Mercurial > repos > yufei-luo > differential_expression_analysis_pipeline_for_rnaseq_data
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/HTseqClean.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,19 @@ +# HTseqClean +# remove extra counts out of genes +# for HTseq output + +# input : rawCounts +# output : cleaned rawCounts + +# created Feb 6th, 2012 +# Modified Feb 16th, 2012 +# Marie-Agnes Dillies + + +HTseqClean <- function( rawCounts ){ + + row2remove <- c("alignment_not_unique", "ambiguous", "no_feature", "not_aligned", "too_low_aQual") + rawCounts <- rawCounts[!rawCounts$Id %in% row2remove,] + rawCounts[is.na(rawCounts)] <- 0 + return(rawCounts) +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/MAplotDE.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,17 @@ +# Marie-Anges Dillies +# MAplotDE +# MAplot of DE genes + +# input : res, alpha,OUT_MAplotDEName +# output : MAplot (png) + +MAplotDE <- function( res, alpha, OUT_MAplotDEName, out = TRUE ){ + + if (out) png( file=OUT_MAplotDEName ) + + plot( res$baseMean, res$log2FoldChange, pch=".", xlab="Mean expression", ylab="log2FC", main="", + log="x", col=ifelse(res$padj < alpha, "red", "black") ) + abline(h=0, col="red") + + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/RNAseqFunctions.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,39 @@ +# Marie-Anges Dillies +# RNAseqFunctions +# when sourced, sources all R functions associated with RNAseq data analysis + +RNAseqFunctions <- function( RfuncDir ){ + + source(paste(RfuncDir, "loadTargetFile.R", sep="")) + source(paste(RfuncDir, "loadCountData.R", sep="")) +# source(paste(RfuncDir, "loadStrandData.R", sep="")) + source(paste(RfuncDir, "HTseqClean.R", sep="")) + source(paste(RfuncDir, "raw2counts.R", sep="")) + source(paste(RfuncDir, "barplotTC.R", sep="")) + source(paste(RfuncDir, "barplotNul.R", sep="")) + source(paste(RfuncDir, "removeNul.R", sep="")) + source(paste(RfuncDir, "densityPlot.R", sep="")) + source(paste(RfuncDir, "boxplotCounts.R", sep="")) + source(paste(RfuncDir, "majSequence.R", sep="")) + source(paste(RfuncDir, "clusterPlot.R", sep="")) + source(paste(RfuncDir, "pairwiseSERE.R", sep="")) + source(paste(RfuncDir, "pairwiseScatterPlots.R", sep="")) +# source(paste(RfuncDir, "pairwiseScatterPlotsAll.R", sep="")) + source(paste(RfuncDir, "plotDispEstimates.R", sep="")) +# source(paste(RfuncDir, "deseqByCond.R", sep="")) +# source(paste(RfuncDir, "edgeRByCond.R", sep="")) +# source(paste(RfuncDir, "fisher.R", sep="")) + source(paste(RfuncDir, "histoRawp.R", sep="")) +# source(paste(RfuncDir, "histoRawpMconds.R", sep="")) + source(paste(RfuncDir, "MAplotDE.R", sep="")) +# source(paste(RfuncDir, "MAplotDEMconds.R", sep="")) + source(paste(RfuncDir, "exportComplete.R", sep="")) +# source(paste(RfuncDir, "exportCompleteEdgeR.R", sep="")) +# source(paste(RfuncDir, "exportCompleteFisher.R", sep="")) +# source(paste(RfuncDir, "exportCompleteMconds.R", sep="")) +# source(paste(RfuncDir, "exportCompleteByCond.R", sep="")) +# source(paste(RfuncDir, "exportCompletePaired.R", sep="")) + source(paste(RfuncDir, "exportDiff.R", sep="")) +# source(paste(RfuncDir, "synthese.R", sep="")) +# source(paste(RfuncDir, "exportDiffByCond.R", sep="")) +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/anadiffGenes2conds.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,196 @@ +# Marie-Anges Dillies, Yufei Luo +# Analyse differentielle de donnees d expression par gene +# avec DESeq +# 2 conditions + +args <- commandArgs() +#print(args[1]) +#print(args[2]) +#print(args[3]) +#print(args[4]) +#print(args[5]) +#print(args[6]) +#output file names +#print(args[7]) # HTML file name +#print(args[8]) # HTML file all images directory +#print(args[9]) # complete xls file name +#print(args[10])# UP xls file name +#print(args[11]) #Down xls file name +#print(args[12]) #the executable scipt (for getting the path) + +library(R2HTML) +library(R.utils) + +#run example: +projectName <- "DESeqAnalysis" +analysisVersion <- "V1" # fitType=local, sharingMode=fit-only, method=blind +rawDir <- "raw" +targetFile <- args[4] +header <- as.integer(args[5]) #si on a header ou pas, si on a, header=1, sinon header=0 +withOutReplicates <- as.integer(args[6]) + +#get the directory to write the results +tab <- splitByPattern(args[7], pattern="/") +res_dir <- "" +for (e in tab[1:length(tab)-1]) { res_dir <- paste(res_dir, e, sep="")} +#get the html output file name +OUT_HTMLname <- args[7] +#get the images directory to write to +OUT_imgDir <- args[8] +#if the directory dosen't existe, we should create it first + +alpha <- 0.05 +adjMethod <- "BH" +outfile <- T +runningScriptTab <- splitByPattern(args[12], pattern="/") +RfuncDir <- "" +for (r in runningScriptTab[1:length(runningScriptTab)-1]) { RfuncDir <- paste(RfuncDir, r, sep="")} #find the path of executable script +RfuncDir <- paste(RfuncDir, "DESeqTools/", sep="") #define the function files path +# Dossier contenant les fonctions +print(RfuncDir) +source( paste(RfuncDir, "RNAseqFunctions.R", sep="/") ) + +# Chargement des packages et des fonctions +library(DESeq) +RNAseqFunctions(RfuncDir) +# Chargement du target file +target <- loadTargetFile( targetFile, header ) +# Chargement des donnees, construction d'une table de comptages par gene +#have changed +rawCounts <- loadCountData( target, header ) +conds <- unique(target$group) +cond1 <- as.character(conds[1]) +cond2 <- as.character(conds[!conds == conds[1]]) +rawCounts <- HTseqClean( rawCounts ) + +# Transformation en matrice de comptages +counts <- raw2counts( rawCounts )[[1]] + +# Nombre de reads par echantillon +OUT_barplotTCName <- paste(OUT_imgDir, "barplotTC.png", sep="/") +barplotTC( counts, target$group, OUT_barplotTCName, out=outfile ) + +# Proportion comptages nuls +OUT_barplotNulName <- paste(OUT_imgDir, "barplotNul.png", sep="/") +barplotNul( counts, target$group, OUT_barplotNulName, out=outfile ) + +# Suppression comptages nuls +counts <- removeNul( counts )[[1]] + +# Density plot +OUT_densityPlotName <- paste(OUT_imgDir, "densityPlot.png", sep="/") +densityPlot( counts, target$group, OUT_densityPlotName, out=outfile ) + +# Boxplot +OUT_boxplotCountsName <- paste(OUT_imgDir, "boxplotCounts.png", sep="/") +boxplotCounts( counts, target$group, type = c("raw", "norm"), OUT_boxplotCountsName, out=outfile ) +# Sequence majoritaire +OUT_majSequenceName <- paste(OUT_imgDir, "majSequence.png", sep="/") +majSequence( counts, target$group, OUT_majSequenceName, out=outfile ) + +# ScatterPlot between two samples +OUT_scatterPlot <- paste(OUT_imgDir, "scatterPlot.png", sep="/") +pairwiseScatterPlots(counts, target, OUT_scatterPlot, out=outfile, pdffile=FALSE) + +# SERE coefficient calculation (Poisson hypothesis for replicates techiques), to know if the variability between the réplicates or the conditons is hight or not. +coef <- pairwiseSERE(counts) +print(coef) +coef +# Creation structure de donnees cds, !! we use newCountDataset because that we have first column not numeric, and DESeq dosen't take non numeric values. +cds <- newCountDataSet( counts, target$group ) + +# Diagnostic for clustering of non-normalized samples +OUT_clusterPlot_before <- paste(OUT_imgDir, "clusteringOfSamplesBefore.png", sep="/") +clusterPlot(cds, OUT_clusterPlot_before, out=outfile) + + +# Normalisation (calcul des lib size factors ) +cds <- estimateSizeFactors( cds ) + +# Estimation de la dispersion +# parametres: + # method: how samples are pooled to estimate dispersion (we use "pooled" + # as default parameter. If no replicates, we use "blind". + # sharingMode: how variance estimate is computed with respect to the fitted line. + #"Maximum" is the most conservative (max between fit andestimation), + #"fit-only" keeps the estimated value, we use "Maximum" as default parameter. + # fitType: refers to the model. "Local" is the published model, + #"parametric" is glm-based (may not converge), now we use "parametric" as default value. + +if(withOutReplicates!=0){ + cds <- estimateDispersions( cds, method="blind")#in this case, without replicates + +} else if(withOutReplicates==0){ + cds <- estimateDispersions( cds, method="pooled", sharingMode="maximum", fitType="parametric") } + +# Analyse differentielle, ajustement BH par defaut +res <- nbinomTest( cds, cond1, cond2) + +# Diagnostic for clustering of normalized samples +OUT_clusterPlot <- paste(OUT_imgDir, "clusteringOfSamples.png", sep="/") +clusterPlot(cds, OUT_clusterPlot, out=outfile) + +# Control plot of dispersion estimates +OUT_plotDispEstimatesName <- paste(OUT_imgDir, "disperssionEstimates.png", sep="/") +plotDispEstimates( cds, OUT_plotDispEstimatesName, out=outfile ) + +# Distribution of raw p-values +OUT_histoRawpName <- paste(OUT_imgDir, "histoRawPvalue.png", sep="/") +histoRawp( res, OUT_histoRawpName, out=outfile ) + +# MAplot showing DE genes +OUT_MAplotDEName <- paste(OUT_imgDir, "MAplotDE.png", sep="/") +MAplotDE( res, alpha, OUT_MAplotDEName, out=outfile ) + +# export complete data +OUT_completeName <- args[9] +complete <- exportComplete( counts, res, target, adjMethod, cond1, cond2, OUT_completeName, out=outfile ) + +# export significant genes +OUT_upName <- args[10] +OUT_downName <- args[11] +diff <- exportDiff( complete, alpha, adjMethod, OUT_upName, OUT_downName, out=outfile ) + +# write all images results into an HTML file +prefixHTMLname <- tab[length(tab)] +#HTMLCSS(file.path(res_dir), filename=prefixHTMLname, CSSfile="R2HTML") +HTMLInitFile(file.path(res_dir), filename=prefixHTMLname, BackGroundColor="white") +HTML.title("<center>Differential Expression DESeq analysis.", HR=1) +HTML.title("<center>BarplotTC: number of RNA-seq reads per sample.", HR=2) + HTMLInsertGraph("barplotTC.png") + +HTML.title("<center>BarplotNul: number of RNA-seq reads that the count is 0 (nul).", HR=2) + HTMLInsertGraph("barplotNul.png") + +HTML.title("<center>DensityPlot: density of each sample.", HR=2) + HTMLInsertGraph("densityPlot.png") + +HTML.title("<center>Boxplot: number of RNA-seq reads distribution per sample.", HR=2) + HTMLInsertGraph("boxplotCounts.png") + +HTML.title("<center>MajorSequence: the proportion of reads associated with the most expressed sequence.", HR=2) + HTMLInsertGraph("majSequence.png") + +HTML.title("<center>ScatterPlot: Scatter plot of samples.", HR=2) + HTMLInsertGraph("scatterPlot.png") + +HTML.title("<center>Clustering Of No-Normalized Samples: Representing the no-normalized samples in Diagnostic.", HR=2) + HTMLInsertGraph("clusteringOfSamplesBefore.png") + +HTML.title("<center>Clustering Of Normalized Samples: Representing the normalized samples in Diagnostic.", HR=2) + HTMLInsertGraph("clusteringOfSamples.png") + +HTML.title("<center>DispersionEstimates: representing dispersion estimates vs mean expression.", HR=2) + HTMLInsertGraph("disperssionEstimates.png") + +HTML.title("<center>HistoRawPValue: histogram of raw p-value.", HR=2) + HTMLInsertGraph("histoRawPvalue.png") + +HTML.title("<center>MAplotDE: the differentially expressed genes (red point).", HR=2) + HTMLInsertGraph("MAplotDE.png") +HTMLEndFile() +absoluPrefixHTMLname <- paste(res_dir, prefixHTMLname, sep="") +outName <- paste(absoluPrefixHTMLname, ".html", sep="") +# change name is to be adapted into Galaxy +file.rename(outName, OUT_HTMLname) +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/barplotNul.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,20 @@ +# barplotNul +# barplot representing null counts per sample + +# input : counts, target, projectName +# output : barplotNul (png) + +# created Feb 7th, 2012 +# modified April 30th, 2012 (target$group instead of target) +# Marie-Anges Dillies + +barplotNul <- function( counts, group, OUT_barplotNulName, out = TRUE ){ + + if (out) png( file=OUT_barplotNulName ) + + N <- apply(counts, 2, function(x){sum(x == 0)})/nrow(counts) + barplot(N, col=as.integer(group)+1, main = "Proportion of null counts per Sample", ylim = c(0,1)) + legend("topright", as.character(unique(group)), lty=1, col=as.integer(unique(group))+1) + + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/barplotTC.