# HG changeset patch # User vladimir-daric # Date 1398416739 14400 # Node ID 8c5de60b3c04c8296bce0269bfe9a9c54faa3064 Uploaded diff -r 000000000000 -r 8c5de60b3c04 DESeq_diff_ann_wrapper.py --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DESeq_diff_ann_wrapper.py Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,108 @@ +#!/usr/bin/env python +# -*- coding: utf-8 -*- + +''' +Created on 10 sep. 2012 + +@author: Vladimir Daric, Rachel Legendre, Coline Billerey, Alban Ott, Claire Wallon +@copyright: eBio ebio@igmors.u-psud.fr +@license: GPL v3 +''' + +from optparse import OptionParser +from os import path, mkdir, getcwd, walk +from shutil import copyfile +import subprocess +import sys + +def stop_err( msg ): + sys.stderr.write( "%s\n" % msg ) + sys.exit() + +def __main__(): + #Parse Command Line + parser = OptionParser() + + # DESeq-count options. + parser.add_option( "-a", "--alpha", type="float", dest="alpha", + default = 0.05 ) + parser.add_option( "-f", "--foldchange", type="float", dest="foldchange", + default = 0.05 ) + parser.add_option( "-b", "--header", action="store_true", dest="header", + default = False ) + parser.add_option( "-r", "--replicates", action="store_true", dest="replicates", + default = False ) + parser.add_option( "-l", "--logbase", type="int", dest="logbase", + default = 10 ) + parser.add_option( "-n", "--geneNumber", type="int", dest="geneNumber", + default = 30 ) + parser.add_option( "-d", "--subfolder", type="string", dest="subfolder") + + parser.add_option( "-c", "--cond1", type="string", dest="cond1") + + parser.add_option( "-C", "--cond2", type="string", dest="cond2") + + parser.add_option( "-s", "--sharingMode", type="string", dest="mode", default = "fit-only" ) + parser.add_option( "-t", "--fitType", type="string", dest="fitType", default = "local" ) + parser.add_option( "-m", "--method", type="string", dest="method", default = "pooled" ) + # Wrapper / Galaxy options. + parser.add_option( '-o', '--out', dest='outfile', help="file containing results for all counts" ) + parser.add_option( '-u', '--up', dest='up_file', help="file containing results for up regulates counts" ) + parser.add_option( '-w', '--down', dest='down_file', help="file containing results for down regulated counts" ) + + (options, args) = parser.parse_args() + #make a folder for results + outfile = options.outfile + workfolder, filename = path.split(outfile) + #mksubdir + newdirname = options.subfolder + if path.exists(newdirname): + raise + try: + mkdir(newdirname) + except: + raise + + #launch script_deseq in subfolder + exebinpath = path.join(path.dirname(__file__), 'script_deseq.R') + + cmd = "%s --OutputDir=%s/DESeq_out --alpha=%f --foldchange=%f --logBase=%d --geneNumber=%d --sharingMode=%s --method=%s --fitType==%s" % (exebinpath, newdirname, options.alpha, options.foldchange, options.logbase, options.geneNumber,options.mode, options.method, options.fitType) + if options.replicates: + cmd += " --IsReplicate=TRUE" + if options.header: + cmd += " --IsHeader=TRUE" + for myarg in args: + + dataset, cond, rep = myarg.split(',') + ## replace group with correct name + if (cond == "1") : + cond = options.cond1 + else : + cond = options.cond2 + cmd += ' --condition=%s --replicate=%s --HTSeqcount=%s' % (cond, rep, dataset) + + try: + proc = subprocess.Popen( args=cmd, shell=True, stdout=None, stderr=subprocess.PIPE ) + returncode = proc.wait() + + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += proc.stderr.read( buffsize ) + if not stderr or len( stderr ) % buffsize != 0: + break + except OverflowError: + pass + if returncode != 0: + raise Exception, stderr + except Exception, e: + stop_err( 'Error running DESeq analyse: %s'% e ) + + ##copy up and down files in galaxy dataset : + copyfile(path.join(newdirname, 'DESeq_out_up_genes.csv'), options.up_file) + copyfile(path.join(newdirname, 'DESeq_out_down_genes.csv'), options.down_file) + copyfile(path.join(path.dirname(__file__), 'deseq_result_html.html'), outfile) + +if __name__=="__main__": + __main__() \ No newline at end of file diff -r 000000000000 -r 8c5de60b3c04 DESeq_diff_ann_wrapper.