217 suppressPackageStartupMessages({
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218 library("GenomicFeatures")
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219 })
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220 txdb <- makeTxDbFromGFF(gtfFile, format="gtf")
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221 k <- keys(txdb, keytype = "GENEID")
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222 df <- select(txdb, keys = k, keytype = "GENEID", columns = "TXNAME")
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223 tx2gene <- df[, 2:1] # tx ID, then gene ID
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224 }
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225 library("tximport")
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226 txiFiles <- as.character(sampleTable[,2])
24
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227 labs <- unname(unlist(filenames_to_labels[sampleTable[,1]]))
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228 names(txiFiles) <- labs
23
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229 txi <- tximport(txiFiles, type=opt$txtype, tx2gene=tx2gene)
17
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230 dds <- DESeqDataSetFromTximport(txi,
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231 sampleTable[,3:ncol(sampleTable),drop=FALSE],
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232 designFormula)
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233 }
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234
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235 if (verbose) cat(paste(ncol(dds), "samples with counts over", nrow(dds), "genes\n"))
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236 # optional outlier behavior
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237 if (is.null(opt$outlier_replace_off)) {
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238 minRep <- 7
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239 } else {
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240 minRep <- Inf
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241 if (verbose) cat("outlier replacement off\n")
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242 }
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243 if (is.null(opt$outlier_filter_off)) {
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244 cooksCutoff <- TRUE
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245 } else {
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246 cooksCutoff <- FALSE
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247 if (verbose) cat("outlier filtering off\n")
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248 }
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249
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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250 # optional automatic mean filtering
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251 if (is.null(opt$auto_mean_filter_off)) {
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252 independentFiltering <- TRUE
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253 } else {
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254 independentFiltering <- FALSE
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255 if (verbose) cat("automatic filtering on the mean off\n")
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256 }
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257
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258 # shrinkage of LFCs
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259 if (is.null(opt$beta_prior_off)) {
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260 betaPrior <- TRUE
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261 } else {
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262 betaPrior <- FALSE
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263 if (verbose) cat("beta prior off\n")
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264 }
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265
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266 # dispersion fit type
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267 if (is.null(opt$fit_type)) {
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268 fitType <- "parametric"
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269 } else {
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270 fitType <- c("parametric","local","mean")[opt$fit_type]
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271 }
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272
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273 if (verbose) cat(paste("using disperion fit type:",fitType,"\n"))
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274
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275 # run the analysis
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276 dds <- DESeq(dds, fitType=fitType, betaPrior=betaPrior, minReplicatesForReplace=minRep)
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277
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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278 # create the generic plots and leave the device open
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279 if (!is.null(opt$plots)) {
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280 if (verbose) cat("creating plots\n")
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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281 pdf(opt$plots)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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282 generateGenericPlots(dds, factors)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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283 }
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284
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285 n <- nlevels(colData(dds)[[primaryFactor]])
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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286 allLevels <- levels(colData(dds)[[primaryFactor]])
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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287
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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288 if (!is.null(opt$countsfile)) {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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289 normalizedCounts<-counts(dds,normalized=TRUE)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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290 write.table(normalizedCounts, file=opt$countsfile, sep="\t", col.names=NA, quote=FALSE)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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291 }
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292
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293 if (is.null(opt$many_contrasts)) {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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294 # only contrast the first and second level of the primary factor
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295 ref <- allLevels[1]
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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296 lvl <- allLevels[2]
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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297 res <- results(dds, contrast=c(primaryFactor, lvl, ref),
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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298 cooksCutoff=cooksCutoff,
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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299 independentFiltering=independentFiltering)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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300 if (verbose) {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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301 cat("summary of results\n")
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iuc
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302 cat(paste0(primaryFactor,": ",lvl," vs ",ref,"\n"))
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iuc
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303 print(summary(res))
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304 }
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
parents:
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305 resSorted <- res[order(res$padj),]
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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306 outDF <- as.data.frame(resSorted)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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307 outDF$geneID <- rownames(outDF)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
parents:
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308 outDF <- outDF[,c("geneID", "baseMean", "log2FoldChange", "lfcSE", "stat", "pvalue", "padj")]
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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309 filename <- opt$outfile
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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310 write.table(outDF, file=filename, sep="\t", quote=FALSE, row.names=FALSE, col.names=FALSE)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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311 if (independentFiltering) {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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312 threshold <- unname(attr(res, "filterThreshold"))
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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313 } else {
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iuc
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314 threshold <- 0
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315 }
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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316 title_suffix <- paste0(primaryFactor,": ",lvl," vs ",ref)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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317 if (!is.null(opt$plots)) {
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318 generateSpecificPlots(res, threshold, title_suffix)
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319 }
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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diff changeset
320 } else {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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321 # rotate through the possible contrasts of the primary factor
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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322 # write out a sorted table of results with the contrast as a suffix
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iuc
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323 # add contrast specific plots to the device
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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324 for (i in seq_len(n-1)) {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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diff changeset
325 ref <- allLevels[i]
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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326 contrastLevels <- allLevels[(i+1):n]
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iuc
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327 for (lvl in contrastLevels) {
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iuc
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328 res <- results(dds, contrast=c(primaryFactor, lvl, ref),
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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329 cooksCutoff=cooksCutoff,
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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diff changeset
330 independentFiltering=independentFiltering)
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iuc
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331 resSorted <- res[order(res$padj),]
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iuc
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332 outDF <- as.data.frame(resSorted)
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iuc
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333 outDF$geneID <- rownames(outDF)
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334 outDF <- outDF[,c("geneID", "baseMean", "log2FoldChange", "lfcSE", "stat", "pvalue", "padj")]
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335 filename <- paste0(opt$outfile,".",primaryFactor,"_",lvl,"_vs_",ref)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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336 write.table(outDF, file=filename, sep="\t", quote=FALSE, row.names=FALSE, col.names=FALSE)
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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diff changeset
337 if (independentFiltering) {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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338 threshold <- unname(attr(res, "filterThreshold"))
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339 } else {
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340 threshold <- 0
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341 }
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342 title_suffix <- paste0(primaryFactor,": ",lvl," vs ",ref)
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343 if (!is.null(opt$plots)) {
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344 generateSpecificPlots(res, threshold, title_suffix)
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345 }
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346 }
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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347 }
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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diff changeset
348 }
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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349
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diff changeset
350 # close the plot device
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diff changeset
351 if (!is.null(opt$plots)) {
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
parents:
diff changeset
352 cat("closing plot device\n")
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
iuc
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diff changeset
353 dev.off()
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354 }
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355
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diff changeset
356 cat("Session information:\n\n")
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diff changeset
357
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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diff changeset
358 sessionInfo()
24930eeff731 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/deseq2 commit 23559e873291b04840f186edb0b4b85e76359e05
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359