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,21 @@ +# barplotTC +# barplot representing total count per sample + +# input : counts, target, projectName +# output : barplotTC (png) + +# created Feb 7th, 2012 +# modified April 30th, 2012 (group instead of target$group) +# Marie-Anges Dillies + +barplotTC <- function( counts, group, OUT_barplotTCName, out = TRUE ){ + + if (out) png( file=OUT_barplotTCName ) + + ylim <- c(0, max(colSums(counts))*1.2) + barplot( colSums(counts), col=as.integer(group)+1, main = "Total Read Count per Sample", ylim=ylim ) + legend( "topright", as.character(unique(group)), lty=1, + col=as.integer(unique(group))+1 ) + + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/boxplotCounts.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,19 @@ +# boxplotCounts +# boxplots representing counts distribution per sample + +# input : counts, target, projectName, type of data (raw or norm) +# output : boxplot (png) + +# created Feb 7th, 2012 +# modified April 30th, 2012 +# Marie-Anges Dillies + +boxplotCounts <- function( counts, group, type = c("raw", "norm"), OUT_boxplotCountsName, out = TRUE ){ + + if (out) png( file=OUT_boxplotCountsName ) + + boxplot( log2(counts+1), col=as.integer(group)+1, main = paste(type[1], " counts distribution", sep="" ) ) + legend( "topright", as.character(unique(group)), lty=1, col=as.integer(unique(group))+1 ) + + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/clusterPlot.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,27 @@ +# clusterPlot +# dendrogram of sample clustering + +# input : counts, outputName, type of data (raw or norm) +# output : dendrogram (jpeg) + +# created Sept 13th, 2012 +# modified Oct 30th, 2012 +# Marie-Agnes Dillies + + +clusterPlot <- function( cds, OUT_clusterPlot, type = "raw", out = TRUE ){ + + if (out) png( file=OUT_clusterPlot ) + + if (type == "norm"){ + cdsblind <- estimateDispersions( cds, method="blind" ) + vsd <- getVarianceStabilizedData( cdsblind ) + } + else { + vsd <- counts(cds) + } + hc <- hclust( dist(t(vsd)), method="ward" ) + plot( hc, xlab = "Euclidean distance, Ward criterion", main=paste("Cluster Dendrogram, ", type, " data", sep="") ) + + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/densityPlot.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,23 @@ +# densityPlot +# density plot of all samples + +# input : counts, target, projectName +# output : densplot (png) + +# created Feb 7th, 2012 +# modified April 30th, 2012 +# Marie-Anges Dillies + +densityPlot <- function( counts, group, OUT_densityPlotName, out = TRUE ){ + + if (out) png( file=OUT_densityPlotName ) + + couleurs <- as.integer( group ) + 1 + ylim <- c(0, max(density(log2(counts)+1)$y)*1.5) + plot( density(log2(counts[,1])+1), main="Density of counts distribution", col=couleurs[1], ylim = ylim ) + for (i in 2:ncol(counts)) + lines( density(log2(counts[,i])+1), col=couleurs[i] ) + legend( "topright", as.character(unique(group)), lty=1, col=as.integer(unique(group))+1 ) + + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/exportComplete.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,20 @@ +# exportComplete +# export complete data and results + +# input : counts res, target +# output : complete data and xls file (in text format) + +# created Feb 14th, 2012 +# modified March 9th, 2012 (order of cond1 and cond2) +# Marie-Anges Dillies + +exportComplete <- function( counts, res, target, adjMethod, cond1, cond2, OUT_completeName, out = T ){ + + complete <- data.frame( res$id, counts, res[,3:ncol(res)] ) + colnames(complete) <- c( "id", as.character(target$label), cond2, cond1, "FC", "log2FC", "rawp", + paste("adjp",adjMethod,sep="") ) + + if (out) + write.table( complete, file=OUT_completeName, sep="\t", row.names=F ) + return( complete ) +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/exportDiff.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,42 @@ +# exportDiff +# export differentially expressed genes + +# input : complete, alpha, adjMethod, projectName +# output : diff genes, up and down in xls files + +# created Feb 14th, 2012 +# Marie-Anges Dillies + +exportDiff <- function( complete, alpha, adjMethod, OUT_upName, OUT_downName, out = T ){ + + diff <- complete[which(complete[,grep("adjp",colnames(complete))] < alpha),] + + gup <- up( diff ) + gdown <- down( diff ) + + if (out){ + gup[,(ncol(gup)-4):ncol(gup)] <- format( gup[,(ncol(gup)-4):ncol(gup)], digits=3, dec=",") + gdown[,(ncol(gdown)-4):ncol(gdown)] <- format( gdown[,(ncol(gdown)-4):ncol(gdown)], digits=3, dec=",") + write.table(gup, file=OUT_upName, row.names=F, sep="\t") + write.table(gdown, file=OUT_downName, row.names=F, sep="\t") + } + return( diff ) +} + + +up <- function( diff ){ + + up <- diff[diff$log2FC > 0,] + up <- up[order(up[,grep("adjp",colnames(up))]),] + + return( up ) +} + + +down <- function( diff ){ + + down <- diff[diff$log2FC < 0,] + down <- down[order(down[,grep("adjp",colnames(down))]),] + + return( down ) +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/histoRawp.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,19 @@ +# Marie-Anges Dillies +# histoRawp +# histogram of raw p-values + +# input : res, OUT_histoRawpName +# output : histogram (png) + + +histoRawp <- function( res, OUT_histoRawpName, out = TRUE ){ + + if (out) png( file=OUT_histoRawpName ) + + ind <- grep("val", colnames(res)) + hist( res[,ind], nclass=50, xlab="Raw p-values", main="", col="skyblue" ) + + if (out) dev.off() +} + +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/loadCountData.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,36 @@ +# loadCountData +# loads counts, one file per lane +# file names from target file + +# input : target +# output : raw count table + +# created Feb 6th, 2012 +# modified May 2nd, 2012 (colnames -> target$label) +# Marie-Agnes Dillies + + +loadCountData <- function(target, header){ + + require(DESeq) + fileNames <- target$files + +if(header!=0){ + #rawCounts <- read.table(as.character(paste(rawDir,target$files[1],sep="/")), sep="\t", header=TRUE) + rawCounts <- read.table(as.character(target$files[1],sep="/"), sep="\t", header=TRUE) +} else if(header==0){ + rawCounts <- read.table(as.character(target$files[1],sep="/"), sep="\t")} + + colnames(rawCounts) <- c("Id", as.character(target$label[1])) + + for (i in 2:length(fileNames)){ + if(header!=0){ + tmp <- read.table(as.character(target$files[i],sep="/"), sep="\t", header=TRUE) + } else if(header==0){ + tmp <- read.table(as.character(target$files[i],sep="/"), sep="\t")} + colnames(tmp) <- c("Id", as.character(target$label[i])) + rawCounts <- merge(rawCounts, tmp, by="Id", all=T) + } + rawCounts[is.na(rawCounts)] <- 0 + return(rawCounts) +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/loadTargetFile.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,17 @@ +# loadTargetFile +# loads file containing sample info + +# input : targetFile Name +# output : target + +# created Feb 6th, 2012 +# Marie-Agnes Dillies + + +loadTargetFile <- function(targetFile, header){ +if(header!=0){ + return(read.table(targetFile, header=T, sep="\t")) + }else if(header==0){ + return(read.table(targetFile, sep="\t")) + } +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/majSequence.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,26 @@ +# majSequence +# compute proportion of reads associated with most expressed sequence + +# input : counts, target, projectName +# output : barplot, % associated with majority gene + +# created Feb 7th, 2012 +# modified Feb 20th, 2012 +# modified April 30th, 2012 +# Marie-Agnes Dillies + + +majSequence <- function( counts, group, OUT_majSequenceName, out = T, position = "topright" ){ + + if (out) png( file=OUT_majSequenceName ) + + maj <- apply(counts, 2, function(x){x <- x[order(x, decreasing=T)]; x[1]*100/sum(x)}) + seqname <- apply(counts, 2, function(x){x <- x[order(x, decreasing=T)]; names(x)[1]}) + + x <- barplot( maj, col=as.integer(group)+1, main = "Proportion of reads from most expressed gene", + ylim = c(0, max(maj)*1.2), cex.main=0.8 ) + for (i in 1:length(seqname)) text( x[i], maj[i]/2, seqname[i], cex=0.8, srt=90, adj=0) + legend( position, as.character(unique(group)), lty=1, col=as.integer(unique(group))+1 ) + + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/pairwiseSERE.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,41 @@ +# pairwiseSERE +# compute pairwise SERE statistics + +# input : counts +# output : matrix of SERE values + +# created october 19th, 2012 +# Marie-Agnes Dillies + + +pairwiseSERE <- function( counts ){ + + sere <- matrix( NA, ncol=ncol(counts), nrow=ncol(counts) ) + for (i in 1:ncol(counts)){ + for (j in 1:ncol(counts)){ + sere[i,j] <- sigfun_Pearson( counts[,c(i,j)] ) + } + } + colnames(sere) <- rownames(sere) <- colnames(counts) + return( formatC(sere, format="f", digits=2) ) +} + +sigfun_Pearson <- function(observed) { + #calculate lambda and expected values + laneTotals<- colSums(observed); + total <- sum(laneTotals) + fullObserved <- observed[rowSums(observed)>0,]; + fullLambda <- rowSums(fullObserved)/total; + fullLhat <- fullLambda > 0; + fullExpected<- outer(fullLambda, laneTotals); + + #keep values + fullKeep <- which(fullExpected > 0); + + #calculate degrees of freedom (nrow*(ncol -1) >> number of parameters - calculated (just lamda is calculated >> thats why minus 1) + #calculate pearson and deviance for all values + oeFull <- (fullObserved[fullKeep] - fullExpected[fullKeep])^2/ fullExpected[fullKeep] # pearson chisq test + dfFull <- length(fullKeep) - sum(fullLhat!=0); + + return(c(sqrt(sum(oeFull)/dfFull))); +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/pairwiseScatterPlots.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,31 @@ +# pairwiseScatterPlots +# scatter plots for pairwise comparaisons of log counts + +# input : counts, target, outputName +# output : scatter plots (pdf: allows multiple figures in one file) + +# created Feb 21th, 2012 +# modified Sept 27th, 2012 (pdf output file) +# modified Oct 30th, 2012 (png) +# Marie-Agnes Dillies + + +pairwiseScatterPlots <- function( counts, target, OUT_scatterPlot, out = TRUE, pdffile = FALSE ){ + + if (out & !pdffile) png( OUT_scatterPlot ) + if (pdffile) pdf( OUT_scatterPlot ) + + conds <- unique(target$group) + # colnames(counts) <- target$label + + for (i in 1:(length(conds)-1)){ + for (j in (i+1):length(conds)){ + cond1 <- conds[i]; cond2 <- conds[j] + pairs( log2(counts[, which(target$group %in% c(as.character(cond1), as.character(cond2)))]+1), + pch=".", cex=0.5, main = paste(cond1, cond2, sep=" vs ") ) + } + } + + if (pdffile) dev.off() + if (out) dev.off() +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/plotDispEstimates.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,22 @@ +# Marie-Anges Dillies +# plotDispEstimates +# scatter plots representing dispersion estimates vs mean expression + +# input : cds, OUT_plotDispEstimatesName +# output : scatterplot (png) + +plotDispEstimates <- function( cds, OUT_plotDispEstimatesName, out = TRUE ){ + + if (out) png( file=OUT_plotDispEstimatesName ) + + plot( + rowMeans( counts(cds, normalized=T) ), + fitInfo(cds)$perGeneDispEsts, + pch=".", log="xy", + xlab = "Mean expression strength", ylab = "Dispersion estimate" ) + + xg <- 10^seq(-.5, 5, length.out=300) + lines( xg, fitInfo(cds)$dispFun(xg), col="red" ) + + if (out) dev.off() +}