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DESeq_diff_ann_wrapper.xml Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,207 @@ + + + R + Bioconductor + DESeq + gplots + plotrix + RColorBrewer + + Uses DESeq R library for differential expression analysis + + + DESeq_diff_ann_wrapper.py + --alpha=$alpha + --out=$outhtml + --up=$up + --down=$down + --foldchange=$foldchange + --logbase=$logbase + --geneNumber=$geneNumber + #if $params.settingsType == "yes": + --sharingMode=$params.mode + --method=$params.method + --fitType=$params.type + #end if + --cond1=$cond1 + --cond2=$cond2 + --subfolder=$outhtml.files_path + #if str( $replicat_opt['options_type_selector'] ) == 'with': + --replicates + #for $i in $replicat_opt.series + ${i.htseq_out},${i.group},${i.label} + #end for + #else + #for $i in $replicat_opt.series + ${i.htseq_out},${i.group},${i.label} + #end for + #end if + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +Summary +------- + +This is a Galaxy wrapper for the DESeq package version 1.14.0 enabling full control of DESeq options (as well as additional graphical outputs/tables). +A major task in the analysis of RNA-seq count data is the detection of differentially expressed genes. Based on read count data for each gene and each sample, the `DESeq`_ package provides methods to test for differential expression based on the negative binomial distribution and a shrinkage estimator for the distribution variance. + +.. _DESeq: http://bioconductor.org/packages/release/bioc/html/DESeq.html + +Input +------ +Two or more count data files. Each file contains the number of reads assigned to each gene for a given sample. One may use for instance the output files of HTSeq-count. + +example: :: + + geneA 12 + geneB 0 + geneC 150 + geneD 3 + + +Output +------- + +This Galaxy wrapper provides an html output and various result tables describing differentially expressed genes. + +.. class:: warningmark + +Warning note : in the absence of biological replicates : use the p-value (not the "adjusted" p-value) as the significance of differential expression. + + +Dependencies +------------ + +.. class:: warningmark + +This tool depends on R (>=3.0.0), DESeq (>=1.14.0), gplots (>=2.12.1), plotrix (>=3.5-3), RColorBrewer (>=1.0-5). + + +.. class:: infomark + +Please contact `eBio`_ for more information + +.. _eBio: http://ebio.u-psud.fr/ + +Citation +--------- + +Simon Anders and Wolfgang Huber (2010): `Differential expression analysis for sequence count data`_. Genome Biology 11:R106 + +.. _Differential expression analysis for sequence count data: http://www.ncbi.nlm.nih.gov/pubmed/20979621 + + + + + + diff -r 000000000000 -r 8c5de60b3c04 GetOptions.R --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/GetOptions.R Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,172 @@ +### +# Created on 25 sep. 2012 + +# @author: Alban Ott +# @copyright: IGM ebio.igmors.u-psud.fr +# @licence: GPL v3 +# + +# Il est interdit de mettre ARGUMENT comme argument long +# spec contains at least 4 columns, as many as 5 columns. +# +# column 1: long name of flag +# +# column 2: short name of flag +# +# column 3: argument flag. 0=without argument, 1=with argument +# +# column 4: mode of argument. one of "logical", "integer", "double", +# "complex", "character", "" for arguments without value (return logical) +# +# column 5 (optional): description of ARGUMENT. +# + + +RetreiveAndDestroy=function(opt,root,stem,regexp,SearchNames,Out,isValue,NbFound,StockNames){ + Bool=lapply(paste(root,SearchNames,stem,sep=""),grepl,opt) + names(Bool)=StockNames + Pos=lapply(Bool,which) + names(Pos)=StockNames + disable=c() + for (i in StockNames){ + nbmatch=length(Pos[[i]]) + if(nbmatch>0){ + NbFound[[i]]=NbFound[[i]]+nbmatch + disable=c(disable,-1*Pos[[i]]) + if(is.null(Out[[i]])){ + if(isValue[[i]]!=0){ + if(regexp=="next"){ + Out[[i]]=opt[Pos[[i]]+1] + disable=c(disable,-1*(Pos[[i]]+1)) + }else{ + Out[[i]]=sub(regexp,"\\1",opt[Pos[[i]]]) + } + }else{ + Out[[i]]=TRUE + } + }else{ + if(isValue[[i]]!=0){ + if(regexp=="next"){ + Out[[i]]=c(Out[[i]],opt[Pos[[i]]+1]) + disable=c(disable,-1*(Pos[[i]]+1)) + }else{ + Out[[i]]=c(Out[[i]],sub(regexp,"\\1",opt[Pos[[i]]])) + } + }else{ + Out[[i]]=c(Out[[i]],TRUE) + } + } + } + } + if(length(disable)>0){ + opt=opt[disable] + } + Out[["ARGUMENT"]]=list() + Out[["ARGUMENT"]][["opt"]]=opt + Out[["ARGUMENT"]][["NbFound"]]=NbFound + return(Out) +} + +getopt = function (spec=NULL,opt=commandArgs()) { + + FindArgs=which(opt=="--args") + if(length(FindArgs)!=1){ + stop(length(FindArgs)," --args found where 1 expected.",call.=F) + } + ExecName=sub("--file=","",opt[FindArgs-1]) + + if(FindArgsmin.columns){ + colNames=c(colNames,"Description") + } + + if(is.null(spec) | !is.matrix(spec) | (DimSpec[2]max.columns)){ + stop('argument "spec" is required and must be a matrix with 4|5 columns.',call.=F) + } + colnames(spec)=colNames + + spec=as.data.frame(spec,stringsAsFactors=F) + #spec validation + if(length(unique(c(spec$ShortName,"ARGUMENT","args")))!