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/raw2counts.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,20 @@ +# raw2counts +# extract counts only from rawCounts +# and add rownames to counts + +# input : rawCounts +# output : counts + +# created Feb 6th, 2012 +# modified April 12, 2012 +# Marie-Agnes Dillies + + +raw2counts <- function( rawCounts, annot=1 ){ + + ex <- 1:annot + counts <- as.matrix( rawCounts[,-ex] ) + rownames(counts) <- rawCounts[,1] + infoCounts <- rawCounts[,ex] + return( list("counts"=counts, "infoCounts"= infoCounts) ) +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/DESeqTools/removeNul.R Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,14 @@ +# removeNul +# remove genes with null counts in all samples + +# input : counts +# output : counts + +# created Feb 7th, 2012 +# Marie-Agnes Dillies + + +removeNul <- function( counts, info = NULL ){ + + return( list(counts[rowSums(counts) > 0,], info[rowSums(counts) > 0,]) ) +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/README.txt Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,82 @@ +---------- +| NAME | +---------- +Differential Expression Analysis Pipeline on RNA-Seq data + + +Description +----------- +This pipeline has been founded by IBISA-APLIBIO(http://www.renabi.fr/platforms/aplibio/), +and has been built by (1)Yufei Luo, (2)Marie-Agnès Dillies, (1)Matthias Zytnicki and (1)Delphine Steinbach. +(1): Bioinformatics plateform URGI, INRA URGI Versailles, France +(2): Plate-forme Transcriptome et Epigenome, Génopole, Institut Pasteur, France + + +This pipeline performs differential expression analysis on two different conditions with arbitrary number of replicates. + +One reference genome (fasta format), one annotation (gtf format) and several RNA-seq samples are required. +Step 0: Upload RNA-seq samples. +Step 1: Clean the annotation file. +Step 2: Clean the RNA-seq files. +Step 3: Map the RNA-seq samples to the genome reference, using Tophat. +Step 4: Convert the bam files (given by Tophat) to sam files. +Step 5: Count the reads per annotation using S-MART (CompareOverlapping). +Step 6: Build the input files for DESeq. +Step 7: Differential expression analysis using DESeq, and output graphical results. + +More information about using this pipeline, please see: http://urgi.versailles.inra.fr/galaxy/u/yluo/p/differential-expression-pipeline-documentation + + +Instructions +------------ +Environment Installation: +0). First, download and install S-MART (eg from Galaxy Tool Shed: http://toolshed.g2.bx.psu.edu/repos/yufei-luo/s_mart). +1). Download and put the pipeline scripts directory under the S-MART directory + (ex. mv DiffExpAnal /home/user/galaxy-dist/tools/s_mart/SMART/.). + +Supplementary Softwares: + * R, under the GNU General Public License (at least 2.14.1 version). + * Python, under the Python License, compatible with the GNU General Public License (better 2.7) + * S-MART (http://urgi.versailles.inra.fr/Tools/S-Mart), a toolbox which handles mapped RNA-Seq and ChIP-Seq data. + * DESeq (http://bioconductor.org/packages/release/bioc/html/DESeq.html), an R package to analyse + count data from high-throughput sequencing assays such as RNA-Seq and test + for differential expression. The package is available via Bioconductor (http://www.bioconductor.org/) + and can be conveniently installed as follows: + Start an R session and type: + source("http://www.bioconductor.org/biocLite.R") + biocLite("DESeq") + * Biobase(http://www.bioconductor.org/packages/2.12/bioc/html/Biobase.html), an + Bioconductor package and can be installed as follows: + source("http://bioconductor.org/biocLite.R") + biocLite("Biobase") + + +Copyright +--------- +Copyright INRA-URGI 20012-2013 + + +Authors +------- +Yufei Luo +Marie-Agnès Dillies +Matthias Zytnicki +Delphine Steinbach + + +Contact +------- +urgi-support@versailles.inra.fr + + +License +------- +This library is distributed under the terms of the CeCILL license +(http://www.cecill.info/index.en.html). +See the LICENSE.txt file. + + +Acknowledgements +---------------- +Yufei Luo was supported by the Plant Breeding and Genetics research division of +the INRA, and by the Groupement d'intérêt scientifique IBISA.
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/bam_to_sam_parallel.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,168 @@ +#!/usr/bin/env python +""" +Yufei LUO +""" +import optparse, os, sys, subprocess, tempfile, shutil, tarfile, random +#from galaxy import eggs +#import pkg_resources; pkg_resources.require( "bx-python" ) +#from bx.cookbook import doc_optparse +#from galaxy import util + +def stop_err( msg ): + sys.stderr.write( '%s\n' % msg ) + sys.exit() + +def toTar(tarFileName, samOutputNames): + dir = os.path.dirname(tarFileName) + tfile = tarfile.open(tarFileName + ".tmp.tar", "w") + currentPath = os.getcwd() + os.chdir(dir) + for file in samOutputNames: + relativeFileName = os.path.basename(file) + tfile.add(relativeFileName) + os.system("mv %s %s" % (tarFileName + ".tmp.tar", tarFileName)) + tfile.close() + os.chdir(currentPath) + + +def __main__(): + #Parse Command Line + parser = optparse.OptionParser() + parser.add_option('-t', '--tar', dest='outputTar', default=None, help='output all SAM results in a tar file.' ) + parser.add_option( '', '--input1', dest='input1', help='The input list of BAM datasets on txt format.' ) + #parser.add_option( '', '--input1', dest='input1', help='The input BAM dataset' ) + parser.add_option( '', '--output1', dest='output1', help='The output list of SAM datasets on txt format.' ) + #parser.add_option( '', '--output1', dest='output1', help='The output SAM dataset' ) + parser.add_option( '', '--header', dest='header', action='store_true', default=False, help='Write SAM Header' ) + ( options, args ) = parser.parse_args() + + + #Parse the input txt file and read a list of BAM files. + file = open(options.input1, "r") + lines = file.readlines() + inputFileNames = [] + samOutputNames = [] + outputName = options.output1 + resDirName = os.path.dirname(outputName) + '/' + #Write output txt file and define all output sam file names. + out = open(outputName, "w") + for line in lines: + tab = line.split() + inputFileNames.append(tab[1]) + samOutName = resDirName + tab[0] + '_samOutput_%s.sam' % random.randrange(0, 10000) + samOutputNames.append(samOutName) + out.write(tab[0] + '\t' + samOutName + '\n') + file.close() + out.close() + + # output version # of tool + try: + tmp_files = [] + tmp = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp) + tmp_stdout = open( tmp, 'wb' ) + proc = subprocess.Popen( args='samtools 2>&1', shell=True, stdout=tmp_stdout ) + tmp_stdout.close() + returncode = proc.wait() + stdout = None + for line in open( tmp_stdout.name, 'rb' ): + if line.lower().find( 'version' ) >= 0: + stdout = line.strip() + break + if stdout: + sys.stdout.write( 'Samtools %s\n' % stdout ) + else: + raise Exception + except: + sys.stdout.write( 'Could not determine Samtools version\n' ) + + + + tmp_dirs = [] + for i in range(len(inputFileNames)): + try: + # exit if input file empty + if os.path.getsize( inputFileNames[i] ) == 0: + raise Exception, 'Initial input txt file is empty.' + # Sort alignments by leftmost coordinates. File <out.prefix>.bam will be created. This command + # may also create temporary files <out.prefix>.%d.bam when the whole alignment cannot be fitted + # into memory ( controlled by option -m ). + tmp_dir = tempfile.mkdtemp() + tmp_dirs.append(tmp_dir) + tmp_sorted_aligns_file = tempfile.NamedTemporaryFile( dir=tmp_dir ) + tmp_sorted_aligns_file_base = tmp_sorted_aligns_file.name + tmp_sorted_aligns_file_name = '%s.bam' % tmp_sorted_aligns_file.name + tmp_files.append(tmp_sorted_aligns_file_name) + tmp_sorted_aligns_file.close() + + command = 'samtools sort %s %s' % ( inputFileNames[i], tmp_sorted_aligns_file_base ) + tmp = tempfile.NamedTemporaryFile( dir=tmp_dir ).name + tmp_stderr = open( tmp, 'wb' ) + proc = subprocess.Popen( args=command, shell=True, cwd=tmp_dir, stderr=tmp_stderr.fileno() ) + returncode = proc.wait() + tmp_stderr.close() + # get stderr, allowing for case where it's very large + tmp_stderr = open( tmp, 'rb' ) + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += tmp_stderr.read( buffsize ) + if not stderr or len( stderr ) % buffsize != 0: + break + except OverflowError: + pass + tmp_stderr.close() + if returncode != 0: + raise Exception, stderr + # exit if sorted BAM file empty + if os.path.getsize( tmp_sorted_aligns_file_name) == 0: + raise Exception, 'Intermediate sorted BAM file empty' + except Exception, e: + stop_err( 'Error sorting alignments from (%s), %s' % ( inputFileNames[i], str( e ) ) ) + + try: + # Extract all alignments from the input BAM file to SAM format ( since no region is specified, all the alignments will be extracted ). + if options.header: + view_options = "-h" + else: + view_options = "" + command = 'samtools view %s -o %s %s' % ( view_options, samOutputNames[i], tmp_sorted_aligns_file_name ) + tmp = tempfile.NamedTemporaryFile( dir=tmp_dir ).name + tmp_stderr = open( tmp, 'wb' ) + proc = subprocess.Popen( args=command, shell=True, cwd=tmp_dir, stderr=tmp_stderr.fileno() ) + returncode = proc.wait() + tmp_stderr.close() + # get stderr, allowing for case where it's very large + tmp_stderr = open( tmp, 'rb' ) + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += tmp_stderr.read( buffsize ) + if not stderr or len( stderr ) % buffsize != 0: + break + except OverflowError: + pass + tmp_stderr.close() + if returncode != 0: + raise Exception, stderr + except Exception, e: + stop_err( 'Error extracting alignments from (%s), %s' % ( inputFileNames[i], str( e ) ) ) + if os.path.getsize( samOutputNames[i] ) > 0: + sys.stdout.write( 'BAM file converted to SAM' ) + else: + stop_err( 'The output file is empty, there may be an error with your input file.' ) + + if options.outputTar != None: + toTar(options.outputTar, samOutputNames) + #clean up temp files + for tmp_dir in tmp_dirs: + if os.path.exists( tmp_dir ): + shutil.rmtree( tmp_dir ) + #print tmp_files + #for tmp in tmp_files: + # os.remove(tmp) + + +if __name__=="__main__": __main__()
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/bam_to_sam_parallel.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,32 @@ +<tool id="bam_to_sam_parallel" name="BAM to SAM" version="1.0.0"> + <description>converts a list of BAM format files to SAM format</description> + <requirements> + <requirement type="package">samtools</requirement> + </requirements> + <command interpreter="python"> bam_to_sam_parallel.py + --input1=$input1 + --output1=$output1 + $header + $tar $outputTarFile + </command> + <inputs> + <param name="input1" type="data" format="txt" label="BAM File LIST to Convert" /> + <param name="header" type="boolean" truevalue="--header" falsevalue="" checked="False" label="Include header in output" /> + <param name="tar" type="boolean" truevalue="-t" falsevalue="" checked="false" label="tar option" help="This option creates a tar file for all out results." /> + </inputs> + <outputs> + <data format="txt" name="output1" label="converted SAM LIST files " /> + <data name="outputTarFile" format="tar"> + <filter>tar</filter> + </data> + </outputs> + <help> + +**What it does** + +This tool uses the SAMTools_ toolkit to produce a SAM file from a BAM file. + +.. _SAMTools: http://samtools.sourceforge.net/samtools.shtml + + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/compareOverlapping_parallel.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,179 @@ +#! /usr/bin/env python +""" +Yufei LUO +""" + +#This program is a wrapp for CompareOverlapping.py. +import optparse, os, sys, subprocess, tempfile, shutil, tarfile, glob +import os, struct, time, random +from optparse import OptionParser +from commons.core.parsing.ParserChooser import ParserChooser +from commons.core.writer.Gff3Writer import Gff3Writer +from SMART.Java.Python.CompareOverlapping import CompareOverlapping +from SMART.Java.Python.structure.Transcript import Transcript +from SMART.Java.Python.structure.Interval import Interval +from SMART.Java.Python.ncList.NCList import NCList +from SMART.Java.Python.ncList.NCListCursor import NCListCursor +from SMART.Java.Python.ncList.NCListFilePickle import NCListFilePickle, NCListFileUnpickle +from SMART.Java.Python.ncList.FileSorter import FileSorter +from SMART.Java.Python.misc.Progress import Progress +from SMART.Java.Python.misc.UnlimitedProgress import UnlimitedProgress +from SMART.Java.Python.misc import Utils + + + +def stop_err( msg ): + sys.stderr.write( "%s\n" % msg ) + sys.exit() + +def toTar(tarFileName, overlapOutputNames): + dir = os.path.dirname(tarFileName) + tfile = tarfile.open(tarFileName + ".tmp.tar", "w") + currentPath = os.getcwd() + os.chdir(dir) + for file in overlapOutputNames: + relativeFileName = os.path.basename(file) + tfile.add(relativeFileName) + os.system("mv %s %s" % (tarFileName + ".tmp.tar", tarFileName)) + tfile.close() + os.chdir(currentPath) + +def __main__(): + description = "Compare Overlapping wrapp script: Get the a list of data which overlap with a reference set. [Category: Data Comparison]" + parser = OptionParser(description = description) + parser.add_option("-i", "--input1", dest="inputFileName1", action="store", type="string", help="input file 1 (for annotation) [compulsory] [format: file in transcript format given by -f]") + parser.add_option("-f", "--format1", dest="format1", action="store", type="string", help="format of file 1 [compulsory] [format: transcript file format]") + parser.add_option("", "--inputTxt", dest="inputTxt", action="store", type="string", help="input, a txt file for a list of input reads files. Should identify all reads files format, given by -g [compulsory]") + #parser.add_option("-j", "--input2", dest="inputFileName2", action="store", default="inputRead", type="string", help="input file 2 [compulsory] [format: file in transcript format given by -g]") + parser.add_option("-g", "--format2", dest="format2", action="store", type="string", help="format of file 2 [compulsory] [format: transcript file format]") + #parser.add_option("-o", "--output", dest="output", action="store", default=None, type="string", help="output file [compulsory] [format: output file in GFF3 format]") + parser.add_option("-S", "--start1", dest="start1", action="store", default=None, type="int", help="only consider the n first nucleotides of the transcripts in file 1 (do not use it with -U) [format: int]") + parser.add_option("-s", "--start2", dest="start2", action="store", default=None, type="int", help="only consider the n first nucleotides of the transcripts in file 2 (do not use it with -u) [format: int]") + parser.add_option("-U", "--end1", dest="end1", action="store", default=None, type="int", help="only consider the n last nucleotides of the transcripts in file 1 (do not use it with -S) [format: int]") + parser.add_option("-u", "--end2", dest="end2", action="store", default=None, type="int", help="only consider the n last nucleotides of the transcripts in file 2 (do not use it with -s) [format: int]") + parser.add_option("-t", "--intron", dest="introns", action="store_true", default=False, help="also report introns [format: bool] [default: false]") + parser.add_option("-E", "--5primeExtension1", dest="fivePrime1", action="store", default=None, type="int", help="extension towards 5' in file 1 [format: int]") + parser.add_option("-e", "--5primeExtension2", dest="fivePrime2", action="store", default=None, type="int", help="extension towards 5' in file 2 [format: int]") + parser.add_option("-N", "--3primeExtension1", dest="threePrime1", action="store", default=None, type="int", help="extension towards 3' in file 1 [format: int]") + parser.add_option("-n", "--3primeExtension2", dest="threePrime2", action="store", default=None, type="int", help="extension towards 3' in file 2 [format: int]") + parser.add_option("-c", "--colinear", dest="colinear", action="store_true", default=False, help="colinear only [format: bool] [default: false]") + parser.add_option("-a", "--antisense", dest="antisense", action="store_true", default=False, help="antisense only [format: bool] [default: false]") + parser.add_option("-d", "--distance", dest="distance", action="store", default=None, type="int", help="accept some distance between query and reference [format: int]") + parser.add_option("-k", "--included", dest="included", action="store_true", default=False, help="keep only elements from file 1 which are included in an element of file 2 [format: bool] [default: false]") + parser.add_option("-K", "--including", dest="including", action="store_true", default=False, help="keep only elements from file 2 which are included in an element of file 1 [format: bool] [default: false]") + parser.add_option("-m", "--minOverlap", dest="minOverlap", action="store", default=None, type="int", help="minimum number of nucleotides overlapping to declare an overlap [format: int] [default: 1]") + parser.add_option("-p", "--pcOverlap", dest="pcOverlap", action="store", default=None, type="int", help="minimum percentage of nucleotides to overlap to declare an overlap [format: int]") + parser.add_option("-O", "--notOverlapping", dest="notOverlapping", action="store_true", default=False, help="also output not overlapping data [format: bool] [default: false]") + parser.add_option("-x", "--exclude", dest="exclude", action="store_true", default=False, help="invert the match [format: bool] [default: false]") + parser.add_option("-v", "--verbosity", dest="verbosity", action="store", default=1, type="int", help="trace level [format: int]") + parser.add_option('', '--tar', dest='outputTar', default=None, help='output all SAM results in a tar file.' ) + parser.add_option( '', '--outTxt', dest='outTxtFile', help='The output list of results files on txt format.[compulsory]' ) + (options, args) = parser.parse_args() + + + #Parse the input txt file and read a list of BAM files. + file = open(options.inputTxt, "r") + lines = file.readlines() + inputFileNames = [] + overlapOutputNames = [] + outputName = options.outTxtFile + resDirName = os.path.dirname(outputName) + "/" + #Write output txt file and define all output sam file names. + out = open(outputName, "w") + for line in lines: + tab = line.split() + inputFileNames.append(tab[1]) + overlapOutName = resDirName + tab[0] + '_overlapOut_%s.gff3' % random.randrange(0, 10000) + overlapOutputNames.append(overlapOutName) + out.write(tab[0] + '\t' + overlapOutName + '\n') + file.close() + out.close() + + #construction the commandes for each input file + cmds = [] + for i in range(len(inputFileNames)): + absFile = sys.argv[0] + absDir = os.path.dirname(absFile) + parentDir = os.path.abspath(os.path.join(absDir, os.path.pardir)) + cmd = "python %s/Java/Python/CompareOverlappingSmallQuery.py " % parentDir + opts = "-i %s -f %s -j %s -g %s -o %s " % (options.inputFileName1, options.format1, inputFileNames[i], options.format2, overlapOutputNames[i]) + #if options.start1 != None: + # opts += "-S %s " % options.start1 + #if options.start2 != None: + # opts += "-s %s " % options.start2 + #if options.end1 != None: + # opts += "-U %s " % options.end1 + #if options.end2 != None: + # opts += "-u %s " % options.end2 + #if options.fivePrime1 != None: + # opts += "-E %s " % options.fivePrime1 + #if options.fivePrime2 != None: + # opts += "-e %s " % options.fivePrime2 + #if options.threePrime1 != None: + # opts += "-N %s " % options.threePrime1 + #if options.threePrime2 != None: + # opts += "-n %s " % options.threePrime2 + #if options.colinear: + # opts += "-c " + #if options.antisense: + # opts +="-a " + #if options.included: + # opts += "-k " + #if options.including: + # opts += "-K " + #if options.pcOverlap != None: + # opts += "-p %s " % options.pcOverlap + if options.notOverlapping: + opts += "-O " + if options.exclude: + opts += "-x " + if options.distance != None: + opts += "-d %s " % options.distance + #if options.minOverlap != None: + # opts += "-m %s " % options.minOverlap + cmd += opts + cmds.append(cmd) + + + print "les commandes sont %s \n" % cmds + + tmp_files = [] + for i in range(len(cmds)): + try: + tmp_out = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_out) + tmp_stdout = open( tmp_out, 'wb' ) + tmp_err = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_err) + tmp_stderr = open( tmp_err, 'wb' ) + proc = subprocess.Popen( args=cmds[i], shell=True, cwd=".", stdout=tmp_stdout, stderr=tmp_stderr ) + returncode = proc.wait() + tmp_stderr.close() + # get stderr, allowing for case where it's very large + tmp_stderr = open( tmp_err, 'rb' ) + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += tmp_stderr.read( buffsize ) + if not stderr or len( stderr ) % buffsize != 0: + break + except OverflowError: + pass + tmp_stdout.close() + tmp_stderr.close() + if returncode != 0: + raise Exception, stderr + except Exception, e: + stop_err( 'Error in :\n' + str( e ) ) + + if options.outputTar != None: + toTar(options.outputTar, overlapOutputNames) + + for tmp_file in tmp_files: + os.remove(tmp_file) + + +if __name__=="__main__": __main__() + +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/compareOverlapping_parallel.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,251 @@ +<tool id="CompareOverlapping_parallel" name="CompareOverlapping_parallel"> + <description>Shrink or extend the sets of genomic coordinates to get the information between starts of reads and starts of genes.</description> + <command interpreter="python"> + compareOverlapping_parallel.py -i $formatType.inputFileName1 + #if $formatType.FormatInputFileName1 == 'bed': + -f bed + #elif $formatType.FormatInputFileName1 == 'gff': + -f gff + #elif $formatType.FormatInputFileName1 == 'gff2': + -f gff2 + #elif $formatType.FormatInputFileName1 == 'gff3': + -f gff3 + #elif $formatType.FormatInputFileName1 == 'sam': + -f sam + #elif $formatType.FormatInputFileName1 == 'gtf': + -f gtf + #end if + + --inputTxt $inputTxt + + -g $format2 + + --outTxt $outTxtFile + + #if $optionNFirstFile1.NFirstForFile1 == 'Yes': + -S $optionNFirstFile1.firstNtFile1 + #end if + #if $optionNFirstFile2.NFirstForFile2 == 'Yes': + -s $optionNFirstFile2.firstNtFile2 + #end if + #if $optionNLastFile1.NLastForFile1 == 'Yes': + -U $optionNLastFile1.lastNtFile1 + #end if + #if $optionNLastFile2.NLastForFile2 == 'Yes': + -u $optionNLastFile2.lastNtFile2 + #end if + + #if $optionExtentionCinqFile1.extentionFile1 == 'Yes': + -E $optionExtentionCinqFile1.extention51 + #end if + #if $optionExtentionCinqFile2.extentionFile2 == 'Yes': + -e $optionExtentionCinqFile2.extention52 + #end if + + #if $optionExtentionTroisFile1.extentionFile1 == 'Yes': + -N $optionExtentionTroisFile1.extention31 + #end if + #if $optionExtentionTroisFile2.extentionFile2 == 'Yes': + -n $optionExtentionTroisFile2.extention32 + #end if + + #if $OptionColinearOrAntiSens.OptionCA == 'Colinear': + -c + #elif $OptionColinearOrAntiSens.OptionCA == 'AntiSens': + -a + #end if + + #if $OptionDistance.Dist == 'Yes': + -d $OptionDistance.distance + #end if + + #if $OptionMinOverlap.MO == 'Yes': + -m $OptionMinOverlap.minOverlap + #end if + + $InvertMatch + $ReportIntron + $NotOverlapping + $tar $outputTarFile + </command> + + <inputs> + + <conditional name="formatType"> + <param name="FormatInputFileName1" type="select" label="Input File Format 1"> + <option value="bed">bed</option> + <option value="gff">gff</option> + <option value="gff2">gff2</option> + <option value="gff3">gff3</option> + <option value="sam">sam</option> + <option value="gtf">gtf</option> + </param> + <when value="bed"> + <param name="inputFileName1" format="bed" type="data" label="Input File 1"/> + </when> + <when value="gff"> + <param name="inputFileName1" format="gff" type="data" label="Input File 1"/> + </when> + <when value="gff2"> + <param name="inputFileName1" format="gff2" type="data" label="Input File 1"/> + </when> + <when value="gff3"> + <param name="inputFileName1" format="gff3" type="data" label="Input File 1"/> + </when> + <when value="sam"> + <param name="inputFileName1" format="sam" type="data" label="Input File 1"/> + </when> + <when value="gtf"> + <param name="inputFileName1" format="gtf" type="data" label="Input File 1"/> + </when> + </conditional> + + <param name="inputTxt" type="data" format="txt" label="A txt file contains a list of several input transcripts files." /> + + <param name="format2" type="text" value="bed" label="format for File 2, you can choose [bed, gff, gff2, gff3, sam, gtf]"/> + + <conditional name="optionNFirstFile1"> + <param name="NFirstForFile1" type="select" label="NFirst for file 1" help="only consider the n first nucleotides of the transcripts in file 1"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="firstNtFile1" type="integer" value="1" label="n first nucleotides for input file 1" /> + </when> + <when value="No"> + </when> + </conditional> + <conditional name="optionNFirstFile2"> + <param name="NFirstForFile2" type="select" label="NFirst for file 2" help="only consider the n first nucleotides of the transcripts in file 2"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="firstNtFile2" type="integer" value="1" label="n first nucleotides for input file 1" /> + </when> + <when value="No"> + </when> + </conditional> + + <conditional name="optionNLastFile1"> + <param name="NLastForFile1" type="select" label="NLast for file 1"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="lastNtFile1" type="integer" value="1" label="n last nucleotides for input file 1" help="only consider the n last nucleotides of the transcripts in file 1"/> + </when> + <when value="No"> + </when> + </conditional> + <conditional name="optionNLastFile2"> + <param name="NLastForFile2" type="select" label="NLast for file 2"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="lastNtFile2" type="integer" value="1" label="n last nucleotides for input file 2" help="only consider the n last nucleotides of the transcripts in file 2"/> + </when> + <when value="No"> + </when> + </conditional> + + <conditional name="optionExtentionCinqFile1"> + <param name="extentionFile1" type="select" label="Extension towards 5 for file 