=DimSpec[1]+2 | length(unique(spec$LongName))!=DimSpec[1]){ + stop('Long|Short names for flags must be unique (Long name : "ARGUMENT" and "args" forbidden).', + "\n","List of duplicated :", + "\n","Short: ",paste(spec$ShortName[duplicated(c(spec$ShortName,"ARGUMENT","args"))],collapse=" "), + "\n","Long: ",paste(spec$ShortName[duplicated(spec$LongName)],collapse=" "),call.=F) + } + if(length(which(nchar(spec$ShortName)>1))!=0){ + stop('Short names flags can\'t be longer than 1 character.') + } + + + #initialize + Out=list() + Short2Long=list() + NbFound=list() + isValue=list() + for (i in 1:DimSpec[1]){ + Short2Long[[spec$ShortName[i]]]=spec$LongName[i] + NbFound[[spec$LongName[i]]]=0 + isValue[[spec$LongName[i]]]=spec$Flag[i] + } + + #Map, retreive and suppress ARGUMENTs and arguments + #Value ARGUMENT --example=value + Out=RetreiveAndDestroy(opt,"^--","=.+$",".+=(.+)$",spec$LongName,Out,isValue,NbFound,spec$LongName) + opt=Out[["ARGUMENT"]][["opt"]] + NbFound=Out[["ARGUMENT"]][["NbFound"]] + Out[["ARGUMENT"]]=NULL + #boolean ARGUMENT --example + Out=RetreiveAndDestroy(opt,"^--","$","$",spec$LongName,Out,isValue,NbFound,spec$LongName) + opt=Out[["ARGUMENT"]][["opt"]] + NbFound=Out[["ARGUMENT"]][["NbFound"]] + Out[["ARGUMENT"]]=NULL + #short name ARGUMENT -t value OR boolean -t + Out=RetreiveAndDestroy(opt,"^-","$","next",spec$ShortName,Out,isValue,NbFound,spec$LongName) + opt=Out[["ARGUMENT"]][["opt"]] + NbFound=Out[["ARGUMENT"]][["NbFound"]] + Out[["ARGUMENT"]]=NULL + #Warn about non mapped ARGUMENTs + if(length(opt)>0){ + PosUnkArg=which(grepl("^-",opt)) + if(length(PosUnkArg)){ + message("Error, argument unreconized :","\n",paste(opt[PosUnkArg],collapse="\n"),"\n\n") + } + if(length(PosUnkArg)>0){ + opt=opt[PosUnkArg*-1] + } + } + #Arguments + Out[["ARGUMENT"]]=opt + + #Validation of ARGUMENTs + for(i in 1:DimSpec[1]){ + if(spec$Flag[i]=="0"){#verify boolean arguments + NbValue=length(Out[[spec$LongName[i]]]) + if(NbValue>1){ + message("Warning : ",spec$LongName[i]," found ",NbValue," times") + } + } + if(length(Out[[spec$LongName[i]]])==0){ + Out[[spec$LongName[i]]]=spec$Default[i] + } + library("methods") + Out[[spec$LongName[i]]]=as(Out[[spec$LongName[i]]],spec$Mod[i]) + } + + return(Out) +} + +# column 3: argument flag. 0=no argument, 1=required argument, 2=optional argument + + + diff -r 000000000000 -r 8c5de60b3c04 README --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/README Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,27 @@ + +This program uses the `DESeq`_ R library to realize a differential analysis on feature count data from RNA-Seq experiments. +As input you have to provide a genomic features count date, such as provided by `HTSeq-count`_. +The script performs a normalisation of data and then the differential analysis (negative binomial test). +Several plots are produced. + + +The wrapper program (DESeq_diff_ann_wrapper.py) and R library GetOptions.R are free software; you can +redistribute it and/or modify it under the terms of the GNU General Public License as published by the +Free Software Foundation; either version 3 of the License, or (at your option) any later version. + +Authors : +Vladimir Daric (eBio), Rachel Legendre (eBio), Coline Billerey (eBio), Alban Ott (IGM), Claire Wallon (eBio) + + +Licenses: + +If **DESeq** is used to obtain results for scientific publications it should be cited as [1]. +References: +[1] Anders, S and Huber, W (2010): `Differential expression analysis for sequence count data`. + +Contact: + ebio@igmors.u-psud.fr + +.. _DESeq: http://bioconductor.org/packages/release/bioc/html/DESeq.html +.. _HTSeq: http://www-huber.embl.de/users/anders/HTSeq/doc/count.html +.. _eBio: http://ebio.u-psud.fr/ \ No newline at end of file diff -r 000000000000 -r 8c5de60b3c04 deseq_result_html.html --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/deseq_result_html.html Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,76 @@ + + + + + + + + +

DESeq analyse results

+

+

+

+ + + diff -r 000000000000 -r 8c5de60b3c04 function_deseq.R --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/function_deseq.R Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,448 @@ +# Home made violin plot (Alban Ott) + +vioplot = function(x, DensCol="#00000060", BoxplotCol="#000000FF", InfFactorSpace=0.4,BoxplotFill="#FFFFFF00", YLAB="log10(normalized count)"){ #x must be a matrix or a dataframe + + # Number of samples : + SampleNumber=length(colnames(x)) + + if(is.vector(x)){ + x=as.matrix(x) + } + YNormalizedDensity=list() + XNormalizedDensity=list() + + #Set a value to -Inf + xNoInfo=x[x!=-Inf] + OverallMinMax=c(min(xNoInfo),max(xNoInfo)) + range=OverallMinMax[2]-OverallMinMax[1] + Inf2Value=OverallMinMax[1]-range*InfFactorSpace + breakInf=OverallMinMax[1]-range*(InfFactorSpace-.1) + xValInf=x + IsInf=xValInf==-Inf + if(sum(IsInf)>0){ + xValInf[xValInf==-Inf]=Inf2Value + } + #Pre compute densities + for (i in 1:dim(xValInf)[2]){ + dens=density(xValInf[,i]) + YNormalizedDensity[[i]]=dens$x + #Normalize density to fit boxplot + XNormalizedDensity[[i]]=dens$y/max(dens$y)/5 + } + + #Print boxplot with a range compatible with density plot + if(sum(IsInf)>0){ + ylim=c(OverallMinMax[1]-range*(InfFactorSpace+0.1),OverallMinMax[2]+range*0.1) + if (SampleNumber > 8) { + boxplot(x, boxwex=0.2, ylim=ylim, border=BoxplotCol, col=BoxplotFill, notch=T, yaxt="n", las=2) + }else { + boxplot(x, boxwex=0.2, ylim=ylim, border=BoxplotCol, col=BoxplotFill, notch=T, yaxt="n") + } + AxisTick=seq(floor(OverallMinMax[1]),round(OverallMinMax[2]),length.out=round(OverallMinMax[2])-floor(OverallMinMax[1])+1) + axis(2,at=c(Inf2Value,AxisTick),c("-Inf",AxisTick),las=1) + axis.break(2,breakInf,style="slash") + + }else{ + if (SampleNumber > 8){ + boxplot(x, boxwex=0.2, border=BoxplotCol, col=BoxplotFill, notch=T, las=2) + }else { + boxplot(x, boxwex=0.2, border=BoxplotCol, col=BoxplotFill, notch=T) + } + + } + mtext(side = 2, YLAB, line = 2) + #Plot densities + for (i in 1:dim(x)[2]){ + points(i+XNormalizedDensity[[i]], YNormalizedDensity[[i]], type="l", lwd=3, col=DensCol) + points(i-XNormalizedDensity[[i]], YNormalizedDensity[[i]], type="l", lwd=3, col=DensCol) + if(sum(IsInf)>0){ + points(i,Inf2Value,pch=8,col=DensCol,lwd=3) + } + } +} + + + +####################################################### +# Histogram of p-value. Use adjusted p-value if +# there are replicats +####################################################### + + +PlotHistPval = function(res, rep, file, ImageSize=1960, PPI=200){ + + png( file=file , width=ImageSize, height=ImageSize, res=PPI ) + if (rep){ + hist(res$padj, breaks=100, col="black", border="black", main="",ylab="Number of elements (e.g. : genes)",xlab="adjusted p-value") + }else{ + hist(res$pval, breaks=100, col="black", border="black", main="",ylab="Number of elements (e.g. : genes)",xlab="raw p-value") + } + invisible(dev.off()) + +} + + + +####################################################### +# Plot violinplot of (un)normalized counts +####################################################### + +PlotViolin = function(cds, norm, file, ImageSize=1960, PPI=200, logBase=10){ + + png( file=file , width=ImageSize, height=ImageSize, res=PPI ) + ## probleme avec les +/-inf + NormCount = log(counts( cds, normalized=norm ), base=logBase) + ## get correct ylab + if ( norm ){ + YLAB=paste("log",logBase,"(normalized count)",sep="") + }else{ + YLAB=paste("log",logBase,"(raw count)",sep="") + } + color_cond1 = colorRampPalette(brewer.pal(3, "Purples"))(sum(conditions(cds)==levels(conditions(cds))[1])) + color_cond2 = colorRampPalette(brewer.pal(3, "RdPu"))(sum(conditions(cds)==levels(conditions(cds))[2])) + colors = c(as.vector(color_cond1),as.vector(color_cond2)) + labels = colnames( NormCount ) + + vioplot(NormCount,BoxplotFill=colors, YLAB=YLAB) + + invisible(dev.off()) + + +} + +############################################################## +# Plot volcano with density plot of foldchange and pvalues +############################################################## + +PlotVolcano = function (res, cond1,cond2 ,alpha, fold, rep, file, ImageSize=1960,logBase=10, InfFactorSpace=0.4,PPI=200){ + + png( file=file , width=ImageSize, height=ImageSize ,res=PPI ) + + + if ( rep ){ + pvalCol=8 + YLAB=paste("-log",logBase,"(adjusted p-value)",sep="") + }else{ + pvalCol=7 + YLAB=paste("-log",logBase,"(raw p-value)",sep="") + } + + ThreslogAlpha = -log(alpha, logBase) + ThreslogFC = log(fold, logBase) + logPVal = -log(res[,pvalCol], logBase) + logFC=log(res$foldChange, logBase) + + #Set values to -/+Inf + MinMaxlogFC=summary(logFC[abs(logFC)!