1"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="extention51" type="integer" value="1" label="in file 1" /> + </when> + <when value="No"> + </when> + </conditional> + + <conditional name="optionExtentionCinqFile2"> + <param name="extentionFile2" type="select" label="Extension towards 5 for file 2"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="extention52" type="integer" value="1" label="in file 2"/> + </when> + <when value="No"> + </when> + </conditional> + + <conditional name="optionExtentionTroisFile1"> + <param name="extentionFile1" type="select" label="Extension towards 3 for file 1"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="extention31" type="integer" value="1" label="in file 1" /> + </when> + <when value="No"> + </when> + </conditional> + + <conditional name="optionExtentionTroisFile2"> + <param name="extentionFile2" type="select" label="Extension towards 3 for file 2"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="extention32" type="integer" value="1" label="in file 2" /> + </when> + <when value="No"> + </when> + </conditional> + + <conditional name="OptionColinearOrAntiSens"> + <param name="OptionCA" type="select" label="Colinear or anti-sens"> + <option value="Colinear">Colinear</option> + <option value="AntiSens">AntiSens</option> + <option value="NONE" selected="true">NONE</option> + </param> + <when value="Colinear"> + </when> + <when value="AntiSens"> + </when> + <when value="NONE"> + </when> + </conditional> + + <conditional name="OptionDistance"> + <param name="Dist" type="select" label="Maximum Distance between two reads"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="distance" type="integer" value="0"/> + </when> + <when value="No"> + </when> + </conditional> + + <conditional name="OptionMinOverlap"> + <param name="MO" type="select" label="Minimum number of overlapping between two reads"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="minOverlap" type="integer" value="1"/> + </when> + <when value="No"> + </when> + </conditional> + <param name="InvertMatch" type="boolean" truevalue="-x" falsevalue="" checked="false" label="Invert match"/> + <param name="ReportIntron" type="boolean" truevalue="-t" falsevalue="" checked="false" label="Report intron"/> + <param name="NotOverlapping" type="boolean" truevalue="-O" falsevalue="" checked="false" label="When there is no overlapping, the number of Overlapping will be set to 0 by defalt."/> + <param name="tar" type="boolean" truevalue="--tar" falsevalue="" checked="false" label="tar option" help="This option creates a tar file for all out results." /> + </inputs> + + <outputs> + <data name="outTxtFile" format="txt" label="overlapping output files "/> + <data name="outputTarFile" format="tar"> + <filter>tar</filter> + </data> + </outputs> + +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/countNumber.pl Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,37 @@ +#!/usr/bin/perl -w +### +###Yufei LUO +### + +use strict; + +my $in_file = $ARGV[0]; +my $out_file = $ARGV[1]; +my $sort_type = $ARGV[2]; # n(umeric) or a(lphanumeric) +my ($line,$ID,$nbOverlaps,%hash); + +open(IN, $in_file); +while ($line = <IN>){ + chomp($line); + $line=~s/\t/|/g; + my @part=split(/\|/,$line); + my @split=split(";",$part[$#part]); + $split[0] =~ m/^(\w+).+$/; + + foreach my $i (@split){ + if ($i=~m/nbOverlaps=(.+)/){ + $nbOverlaps=$1; + } + if ($i=~m/gene_id=(.+)/){ + $ID=$1; + $hash{$ID}=$nbOverlaps; + } + } +} +close(IN); + +open(OUT, ">$out_file"); +foreach my $key ( sort keys %hash) { + print OUT "$key\t$hash{$key}\n"; +} +close(OUT);
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/countNumber.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,16 @@ +<tool id="countNumber" name="countNumber"> + <description>Calculate the number of reads(annotations) overlapping for each transcript.</description> + <command interpreter="perl"> countNumber.pl $input $outputCSV + </command> + + <inputs> + <param name="input" type="data" format="gff3" label="Please choose your gff3 format file (which contains the number of overlaps)."/> + </inputs> + + <outputs> + <data format="csv" name="outputCSV" label="countNumber Output"/> + </outputs> + + <help> + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/countNumber_parallel.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,98 @@ +#! /usr/bin/env python +""" +Yufei LUO +""" + +import optparse, os, sys, subprocess, tempfile, shutil, tarfile, random +from optparse import OptionParser + +def stop_err(msg): + sys.stderr.write('%s\n' % msg) + sys.exit() + +def toTar(tarFileName, outCountNames): + dir = os.path.dirname(tarFileName) + tfile = tarfile.open(tarFileName + ".tmp.tar", "w") + currentPath = os.getcwd() + os.chdir(dir) + for file in outCountNames: + relativeFileName = os.path.basename(file) + tfile.add(relativeFileName) + os.system("mv %s %s" % (tarFileName + ".tmp.tar", tarFileName)) + tfile.close() + os.chdir(currentPath) + + +def __main__(): + #Parse Command Line + parser = optparse.OptionParser() + parser.add_option("-i", "--input", dest="inputFile", help="input txt file, a list of overlapping results files.") + parser.add_option("-o", "--output", dest="outputFile", help="Out txt file.") + parser.add_option("-t", "--tar", dest="outputTar", default=None, help="output all count results in a tar file.") + (options, args) = parser.parse_args() + + #Parse the input txt file and read a list of transcripts files. + file = open(options.inputFile, "r") + lines = file.readlines() + inputFileNames = [] + outCountNames = [] + outputName = options.outputFile + resDirName = os.path.dirname(outputName) + '/' + + #Write output txt file and define all output count file names + out = open(outputName, "w") + out.write("label\tfiles\tgroup\n") + for line in lines: + tab = line.split() + inputFileNames.append(tab[1]) + outCountName = resDirName + tab[0] + "_outCount_%s.csv" % random.randrange(0, 10000) + outCountNames.append(outCountName) + out.write(tab[0] + '\t' + outCountName + '\t' + tab[0][5] + '\n') + file.close() + out.close() + + #Construct the lines commands + cmds = [] + for i in range(len(inputFileNames)): + cmd = "perl countNumber.pl " + opts = "%s %s " % (inputFileNames[i], outCountNames[i]) + cmd += opts + cmds.append(cmd) + + tmp_files = [] + for i in range(len(cmds)): + try: + tmp_out = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_out) + tmp_stdout = open(tmp_out, 'wb') + tmp_err = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_err) + tmp_stderr = open(tmp_err, 'wb') + proc = subprocess.Popen(args=cmds[i], shell=True, cwd=".", stdout=tmp_stdout, stderr=tmp_stderr) + returncode = proc.wait() + tmp_stderr.close() + #get stderr, allowing for case where it's very large + tmp_stderr = open(tmp_err, 'rb') + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += tmp_stderr.read(buffsize) + if not stderr or len(stderr) % buffsize != 0: + break + except OverflowError: + pass + tmp_stdout.close() + tmp_stderr.close() + if returncode != 0: + raise Exception, stderr + except Exception, e: + stop_err('Error in :\n' + str(e)) + + if options.outputTar != None: + toTar(options.outputTar, outCountNames) + + for tmp_file in tmp_files: + os.remove(tmp_file) + +if __name__=="__main__":__main__()
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/countNumber_parallel.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,19 @@ +<tool id="countNumber_parallel" name="countNumber_parallel"> + + <description>Calculate the number of reads(annotations) overlapping for each transcript.</description> + <command interpreter="python"> countNumber_parallel.py -i $inputTxt -o $outputTxt $tar $outputTarFile + </command> + + <inputs> + <param name="inputTxt" type="data" format="txt" label="Please choose your txt format file (which contains a list of gff3 overlapping results files)."/> + <param name="tar" type="boolean" truevalue="-t" falsevalue="" checked="False" label="tar option" help="This option creates a tar file for all out results" /> + </inputs> + + <outputs> + <data format="txt" name="outputTxt" label="countNumber Output"/> + <data name="outputTarFile" format="tar"> + <filter>tar</filter> + </data> + </outputs> + +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/deseq.sh Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,25 @@ +#! /bin/sh +### +###Yufei LUO +### + + +#Arguments : +#$1=targetFile(the list of files) +#$2=with or without header +#$3=with or without replicates +#$4=OUT_HTML.html +#$5=OUT_HTML images directory +#$6=OUT_complete.xls +#$7=OUT_up.xls +#$8=OUT_down.xls + +#run example: +#bash deseq.sh DESeqTools/targetTest.txt 1 1 testOUT_HTML.html /tmp/ testOUT_complet.xls testOUT_up.xls testOUT_down.xls + +#echo $5 +#mkdir -p $5 #First, create the images tmp directory given by Galaxy, -p option can create the parent directory which dosen't exist. + +mkdir -p $5 +MY_PATH=`dirname $0` +cat $MY_PATH/DESeqTools/anadiffGenes2conds.R | R --slave --args $1 $2 $3 $4 $5 $6 $7 $8 $0 < $MY_PATH/DESeqTools/anadiffGenes2conds.R
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/deseq.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,28 @@ +<tool id="DESEQ" name="DESEQ for differential expression analysis"> + <description>Differential expression analysis for reads count data</description> + <requirements> + <requirement type="package">R</requirement> + <requirement type="package">Biobase</requirement> + <requirement type="package">DESeq</requirement> + </requirements> + + <command interpreter="bash"> deseq.sh $inputFile $header $withOutReplicates $outHTML $outHTML.files_path $outComplete $outUP $outDown 2> $log </command> + + <inputs> + <param name="inputFile" type="data" label="Input File list" format="txt"/> + <param name="header" type="boolean" truevalue="1" falsevalue="0" checked="false" label="If there is a header for your count files, please choose this case."/> + <param name="withOutReplicates" type="boolean" truevalue="1" falsevalue="0" checked="false" label="If your data has not replicates, please choose this case."/> + + </inputs> + + <outputs> + <data format="HTML" name="outHTML" label="[DESEQ] Output HTML File" help="This output file shows all results images by DESeq analysis"/> + <data format="tabular" name="outComplete" label="[DESEQ] Output complete File"/> + <data format="tabular" name="outUP" label="[DESEQ] Output up File" help="This output file shows the genes of group1 which are overexpressed than those of group2"/> + <data format="tabular" name="outDown" label="[DESEQ] Output down File" help="This output file shows the genes of group1 which are less expressed than those of group2"/> + <data format="txt" name="log" label="[DESEQ] Output log File"/> + </outputs> + + <help> + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/fastq_groomer_parallel.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,121 @@ +#!