=Inf])[c("Min.","Max.")] + range=MinMaxlogFC[2]-MinMaxlogFC[1] + Inf2Value=c(MinMaxlogFC[1]-range*InfFactorSpace, MinMaxlogFC[2]+range*InfFactorSpace) + breakInf=c(MinMaxlogFC[1]-range*(InfFactorSpace-0.1), MinMaxlogFC[2]+range*(InfFactorSpace-0.1)) + logFC[logFC==-Inf]=Inf2Value[1] + logFC[logFC== Inf]=Inf2Value[2] + + + #Precompute densities + logFCDens=density(logFC) + logPvalDens=density(logPVal) + tmp=logPvalDens$x + logPvalDens$x=logPvalDens$y + logPvalDens$y=tmp + #Compute xlim and ylim to align graphics + marOutside=0.05 + XLIM=c(min(logFCDens$x)-(max(logFCDens$x)-min(logFCDens$x))*marOutside,max(logFCDens$x)+(max(logFCDens$x)-min(logFCDens$x))*marOutside) + YLIM=c(min(logPvalDens$y)-(max(logPvalDens$y)-min(logPvalDens$y))*marOutside,max(logPvalDens$y)+(max(logPvalDens$y)-min(logPvalDens$y))*marOutside) + + + plot.new() + split.screen(rbind( + c(0,0.7,0,0.7), + c(0,0.7,0.7,1), + c(0.7,1,0,0.7) + )) + screen(1) + # c(bottom, left, top, right) default : par(mar=c(5,6,3,4)+.1) + par(mar=c(5,6,0,0)+.1) + # volcano plot + VolcanoCol=ifelse((log(res$foldChange, logBase) > ThreslogFC | log(res$foldChange, logBase) < -ThreslogFC)&(logPVal>ThreslogAlpha), "red","black") + plot(logFC,logPVal,pch=20,cex=0.3,xlim=XLIM,ylim=YLIM,xaxt="n",col=VolcanoCol,xlab=paste("log",logBase,"(FoldChange ",paste(cond2,cond1,sep="/"),")",sep=""),ylab=YLAB,bty="l") + + AxisTick=seq(floor(MinMaxlogFC[1]),round(MinMaxlogFC[2]),length.out=round(MinMaxlogFC[2])-floor(MinMaxlogFC[1])+1) + axis(1,at=c(Inf2Value[1],AxisTick,Inf2Value[2]),c("-Inf",AxisTick,"+Inf"),las=1) + axis.break(1,breakInf[1],style="slash") + axis.break(1,breakInf[2],style="slash") + + #pval threshold dashed line + abline(h=ThreslogAlpha,col="red", lty=2, lwd=2 ) + + screen(2) + par(mar=c(0,6,3,0)+.1) + plot(logFCDens, col="black",main ="",xaxt="n",bty="n",ylab=paste("Density of\nlog",logBase,"(FoldChange)",sep=""),xlab="",xlim=XLIM) + + screen(3) + par(mar=c(5,0,0,4)+.1) + plot(logPvalDens,main="", col="black",yaxt="n",bty="n",xlab=paste("Density of\n",YLAB,sep=""),ylab="",ylim=YLIM) + + invisible(dev.off()) + +} + +####################################################### +# Plot mean of normalized counts vs log2 of foldchange +####################################################### + +MAPlot = function(res, OutPrefix, file, ImageSize=1960, PPI=200){ + + png( file=file , width=ImageSize, height=ImageSize, res=PPI ) + plotMA(res) + + invisible(dev.off()) +} + + +####################################################### +# Plot estimation of dispersion +####################################################### + + +EstDispPlot = function(cds, method, file, ImageSize=1960, PPI=200){ + + png( file=file , width=ImageSize, height=ImageSize, res=PPI ) + + if (method == "per-condition"){ + + #print(ls( cds@fitInfo)) + par(mfcol=c(1,2)) + for (cond in (ls(cds@fitInfo))){ + plotDispEsts(cds, name=cond,main=paste("Estimate dispersion plot for ",cond,sep="")) + } + + }else{ + plotDispEsts(cds) + } + + + invisible(dev.off()) +} + +####################################################### +# Histogram of null counts proportion per condition +####################################################### + + +NullCondPlot = function(cds, file ,ImageSize=1960, PPI=200){ + + png( file=file , width=ImageSize/2, height=ImageSize, res=PPI ) + + + CountsNorm_cond=list() + CountsNull_cond=list() + ## for each condition + for (cond in rev(levels(conditions(cds)))){ + # we count number of feature with no count + CountsNorm_cond[[cond]]=counts( cds, normalized=TRUE )[ , conditions(cds)==cond ] + # if there are no replicate dim is null and return R error + if (is.null(dim(CountsNorm_cond[[cond]]))) { + CountsNull_cond[[cond]]=sum(CountsNorm_cond[[cond]] == 0)/length(CountsNorm_cond[[cond]]) + } + else { + CountsNull_cond[[cond]]=sum(apply(CountsNorm_cond[[cond]],1,sum) == 0)/nrow(CountsNorm_cond[[cond]]) + } + + } + # we plot number of feature with no count + colors = c("#756bb1","#fa9fb5") + barplot(as.numeric(CountsNull_cond), ylab="Proportion of null count elements (e.g. : genes)", names.arg = names(CountsNull_cond), col=colors, ylim=c(0,1)) + + invisible(dev.off()) +} + + +####################################################### +# Histogram of total reads count per sample +####################################################### + + +TotReadSamplePlot = function(cds, file ,ImageSize=1960, PPI=200){ + + png( file=file , width=ImageSize, height=ImageSize, res=PPI ) + + RawCount = counts( cds, normalized=FALSE) + ## choice of x colors according to number of replicate in Ramp palette + color_cond1 = colorRampPalette(brewer.pal(3, "Purples"))(sum(conditions(cds)==levels(conditions(cds))[1])) + color_cond2 = colorRampPalette(brewer.pal(3, "RdPu"))(sum(conditions(cds)==levels(conditions(cds))[2])) + colors = c(as.vector(color_cond1),as.vector(color_cond2)) + #if there are many replicates, x labels are drawn vertically + if (length(sampleNames(cds)) > 8){ + barplot(colSums(RawCount), ylab="", col=colors, ylim=c(0,max(colSums(RawCount))*1.2 ), las=2, yaxt="n") + ## As there are millions of reads, y labels are drawn horizontally + axis(2, las=0) + mtext("Total