/usr/bin/env python +""" +Yufei LUO +""" + + +import sys, os, optparse, random +from galaxy_utils.sequence.fastq import fastqReader, fastqVerboseErrorReader, fastqAggregator, fastqWriter + +def stop_err(msg): + sys.stderr.write("%s\n" % msg) + sys.exit() + +def main(): + + input_filename = sys.argv[1] #a txt file + input_type = sys.argv[2] + output_filename = sys.argv[3] #a txt file + output_type = sys.argv[4] + force_quality_encoding = sys.argv[5] + summarize_input = sys.argv[6] == 'summarize_input' + pairedEnd_input = sys.argv[7] + if pairedEnd_input == 'None': + pairedEnd_input = None + else: + output_pairedEndFileName = sys.argv[8] + + if force_quality_encoding == 'None': + force_quality_encoding = None + + #Parse the input txt file and read a list of fastq files + file = open(input_filename, "r") + lines = file.readlines() + inputFileNames = [] + outGroomerNames = [] + resDirName = os.path.dirname(output_filename) + "/" + #Write output txt file and define all output groomer file names + outFile = open(output_filename, "w") + for line in lines: + tab = line.split() + inputFileNames.append(tab[1]) + outGroomerName = resDirName + tab[0] + '_outGroomer_%s.fastq' % random.randrange(0, 10000) + outGroomerNames.append(outGroomerName) + outFile.write(tab[0] + '\t' + outGroomerName + '\n') + outFile.close() + file.close() + + if pairedEnd_input != None: + inPairedFile = open(pairedEnd_input, "r") + lines = inPairedFile.readlines() + inputPairedEndFileNames = [] + outGroomerPairedEndNames = [] + outPairedEndFile = open(output_pairedEndFileName, "w") + for line in lines: + tab = line.split() + inputPairedEndFileNames.append(tab[1]) + outGroomerPairedEndName = resDirName + tab[0] + '_outGroomer_pairedEnd_%s.fastq' % random.randrange(0, 10000) + outGroomerPairedEndNames.append(outGroomerPairedEndName) + outPairedEndFile.write(tab[0] + '\t' + outGroomerPairedEndName + '\n') + outPairedEndFile.close() + inPairedFile.close() + + # Write output file + aggregator = fastqAggregator() + for i in range(len(outGroomerNames)): + out = fastqWriter( open( outGroomerNames[i], 'wb' ), format = output_type, force_quality_encoding = force_quality_encoding ) + read_count = None + if summarize_input: + reader = fastqVerboseErrorReader + else: + reader = fastqReader + for read_count, fastq_read in enumerate( reader( open( inputFileNames[i] ), format = input_type, apply_galaxy_conventions = True ) ): + if summarize_input: + aggregator.consume_read( fastq_read ) + out.write( fastq_read ) + out.close() + + if read_count is not None: + print "Groomed %i %s reads into %s reads." % ( read_count + 1, input_type, output_type ) + if input_type != output_type and 'solexa' in [ input_type, output_type ]: + print "Converted between Solexa and PHRED scores." + if summarize_input: + print "Based upon quality and sequence, the input data is valid for: %s" % ( ", ".join( aggregator.get_valid_formats() ) or "None" ) + ascii_range = aggregator.get_ascii_range() + decimal_range = aggregator.get_decimal_range() + print "Input ASCII range: %s(%i) - %s(%i)" % ( repr( ascii_range[0] ), ord( ascii_range[0] ), repr( ascii_range[1] ), ord( ascii_range[1] ) ) #print using repr, since \x00 (null) causes info truncation in galaxy when printed + print "Input decimal range: %i - %i" % ( decimal_range[0], decimal_range[1] ) + else: + print "No valid FASTQ reads were provided." + + + # Write output pairedEnd file + if pairedEnd_input != None: + aggregator = fastqAggregator() + for i in range(len(outGroomerPairedEndNames)): + outPair = fastqWriter(open(outGroomerPairedEndNames[i], 'wb'), format = output_type, force_quality_encoding = force_quality_encoding) + read_count = None + if summarize_input: + reader = fastqVerboseErrorReader + else: + reader = fastqReader + for read_count, fastq_reader in enumerate(reader(open(inputPairedEndFileNames[i]), format=input_type, apply_galaxy_conventions=True)): + if summarize_input: + aggregator.consume_read(fastq_read) + outPair.write(fastq_read) + outPair.close() + + if read_count is not None: + print "Groomed %i %s reads into %s reads." % ( read_count + 1, input_type, output_type ) + if input_type != output_type and 'solexa' in [ input_type, output_type ]: + print "Converted between Solexa and PHRED scores." + if summarize_input: + print "Based upon quality and sequence, the input data is valid for: %s" % ( ", ".join( aggregator.get_valid_formats() ) or "None" ) + ascii_range = aggregator.get_ascii_range() + decimal_range = aggregator.get_decimal_range() + print "Input ASCII range: %s(%i) - %s(%i)" % ( repr( ascii_range[0] ), ord( ascii_range[0] ), repr( ascii_range[1] ), ord( ascii_range[1] ) ) #print using repr, since \x00 (null) causes info truncation in galaxy when printed + print "Input decimal range: %i - %i" % ( decimal_range[0], decimal_range[1] ) + else: + print "No valid paired-end FASTQ reads were provided." + +if __name__ == "__main__": main()
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/fastq_groomer_parallel.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,122 @@ +<tool id="fastq_groomer_parallel" name="FASTQ Groomer parallel" version="1.0.0"> + <description>convert between various FASTQ quality formats for a list of inputs</description> + <command interpreter="python">fastq_groomer_parallel.py '$input_file' '$input_type' '$output_file' +#if str( $options_type['options_type_selector'] ) == 'basic': +#if str( $input_type ) == 'cssanger': +'cssanger' +#else: +'sanger' +#end if +'ascii' 'summarize_input' +#else: +'${options_type.output_type}' '${options_type.force_quality_encoding}' '${options_type.summarize_input}' +#end if +#if $OptionPairedEnd.pairedEnd == "Yes": +'$OptionPairedEnd.pairedEnd_input' '$output_pairedEndFile' +#else: +'None' 'None' +#end if +</command> + <inputs> + <param name="input_file" type="data" format="txt" label="The File list to groom" /> + <param name="input_type" type="select" label="Input FASTQ quality scores type"> + <option value="solexa">Solexa</option> + <option value="illumina">Illumina 1.3-1.7</option> + <option value="sanger" selected="True">Sanger</option> + <option value="cssanger">Color Space Sanger</option> + </param> + <conditional name="options_type"> + <param name="options_type_selector" type="select" label="Advanced Options"> + <option value="basic" selected="True">Hide Advanced Options</option> + <option value="advanced">Show Advanced Options</option> + </param> + <when value="basic"> + <!-- no options --> + </when> + <when value="advanced"> + <param name="output_type" type="select" label="Output FASTQ quality scores type" help="Galaxy tools are designed to work with the Sanger Quality score format."> + <option value="solexa">Solexa</option> + <option value="illumina">Illumina 1.3+</option> + <option value="sanger" selected="True">Sanger (recommended)</option> + <option value="cssanger">Color Space Sanger</option> + </param> + <param name="force_quality_encoding" type="select" label="Force Quality Score encoding"> + <option value="None">Use Source Encoding</option> + <option value="ascii" selected="True">ASCII</option> + <option value="decimal">Decimal</option> + </param> + <param name="summarize_input" type="select" label="Summarize input data"> + <option value="summarize_input" selected="True">Summarize Input</option> + <option value="dont_summarize_input">Do not Summarize Input (faster)</option> + </param> + </when> + </conditional> + + <conditional name="OptionPairedEnd"> + <param name="pairedEnd" type="select" label="For paired-end analysis."> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="pairedEnd_input" type="data" format="txt" label="input paired-end files list"/> + </when> + <when value="No"> + </when> + </conditional> + + </inputs> + + <outputs> + <data name="output_file" format="txt"> + </data> + <data format="txt" name="output_pairedEndFile" label="output Paired-end fastq files"> + <filter>(OptionPairedEnd['pairedEnd']=='Yes')</filter> + </data> + </outputs> + <help> +**What it does** + +This tool offers several conversions options relating to the FASTQ format. + +When using *Basic* options, the output will be *sanger* formatted or *cssanger* formatted (when the input is Color Space Sanger). + +When converting, if a quality score falls outside of the target score range, it will be coerced to the closest available value (i.e. the minimum or maximum). + +When converting between Solexa and the other formats, quality scores are mapped between Solexa and PHRED scales using the equations found in `Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res. 2009 Dec 16.`_ + +When converting between color space (csSanger) and base/sequence space (Sanger, Illumina, Solexa) formats, adapter bases are lost or gained; if gained, the base 'G' is used as the adapter. You cannot convert a color space read to base space if there is no adapter present in the color space sequence. Any masked or ambiguous nucleotides in base space will be converted to 'N's when determining color space encoding. + +----- + +**Quality Score Comparison** + +:: + + SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS + ...............................IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII + ..........................XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX + !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~ + | | | | | | + 33 59 64 73 104 126 + + S - Sanger Phred+33, 93 values (0, 93) (0 to 60 expected in raw reads) + I - Illumina 1.3 Phred+64, 62 values (0, 62) (0 to 40 expected in raw reads) + X - Solexa Solexa+64, 67 values (-5, 62) (-5 to 40 expected in raw reads) + +Diagram adapted from http://en.wikipedia.org/wiki/FASTQ_format + +.. class:: infomark + +Output from Illumina 1.8+ pipelines are Sanger encoded. + +------ + +**Citation** + +If you use this tool, please cite `Blankenberg D, Gordon A, Von Kuster G, Coraor N, Taylor J, Nekrutenko A; Galaxy Team. Manipulation of FASTQ data with Galaxy. Bioinformatics. 2010 Jul 15;26(14):1783-5. <http://www.ncbi.nlm.nih.gov/pubmed/20562416>`_ + + +.. _Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res. 2009 Dec 16.: http://www.ncbi.nlm.nih.gov/pubmed/20015970 + + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/gsnap.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,49 @@ +<tool id="gsnap" name="gsnap"> + + <description>GSNAP version 2012-12-20. + GMAP: A Genomic Mapping and Alignment Program for mRNA and EST Sequences, and + GSNAP: Genomic Short-read Nucleotide Alignment Program + </description> + + <requirements> + <requirement type="package">gmap</requirement> + </requirements> + + + <command interpreter="python"> wrappGSNAP.py + -d $genomeName -D $genomeName.files_path -k $kmer -i $inputFasta -q $inputFastq -A $outputFormat + + #if $optionPairedEnd.paire == 'Yes': + -p $optionPairedEnd.pairedEndFile + #end if + + > $outputSam + + </command> + + <inputs> + <param name="inputFasta" type="data" format="fasta" label="Reference genome file, fasta format."/> + <param name="genomeName" type="text" value="Arabidopsis_Thaliana" label="Please give the reference genome a name! (Ex. Arabidopsis_Thaliana)"/> + <param name="kmer" type="integer" value="12" label="Choose kmer value (<=16), a big kmer value can take more RAM(4Go)." /> + <param name="inputFastq" type="data" format="fastq" label="Input fastq file."/> + <param name="outputFormat" type="text" format="sam" label="Choose an output format [sam, goby (need to re-compile with appropriate options)]."/> + + <conditional name="optionPairedEnd"> + <param name="paire" type="select" label="pairedEnd fastq file"> + <option value="Yes">Yes</option> + <option value="No" selected="true">No</option> + </param> + <when value="Yes"> + <param name="pairedEndFile" type="data" format="fastq"/> + </when> + <when value="No"> + </when> + </conditional> + + </inputs> + + <outputs> + <data format="sam" name="outputSam" label="gsnap Output"/> + </outputs> + +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/listInputs.pl Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,18 @@ +#!/usr/bin/perl -w +### +###Yufei LUO +### + + + +use strict; + +my $in_file1 = $ARGV[0]; +my $in_file2 = $ARGV[1]; +my $out_file = $ARGV[2]; + +open(OUT, ">$out_file"); +print OUT "label\tfiles\tgroup\n"; +print OUT "fileID=1\t$in_file1\tgroup1\n"; +print OUT "fileID=2\t$in_file2\tgroup2\n"; +close(OUT);
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/listInputs.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,25 @@ +<tool id="listInputs" name="listInputs"> + <description>Give a list of input files from different conditions/groups for DESeq analysis, DESeq can then charge these input files from the given list.</description> + <command interpreter="perl"> listInputs.pl $inputFromGroup1 $inputFromGroup2 $output + </command> + + <inputs> + <param name="inputFromGroup1" type="data" format="tabular" label="Please choose your file from group1."/> + <param name="inputFromGroup2" type="data" format="tabular" label="Please choose your file from group2."/> + </inputs> + + <outputs> + <data format="txt" name="output" label="listInputs Output"/> + </outputs> + + <help> + This tool can facilate the the chargement for DESeq tool. + Example: + From group1, we have input1. + From group2, we have input2. + This tool will give us a list like: + fileID=1 input1 group1 + fileID=2 input2 group2 + Where the value of fileID is unique for each input file. + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/loadHTSeqResultFiles.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,46 @@ +#!/usr/bin/env python + +""" +Yufei LUO +""" + + + +import optparse, sys + + +def __main__(): + #Parse Command Line + parser = optparse.OptionParser() + parser.add_option('-i', '--inputs', dest='inputFiles', default=None, help='several input files. (seperated by @ or @@' ) + parser.add_option( '-o', '--output', dest='outputFile', default=None, help='The output list of HTSeq results files(.tabular) on txt format.' ) + ( options, args ) = parser.parse_args() + + + out = open(options.outputFile, 'w') + out.write("label\tfiles\tgroup\n") + if options.inputFiles == None: + raise Exception, 'input file name is not defined!' + + groupCount = 1 + fileCount = 0 + + inputFiles = sys.argv[6:] + print '\n\nthe length of inputfiles is : %s \n' % len(inputFiles) + i = 0 + while i < (len(inputFiles)-1): + if inputFiles[i] == "@": + i += 1 + fileCount = 1 + groupCount += 1 + out.write("Group%s_%s\t%s\t%s\n" % (groupCount, fileCount, inputFiles[i], groupCount)) + else: + fileCount += 1 + out.write("Group%s_%s\t%s\t%s\n" % (groupCount, fileCount, inputFiles[i], groupCount)) + i += 1 + + out.close() + + + +if __name__=="__main__": __main__()