Read Count per Sample", side=2, line=2.5) + } + else { + barplot(colSums(RawCount), ylab="", names.arg = colnames(RawCount), col=colors, ylim=c(0,max(colSums(RawCount))*1.2 ) ) + ## As there are millions of reads, y labels are drawn horizontally + axis(2, las=0) + mtext("Total Read Count per Sample", side=2, line=2.5) + } + invisible(dev.off()) +} + + +################################################################### +# heatmap of expression data of the 30 most highly expressed genes +################################################################### + +HeatCountPlot=function(cds, vsd, file, geneNumber=30, ImageSize=1960, PPI=200){ + + png( file=file , width=ImageSize, height=ImageSize, res=PPI ) + + select = order(rowMeans(counts(cds)), decreasing=TRUE)[1:geneNumber] + hmcol = colorRampPalette(brewer.pal(9, "GnBu"))(100) + heatmap.2(exprs(vsd)[select,], col = hmcol, trace="none", margin=c(10, 6)) + + invisible(dev.off()) +} + +####################################################### +# Heatmap of Euclidean distances between the samples +####################################################### + +HeatDistPlot=function(vsd, cdsBlind, file, ImageSize=1960, PPI=200){ + + png( file=file , width=ImageSize, height=ImageSize, res=PPI ) + + dists = dist(t(exprs(vsd))) + mat = as.matrix(dists) + rownames(mat) = colnames(mat) = with(pData(cdsBlind), paste(condition,sampleNames(cdsBlind), sep=" : ")) + hmcol = colorRampPalette(brewer.pal(9, "GnBu"))(100) + heatmap.2(mat, trace="none", col = rev(hmcol), margin=c(13, 13)) + + invisible(dev.off()) +} + +####################################################### +# Export up and down genes according to p-value and +# Foldchange, and export complete results +####################################################### + + +WriteResults = function (res, rawCounts, normCounts, cond1, cond2, alpha, foldchange, rep, OutPrefix, logBase=10){ + + ## re-compute logBase(FC) passed in argument + res[,6] = log(res[,5],base=logBase) + + ## get results not failed pvalue threshold + if (rep == TRUE){ + res_sig = res[res$padj < alpha,] + }else{ + res_sig = res[res$pval < alpha,] + } + + # select up genes + up = res_sig[res_sig$foldChange > foldchange,] + up = up[rev(order(up$foldChange)),] + + # select down genes + down = res_sig[res_sig$foldChange < 1/foldchange,] + down = down[order(down$foldChange),] + + # change any column names for more comprehension + names = colnames(res) + names[3] = paste("baseMean_",cond1,sep="") + names[4] = paste("baseMean_",cond2,sep="") + names[5] = paste("foldChange_",paste(cond2,cond1,sep="/"),sep="") + names[6] = paste("log",logBase,"FoldChange",sep="") + colnames(res) = names + colnames(up) = names + colnames(down) = names + + # export up genes + write.table( format(up,scientific=FALSE,digits=3,dec=','), file=paste(OutPrefix,"_up_genes.csv",sep=""), sep="\t", row.names=F, quote=F) + # export down genes + write.table( format(down,scientific=FALSE,digits=3,dec=','), file=paste(OutPrefix,"_down_genes.csv",sep=""), sep="\t", row.names=F, quote=F) + + #rename col names of raw counts + names = NULL + for(name in colnames(rawCounts)){ + names = c(names,paste("raw_",name,sep="")) + } + colnames(rawCounts) = names + + #rename col names of normalized counts + names = NULL + for(name in colnames(normCounts)){ + names = c(names,paste("norm_",name,sep="")) + } + colnames(normCounts) = names + + ## merge raw and normalized counts + counts = merge(rawCounts, normCounts,by="row.names",all=T) + all_results = data.frame(res$id, counts[,2:ncol(counts)], res[,3:ncol(res)]) + colnames(all_results)[1] <- "ID" + ## export complete results + write.table( format(all_results,scientific=FALSE,digits=3,dec=','), file=paste(OutPrefix,"_complete.csv",sep=""), sep="\t", row.names=F, quote=F) + + +} + +####################################################### +# create a raw count matrix from argument's sample +####################################################### + + +CreateCountMatrix = function(sample, header, rep){ + + #Read the HTseq-count files dans concatenate them in different columns. + names = sample$files + rawCounts=data.frame(Id=character(),stringsAsFactors=T) + for (i in 1:length(names)){ + tmp = read.table(names[i], sep="\t", header=header, as.is=T,col.names=c("Id", sample$replicate[i]))#read + rawCounts = merge(rawCounts, tmp, by="Id", all=T)#concatenate + } + colnames(rawCounts) = c("Id", sample$replicate)#rename + + rawCounts[is.na(rawCounts)] = 0 + + #Clean up the HTseq-count from overview lines + row2remove = c("alignment_not_unique", "ambiguous", "no_feature", "not_aligned", "too_low_aQual") + rawCounts = rawCounts[!rawCounts$Id %in% row2remove,] + rawCounts[is.na(rawCounts)] = 0 + + #convert Id columns to row names + rownames(rawCounts)=rawCounts[,1] + rawCounts=rawCounts[,-1] + + return(rawCounts) +} + + + +####################################################### +# Launch DE analysis with(out) replicates +####################################################### +# parameters: +# method: how samples are pooled to estimate dispersion. If no replicates use "blind" +# sharingMode: how variance estimate is computed with respect to the fitted line. +# "Maximum" is the most conservative (takes care of outliers, i.e., genes with dispersion much larger than the fitted values), +# "fit-only" keeps the estimated value. Use this only with very few replicates, and when you are not too concerned about false positives from dispersion outliers, i.e. genes with an unusually high variability. +# "gene-est-only" : No fitting or sharing, use only the empirical value. +#sharingMode = c( "maximum", "fit-only", "gene-est-only" ) +# fitType: refers to the model. "Local" is the published model, "parametric" is glm-based (may not converge) +#in this case, without replicates +#fitType = c("parametric", "local") + + +DE_analysis = function(countTable, design, rep, mode="maximum", method="pooled", fitType="parametric"){ + + cds = newCountDataSet( countTable, design$condition) + cds = estimateSizeFactors( cds ) + + if (rep){ + cds = estimateDispersions( cds, fitType=fitType, sharingMode=mode, method=method) + # If there are no replicates, we force Mode and method as described in DESeq manual + } else { + cds = estimateDispersions( cds, fitType=fitType, sharingMode="fit-only", method="blind") + } + return(cds) +} + diff -r 000000000000 -r 8c5de60b3c04 script_deseq.R --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/script_deseq.R Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,140 @@ +#!/usr/bin/env Rscript + +################################### +# @author: Vladimir Daric, Rachel Legendre, Coline Billerey, Alban Ott, Claire Wallon +# @copyright: eBio ebio@igmors.u-psud.fr +# @license: GPL v3 + + + +####################################################### +# GetExecutingDirectory (for galaxy) +####################################################### + +getScriptDirectory = function() { + cheminScript = unlist(strsplit(commandArgs()[4],split="="))[2] + cheminScriptSplit = unlist(strsplit(cheminScript,split="/")) + return(paste(cheminScriptSplit[1:(length(cheminScriptSplit)-1)],collapse="/")) +} + + +#################################################### +# MAIN PROGRAM +#################################################### + + +scriptDirectory = getScriptDirectory() +source(paste(scriptDirectory,"/GetOptions.R",sep="")) +source(paste(scriptDirectory,"/function_deseq.R",sep="")) + +optArgs=getopt( + rbind( + c('IsReplicate' , 'r', 0, 'logical' , "FALSE" , "Is there any replicates ?"), + c('help' , 'h', 0, 'logical' , "FALSE" , "Display help"), + c('IsHeader' , 'b', 0, 'logical' , "FALSE" , "Is there a header in the HTSeq-count file ?"), + c('alpha' , 'a', 1, 'numeric' , "0.05" , "p-value threshold"), + c('foldchange' , 'f', 1, 'numeric' , "2" , "foldchange threshold"), + c('HTSeqcount' , 'c', 1, 'character', "" , "HTSeq-count files"), + c('condition' , 'g', 1, 'character', "" , "Biological conditions"), + c('replicate' , 'l', 1, 'character', "" , "Names of each replicate"), + c('OutputDir' , 'p', 1, 'character', "DESeq_out" , "Output directory"), + c('ImageSize' , 's', 1, 'numeric' , "1960" , "Image size"), + c('PPI' , 'i', 1, 'numeric' , "200" , "Image resolution"), + c('logBase' , 'o', 1, 'numeric' , "10" , "log base for image and result"), + c('geneNumber' , 'z', 1, 'numeric' , "30" , "Gene number for heatmap of most highly expressed genes."), + c('sharingMode' , 'v', 1, 'character', "maximum" , "Sharing mode of estimate dispersion fonction (maximum, fit-only, gene-est-only)"), + c('method' , 'w', 1, 'character', "pooled" , "Method of estimate dispersion fonction (pooled, per-condition or blind)"), + c('fitType' , 't', 1, 'character', "parametric" , "Fit type of estimate dispersion fonction (local or parametric)") + ) +) +attach(optArgs) +sample = data.frame(replicate=replicate, files=HTSeqcount, condition=condition,stringsAsFactors=F) + +if (help) { + #AO : deprecated + cat("DESeqTools version eBio + Commande : script_deseq.R [options] file1 file2 file3 file4 .... + Exemple: ./script_deseq.R --condition=Cond1 --replicate=replicate1 -HTSeqcount=file_count1.txt + --condition=Cond1 --replicate=replicate2 -HTSeqcount=file_count2.txt + --condition=Cond2 --replicate=replicate1 -HTSeqcount=file_count3.txt + --condition=Cond2 --replicate=replicate2 -HTSeqcount=file_count4.txt") + stop("",call.