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/loadHTSeqResultFiles.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,29 @@ +<tool id="load_HTSeqResultFiles" name="load HTSeqResultFiles" > + <description>To load several HTSeq result files from different conditions.</description> + <command interpreter="python"> loadHTSeqResultFiles.py -o $htseqRes_out + -i + #for $i in $condition_groups + #for $j in $i.replicates + $j.tabular_file + #end for + @ + #end for + +</command> + <inputs> + <repeat name="condition_groups" title="Condition group" min="2"> + <repeat name="replicates" title="Replicate"> + <param name="tabular_file" format="tabular" type="data" label="TABULAR file."/> + </repeat> + </repeat> + </inputs> + + <outputs> + <data format="txt" name="htseqRes_out" label="HTSeq result files" help="This program gives you a list of files you choose for the following data analysis."/> + +</outputs> +<help> +</help> + +</tool> +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/loadMultiFastqFiles.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,76 @@ +#!/usr/bin/env python + +""" +Yufei LUO +""" + + + +import optparse, sys + + +def __main__(): + #Parse Command Line + parser = optparse.OptionParser() + parser.add_option('-i', '--inputs', dest='inputFiles', default=None, help='several input files. (seperated by @ or @@' ) + parser.add_option( '-o', '--output', dest='outputSingleFile', default=None, help='The output list of fastq files on txt format.' ) + parser.add_option( '', '--pairedEnd', dest='outputPaireFile', default=None, help='paired end option help to upload the corresponding paired end complementary fastq files' ) + ( options, args ) = parser.parse_args() + + + + if options.outputSingleFile == None: + raise Exception, 'OutSingleFile txt file name is not defined!' + else: + outSingle = open(options.outputSingleFile, 'w') + + if options.inputFiles == None: + raise Exception, 'input file name is not defined!' + + groupCount = 1 + fileCount = 0 + + if options.outputPaireFile == None: + inputFiles = sys.argv[4:] + i = 0 + while i < (len(inputFiles)-1): + if inputFiles[i] == "@": + i += 1 + fileCount = 1 + groupCount += 1 + outSingle.write("Group%s_%s\t%s\n" % (groupCount, fileCount, inputFiles[i])) + + else: + fileCount += 1 + outSingle.write("Group%s_%s\t%s\n" % (groupCount, fileCount, inputFiles[i])) + + i += 1 + else: + inputFiles = sys.argv[6:] + print '\n\nthe length of inputfiles is : %s \n' % len(inputFiles) + outPaire = open(options.outputPaireFile, 'w') + i = 0 + while i < (len(inputFiles)-1): + if inputFiles[i] == "@@": + i += 1 + outPaire.write("Group%s_%s\t%s\n" % (groupCount, fileCount, inputFiles[i])) + elif inputFiles[i] == "@": + i += 1 + fileCount = 1 + groupCount += 1 + outSingle.write("Group%s_%s\t%s\n" % (groupCount, fileCount, inputFiles[i])) + else: + fileCount += 1 + outSingle.write("Group%s_%s\t%s\n" % (groupCount, fileCount, inputFiles[i])) + + i += 1 + + + + outPaire.close() + + outSingle.close() + + + +if __name__=="__main__": __main__()
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/loadMultiFastqFiles.sh Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,24 @@ +#!/bin/bash + +OUTFile=${1} +shift +groupCount=1 +replicateNumber=1 + +arrayZ=( $@ ) +#remove the last symble '@' given by commande line +unset arrayZ[${#arrayZ[@]}-1] + +for FILE in ${arrayZ[@]} +do + #if a new group of fastq, re-count the replicateNumber + if echo $FILE | grep -q "@" + then + groupCount=$(($groupCount + 1)) + replicateNumber=1 + else + echo -e "Group${groupCount}_${replicateNumber}\t${FILE}" >>${OUTFile} + replicateNumber=$(($replicateNumber + 1)) + fi +done +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/loadMultiFastqFiles.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,75 @@ +<tool id="load_multiFASTQFiles" name="load_multiFASTQfiles" > + <description>To load several FASTQ files from different conditions.</description> + <command interpreter="python"> loadMultiFastqFiles.py -o $multiFASTQfiles_out +#if $single_end_paired_end.mapping_mode == 'single': + -i + #for $i in $single_end_paired_end.condition_groups + #for $j in $i.replicates + $j.fastq_file + #end for + @ + #end for + +#elif $single_end_paired_end.mapping_mode == 'paired': + + --pairedEnd $multiFASTQfiles_paired_end_out + -i + #for $i in $single_end_paired_end.condition_groups + #for $j in $i.replicates + $j.fastq_file + @@ + $j.fastq_paired_end_file + #end for + @ + #end for +#end if + +</command> + <inputs> + <conditional name="single_end_paired_end"> + <param name="mapping_mode" type="select" label="The uploading fastq files for single-end or paired-end mapping mode."> + <option value="single">Single-End</option> + <option value="paired">Paire-End</option> + </param> + <when value="single"> + <repeat name="condition_groups" title="Condition group" min="2"> + <repeat name="replicates" title="Replicate"> + <param name="fastq_file" format="fastq" type="data" label="FASTQ file. Can show the sequences quality."/> + </repeat> + </repeat> + </when> + <when value="paired"> + <repeat name="condition_groups" title="Condition group" min="2"> + <repeat name="replicates" title="Replicate"> + <param name="fastq_file" format="fastq" type="data" label="FASTQ file. Can show the sequences quality."/> + <param name="fastq_paired_end_file" format="fastq" type="data" label="fastq paired end complementary file" help="Add the corresponding paired end file for paired end mapping"/> + </repeat> + </repeat> + </when> + + </conditional> + </inputs> + + <outputs> + <data format="txt" name="multiFASTQfiles_out" label="loadMultiFASTQFiles result" help="This program gives you a list of files you choose for the following data analysis."/> + <data format="txt" name="multiFASTQfiles_paired_end_out" label="loadMultiFASTQFiles for paired end result" help="This program gives you a list of files you choose for the following data analysis."> + <filter>(single_end_paired_end['mapping_mode']=='paired')</filter> + + </data> +</outputs> +<help> + **This tool is to help upload several data for differential expression pipeline. Before click 'Execute', you should Click** Ctrl + here_ **first to open the pipeline in a new page.** + + .. _here: http://127.0.0.1:8085/u/yufei-luo/w/differentialexpressiondeseq-with-replicates +</help> + +</tool> + + + + + + + + +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/tophat_parallel.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,327 @@ +#!/usr/bin/env python +""" +Yufei LUO +""" + +import optparse, os, shutil, subprocess, sys, tempfile, fileinput, tarfile,random + +def stop_err( msg ): + sys.stderr.write( "%s\n" % msg ) + sys.exit() + +def toTar(tarFileName, accepted_hits_outputNames): + fileName = os.path.splitext(tarFileName)[0] + fileNameBaseName = os.path.basename(fileName) + dir = os.path.dirname(tarFileName) + tfile = tarfile.open(tarFileName + ".tmp.tar", "w") + currentPath = os.getcwd() + os.chdir(dir) + for file in accepted_hits_outputNames: + relativeFileName = os.path.basename(file) + tfile.add(relativeFileName) + os.system("mv %s %s" % (tarFileName + ".tmp.tar", tarFileName)) + tfile.close() + os.chdir(currentPath) + + +def __main__(): + #Parse Command Line + parser = optparse.OptionParser() + parser.add_option('-o', '--outputTxtFile', dest='outputTxtFile', help='for Differential expression analysis pipeline, new output option gives a txt output containing the list of mapping results.') + parser.add_option('-t', '--tar', dest='outputTar', default=None, help='output all accepted hits results in a tar file.' ) + parser.add_option( '-p', '--num-threads', dest='num_threads', help='Use this many threads to align reads. The default is 1.' ) + parser.add_option( '-C', '--color-space', dest='color_space', action='store_true', help='This indicates color-space data' ) + parser.add_option( '-J', '--junctions-output', dest='junctions_output_file', default='junctions_output.bed', help='Junctions output file; formate is BED.' ) + parser.add_option( '-H', '--hits-output', dest='accepted_hits_output_file', default='hits_output_%s.bam' % random.randrange(0, 10000), help='Accepted hits output file; formate is BAM.' ) + parser.add_option( '', '--own-file', dest='own_file', help='' ) + parser.add_option( '-D', '--indexes-path', dest='index_path', help='Indexes directory; location of .ebwt and .fa files.' ) + parser.add_option( '-r', '--mate-inner-dist', dest='mate_inner_dist', help='This is the expected (mean) inner distance between mate pairs. \ + For, example, for paired end runs with fragments selected at 300bp, \ + where each end is 50bp, you should set -r to be 200. There is no default, \ + and this parameter is required for paired end runs.') + parser.add_option( '', '--mate-std-dev', dest='mate_std_dev', help='Standard deviation of distribution on inner distances between male pairs.' ) + parser.add_option( '-a', '--min-anchor-length', dest='min_anchor_length', + help='The "anchor length". TopHat will report junctions spanned by reads with at least this many bases on each side of the junction.' ) + parser.add_option( '-m', '--splice-mismatches', dest='splice_mismatches', help='The maximum number of mismatches that can appear in the anchor region of a spliced alignment.' ) + parser.add_option( '-i', '--min-intron-length', dest='min_intron_length', + help='The minimum intron length. TopHat will ignore donor/acceptor pairs closer than this many bases apart.' ) + parser.add_option( '-I', '--max-intron-length', dest='max_intron_length', + help='The maximum intron length. When searching for junctions ab initio, TopHat will ignore donor/acceptor pairs farther than this many bases apart, except when such a pair is supported by a split segment alignment of a long read.' ) + parser.add_option( '-F', '--junction_filter', dest='junction_filter', help='Filter out junctions supported by too few alignments (number of reads divided by average depth of coverage)' ) + parser.add_option( '-g', '--max_multihits', dest='max_multihits', help='Maximum number of alignments to be allowed' ) + parser.add_option( '', '--initial-read-mismatches', dest='initial_read_mismatches', help='Number of mismatches allowed in the initial read mapping' ) + parser.add_option( '', '--seg-mismatches', dest='seg_mismatches', help='Number of mismatches allowed in each segment alignment for reads mapped independently' ) + parser.add_option( '', '--seg-length', dest='seg_length', help='Minimum length of read segments' ) + parser.add_option( '', '--library-type', dest='library_type', help='TopHat will treat the reads as strand specific. Every read alignment will have an XS attribute tag. Consider supplying library type options below to select the correct RNA-seq protocol.' ) + parser.add_option( '', '--allow-indels', action="store_true", help='Allow indel search. Indel search is disabled by default.