=F) +} + +## Supress warning +options(warn=-1) + +if (length(unique(condition))!=2){ + stop("Please specify exactly TWO and ONLY two diferent condition names",call.=F) +} + +if (length(condition)!=length(HTSeqcount) || length(replicate) != length(HTSeqcount)){ + stop("You must specify as many conditions and replicates as files. In your input I\'ve found ", + length(HTSeqcount)," files, ", + length(condition)," conditions, ", + length(replicate)," replicates", call.=F) +} + +# Chargement des packages et des fonctions +suppressPackageStartupMessages(library("DESeq")) +suppressPackageStartupMessages(library("RColorBrewer")) +suppressPackageStartupMessages(library("gplots")) +suppressPackageStartupMessages(library("plotrix")) + + +## conditions +conds <- unique(sample$condition) + +######################################################## +# Counts matrix creation +countTable = CreateCountMatrix(sample, IsHeader, IsReplicate) + +# suppress non expressed transcript in all conditions +countTable=countTable[apply(countTable,1,sum)!=0,] + +# Make relations between names and conditions +design = data.frame(names = sample$replicate, condition = sample$condition ) + +# DESeq main analysis, NB : type of analysis depend on IsReplicate +cds = DE_analysis(countTable,design,IsReplicate,mode=sharingMode, method=method, fitType=fitType) +DESeqResults = nbinomTest( cds,conds[1],conds[2]) + +# Plot hist of null counts +NullCondPlot(cds,file=paste(OutputDir,"_NullCondPlot.png", sep=""),ImageSize=ImageSize, PPI=PPI) + +# plot of total read count per sample +TotReadSamplePlot(cds, file=paste(OutputDir,"_TotSamplePlot.png", sep="") ,ImageSize=ImageSize, PPI=PPI) + +# plot hist of pvalues +# |_->if there is replicate : adjusted pvalue +# |_->if there is no replicate : raw pvalue +PlotHistPval(DESeqResults,IsReplicate,file=paste(OutputDir,"_histPval.png", sep=""),ImageSize=ImageSize, PPI=PPI) + +## Violin plot for raw data +PlotViolin(cds, FALSE, paste(OutputDir,"_RawViolin.png", sep=""), ImageSize=ImageSize, PPI=PPI, logBase=logBase) + +## Violin plot for raw data +PlotViolin(cds, TRUE, paste(OutputDir,"_NormViolin.png", sep=""), ImageSize=ImageSize, PPI=PPI, logBase=logBase) + +# plot volcano +PlotVolcano(res=DESeqResults, cond1=conds[1], cond2=conds[2], alpha=alpha, fold=foldchange, rep=IsReplicate, file=paste(OutputDir,"_volcanoplot.png", sep=""), ImageSize=ImageSize, PPI=PPI, logBase=logBase) + +# plot MA and dispersion +MAPlot(DESeqResults,file=paste(OutputDir,"_MAplot.png", sep=""), ImageSize=ImageSize, PPI=PPI) +EstDispPlot(cds, method, file=paste(OutputDir,"_EstDispPlot.png", sep=""), ImageSize=ImageSize, PPI=PPI) + +# compute clustering +if (IsReplicate){ + cdsBlind = estimateDispersions( cds, method = "blind",sharingMode=sharingMode, fitType=fitType ) +}else{ + cdsBlind = estimateDispersions( cds, method = "blind",sharingMode='fit-only', fitType=fitType ) +} +vsd = varianceStabilizingTransformation( cdsBlind ) + +# plot clustering and correlation +HeatCountPlot(cds,vsd,file=paste(OutputDir,"_HeatCountPlot.png", sep=""), geneNumber, ImageSize=ImageSize, PPI=PPI) +HeatDistPlot(vsd,cdsBlind,file=paste(OutputDir,"_HeatDistPlot.png", sep=""), ImageSize=ImageSize, PPI=PPI) + +## export result +WriteResults (DESeqResults, countTable, counts( cds, normalized=TRUE ), conds[1], conds[2], alpha, foldchange, IsReplicate, OutputDir, logBase=logBase) + diff -r 000000000000 -r 8c5de60b3c04 test/.svn/all-wcprops --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test/.svn/all-wcprops Fri Apr 25 05:05:39 2014 -0400 @@ -0,0 +1,41 @@ +K 25 +svn:wc:ra_dav:version-url +V 79 +/depot/!svn/ver/2847/trunk/eBio/Galaxy/galaxy-dist/tools/ebio.tools/RnaSeq/test +END +B1_count.txt +K 25 +svn:wc:ra_dav:version-url +V 92 +/depot/!svn/ver/2847/trunk/eBio/Galaxy/galaxy-dist/tools/ebio.tools/RnaSeq/test/B1_count.txt +END +A1_count.txt +K 25 +svn:wc:ra_dav:version-url +V 92 +/depot/!svn/ver/2847/trunk/eBio/Galaxy/galaxy-dist/tools/ebio.tools/RnaSeq/test/A1_count.txt +END +B2_count.txt +K 25 +svn:wc:ra_dav:version-url +V 92 +/depot/!svn/ver/2847/trunk/eBio/Galaxy/galaxy-dist/tools/ebio.tools/RnaSeq/test/B2_count.txt +END +A2_count.txt +K 25 +svn:wc:ra_dav:version-url +V 92 +/depot/!svn/ver/2847/trunk/eBio/Galaxy/galaxy-dist/tools/ebio.tools/RnaSeq/test/A2_count.txt +END +B3_count.txt 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