(Not used since version 1.3.0)' ) + parser.add_option( '', '--max-insertion-length', dest='max_insertion_length', help='The maximum insertion length. The default is 3.' ) + parser.add_option( '', '--max-deletion-length', dest='max_deletion_length', help='The maximum deletion length. The default is 3.' ) + + # Options for supplying own junctions + parser.add_option( '-G', '--GTF', dest='gene_model_annotations', help='Supply TopHat with a list of gene model annotations. \ + TopHat will use the exon records in this file to build \ + a set of known splice junctions for each gene, and will \ + attempt to align reads to these junctions even if they \ + would not normally be covered by the initial mapping.') + parser.add_option( '-j', '--raw-juncs', dest='raw_juncs', help='Supply TopHat with a list of raw junctions. Junctions are \ + specified one per line, in a tab-delimited format. Records \ + look like: <chrom> <left> <right> <+/-> left and right are \ + zero-based coordinates, and specify the last character of the \ + left sequenced to be spliced to the first character of the right \ + sequence, inclusive.') + parser.add_option( '', '--no-novel-juncs', action="store_true", dest='no_novel_juncs', help="Only look for junctions indicated in the \ + supplied GFF file. (ignored without -G)") + parser.add_option( '', '--no-novel-indels', action="store_true", dest='no_novel_indels', help="Skip indel search. Indel search is enabled by default.") + # Types of search. + parser.add_option( '', '--microexon-search', action="store_true", dest='microexon_search', help='With this option, the pipeline will attempt to find alignments incident to microexons. Works only for reads 50bp or longer.') + parser.add_option( '', '--closure-search', action="store_true", dest='closure_search', help='Enables the mate pair closure-based search for junctions. Closure-based search should only be used when the expected inner distance between mates is small (<= 50bp)') + parser.add_option( '', '--no-closure-search', action="store_false", dest='closure_search' ) + parser.add_option( '', '--coverage-search', action="store_true", dest='coverage_search', help='Enables the coverage based search for junctions. Use when coverage search is disabled by default (such as for reads 75bp or longer), for maximum sensitivity.') + parser.add_option( '', '--no-coverage-search', action="store_false", dest='coverage_search' ) + parser.add_option( '', '--min-segment-intron', dest='min_segment_intron', help='Minimum intron length that may be found during split-segment search' ) + parser.add_option( '', '--max-segment-intron', dest='max_segment_intron', help='Maximum intron length that may be found during split-segment search' ) + parser.add_option( '', '--min-closure-exon', dest='min_closure_exon', help='Minimum length for exonic hops in potential splice graph' ) + parser.add_option( '', '--min-closure-intron', dest='min_closure_intron', help='Minimum intron length that may be found during closure search' ) + parser.add_option( '', '--max-closure-intron', dest='max_closure_intron', help='Maximum intron length that may be found during closure search' ) + parser.add_option( '', '--min-coverage-intron', dest='min_coverage_intron', help='Minimum intron length that may be found during coverage search' ) + parser.add_option( '', '--max-coverage-intron', dest='max_coverage_intron', help='Maximum intron length that may be found during coverage search' ) + + # Wrapper options. + parser.add_option( '-1', '--input1', dest='input1', help='A list of the (forward or single-end) reads files of Sanger FASTQ format, txt format' ) + #parser.add_option( '-1', '--input1', dest='input1', help='The (forward or single-end) reads file in Sanger FASTQ format' ) + #parser.add_option( '-2', '--input2', dest='input2', help='The reverse reads file in Sanger FASTQ format' ) + parser.add_option( '-2', '--input2', dest='input2', help='The list of reverse reads file in Sanger FASTQ format' ) + parser.add_option( '', '--single-paired', dest='single_paired', help='' ) + parser.add_option( '', '--settings', dest='settings', help='' ) + + (options, args) = parser.parse_args() + + # output version # of tool + try: + tmp_files = [] + tmp = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp) + tmp_stdout = open( tmp, 'wb' ) + proc = subprocess.Popen( args='tophat -v', shell=True, stdout=tmp_stdout ) + tmp_stdout.close() + returncode = proc.wait() + stdout = open( tmp_stdout.name, 'rb' ).readline().strip() + if stdout: + sys.stdout.write( '%s\n' % stdout ) + else: + raise Exception + except: + sys.stdout.write( 'Could not determine Tophat version\n' ) + + # Color or base space + space = '' + if options.color_space: + space = '-C' + + + #reads = options.input1 + file = open(options.input1,"r") + lines = file.readlines() + inputFileNames = [] + accepted_hits_outputNames = [] + outputName = options.outputTxtFile + resDirName = os.path.dirname(outputName) + '/' + out = open(outputName, "w") + for line in lines: + tab = line.split() + inputFileNames.append(tab[1]) + aHitOutName = resDirName + tab[0] + '_' + options.accepted_hits_output_file + accepted_hits_outputNames.append(aHitOutName) + out.write(tab[0] + '\t' + aHitOutName + '\n') + file.close() + out.close() + + if options.input2: + revFile = open(options.input2,"r") + lines = revFile.readlines() + inputRevFileNames = [] + for line in lines: + revTab = line.split() + inputRevFileNames.append(revTab[1]) + revFile.close() + + + # Creat bowtie index if necessary. + tmp_index_dirs = [] + index_paths = [] + tmp_index_dir = tempfile.mkdtemp() + tmp_index_dirs.append(tmp_index_dir) + if options.own_file: + index_path = os.path.join( tmp_index_dir, '.'.join( os.path.split( options.own_file )[1].split( '.' )[:-1] ) ) + index_paths.append(index_path) + try: + os.link( options.own_file, index_path + '.fa' ) + except: + # Tophat prefers (but doesn't require) fasta file to be in same directory, with .fa extension + pass + cmd_index = 'bowtie-build %s -f %s %s' % ( space, options.own_file, index_path ) + try: + tmp = tempfile.NamedTemporaryFile( dir=tmp_index_dir ).name + tmp_stderr = open( tmp, 'wb' ) + proc = subprocess.Popen( args=cmd_index, shell=True, cwd=tmp_index_dir, stderr=tmp_stderr.fileno() ) + returncode = proc.wait() + tmp_stderr.close() + # get stderr, allowing for case where it's very large + tmp_stderr = open( tmp, 'rb' ) + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += tmp_stderr.read( buffsize ) + if not stderr or len( stderr ) % buffsize != 0: + break + except OverflowError: + pass + tmp_stderr.close() + if returncode != 0: + raise Exception, stderr + except Exception, e: + if os.path.exists( tmp_index_dir ): + shutil.rmtree( tmp_index_dir ) + stop_err( 'Error indexing reference sequence\n' + str( e ) ) + else: + for file in inputFileNames: + tmp_index_dir = tempfile.mkdtemp() + index_path = tmp_index_dir + '/' + os.path.basename(file).split('.')[0] + index_paths.append(index_path) + tmp_index_dirs.append(tmp_index_dir) + + + + # Build tophat command. + cmds = [] + # for inputFileName in inputFileNames: + for i in range(len(inputFileNames)): + cmd = 'tophat %s %s %s ' + input_files = inputFileNames[i] + if options.input2: + input_files += ' ' + inputRevFileNames[i] + opts = '-p %s %s' % ( options.num_threads, space ) + if options.single_paired == 'paired': + opts += '-r %s ' % options.mate_inner_dist + if options.settings == 'preSet': + if options.own_file: + cmd = cmd % ( opts, index_paths[0], input_files ) #here add paired end file + else: + cmd = cmd % ( opts, index_paths[i], input_files ) #here add paired end file + else: + try: + if int( options.min_anchor_length ) >= 3: + opts += '-a %s ' % options.min_anchor_length + else: + raise Exception, 'Minimum anchor length must be 3 or greater' + opts += '-m %s ' % options.splice_mismatches + opts += '-i %s ' % options.min_intron_length + opts += '-I %s ' % options.max_intron_length + if float( options.junction_filter ) != 0.0: + opts += '-F %s ' % options.junction_filter + opts += '-g %s ' % options.max_multihits + # Custom junctions options. + if options.gene_model_annotations: + opts += '-G %s ' % options.gene_model_annotations + if options.raw_juncs: + opts += '-j %s ' % options.raw_juncs + if options.no_novel_juncs: + opts += '--no-novel-juncs ' + if options.library_type: + opts += '--library-type %s ' % options.library_type + if options.no_novel_indels: + opts += '--no-novel-indels ' + else: + if options.max_insertion_length: + opts += '--max-insertion-length %i ' % int( options.max_insertion_length ) + if options.max_deletion_length: + opts += '--max-deletion-length %i ' % int( options.max_deletion_length ) + # Max options do not work for Tophat v1.2.0, despite documentation to the contrary. (Fixed in version 1.3.1) + # need to warn user of this fact + #sys.stdout.write( "Max insertion length and max deletion length options don't work in Tophat v1.2.0\n" ) + + # Search type options. + if options.coverage_search: + opts += '--coverage-search --min-coverage-intron %s --max-coverage-intron %s ' % ( options.min_coverage_intron, options.max_coverage_intron ) + else: + opts += '--no-coverage-search ' + if options.closure_search: + opts += '--closure-search --min-closure-exon %s --min-closure-intron %s --max-closure-intron %s ' % ( options.min_closure_exon, options.min_closure_intron, options.max_closure_intron ) + else: + opts += '--no-closure-search ' + if options.microexon_search: + opts += '--microexon-search ' + if options.single_paired == 'paired': + opts += '--mate-std-dev %s ' % options.mate_std_dev + if options.initial_read_mismatches: + opts += '--initial-read-mismatches %d ' % int( options.initial_read_mismatches ) + if options.seg_mismatches: + opts += '--segment-mismatches %d ' % int( options.seg_mismatches ) + if options.seg_length: + opts += '--segment-length %d ' % int( options.seg_length ) + if options.min_segment_intron: + opts += '--min-segment-intron %d ' % int( options.min_segment_intron ) + if options.max_segment_intron: + opts += '--max-segment-intron %d ' % int( options.max_segment_intron ) + if options.own_file: + cmd = cmd % ( opts, index_paths[0], input_files ) #here to add paired end file + else: + cmd = cmd % ( opts, index_paths[i], input_files ) #here to add paired end file + except Exception, e: + # Clean up temp dirs + if os.path.exists( tmp_index_dir ): + shutil.rmtree( tmp_index_dir ) + stop_err( 'Something is wrong with the alignment parameters and the alignment could not be run\n' + str( e ) ) + + cmds.append(cmd) + + # Run the command line for each file. + for i in range(len(cmds)): + try: + tmp_out = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_out) + tmp_stdout = open( tmp_out, 'wb' ) + tmp_err = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_err) + tmp_stderr = open( tmp_err, 'wb' ) + proc = subprocess.Popen( args=cmds[i], shell=True, cwd=".", stdout=tmp_stdout, stderr=tmp_stderr ) + returncode = proc.wait() + tmp_stderr.close() + # get stderr, allowing for case where it's very large + tmp_stderr = open( tmp_err, 'rb' ) + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += tmp_stderr.read( buffsize ) + if not stderr or len( stderr ) % buffsize != 0: + break + except OverflowError: + pass + tmp_stdout.close() + tmp_stderr.close() + if returncode != 0: + raise Exception, stderr + + # Copy output files from tmp directory to specified files. + #shutil.copyfile( os.path.join( "tophat_out", "junctions.bed" ), junctions_outputNames[i] ) + shutil.copyfile( os.path.join( "tophat_out", "accepted_hits.bam" ), accepted_hits_outputNames[i] ) + # TODO: look for errors in program output. + except Exception, e: + stop_err( 'Error in tophat:\n' + str( e ) ) + + if options.outputTar != None: + toTar(options.outputTar, accepted_hits_outputNames) + + + # Clean up temp dirs + for tmp_index_dir in tmp_index_dirs: + if os.path.exists( tmp_index_dir ): + shutil.rmtree( tmp_index_dir ) + + for tmp in tmp_files: + os.remove(tmp) + + +if __name__=="__main__": __main__()
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/tophat_parallel.xml Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,577 @@ +<tool id="tophat_parallel" name="Tophat parallel for Illumina" version="1.0.0"> + <description>Find splice junctions using RNA-seq data, can have several input RNA-seq data.</description> + <version_command>tophat --version</version_command> + <requirements> + <requirement type="package">tophat</requirement> + </requirements> + <command interpreter="python"> + tophat_parallel.py + ## Change this to accommodate the number of threads you have available. + --num-threads="4" + + ## Provide outputs. + -o $outputFileName + ##--junctions-output=$junctions + ##--hits-output=$accepted_hits + + ## Handle reference file. + #if $refGenomeSource.genomeSource == "history": + --own-file=$refGenomeSource.ownFile + #else: + --indexes-path="${ filter( lambda x: str( x[0] ) == str( $refGenomeSource.index ), $__app__.tool_data_tables[ 'tophat_indexes' ].get_fields() )[0][-1] }" + #end if + + ## Are reads single-end or paired? + --single-paired=$singlePaired.sPaired + + ## First input file always required. + --input1=$input1 + + ## Set params based on whether reads are single-end or paired. + #if $singlePaired.sPaired == "single": + --settings=$singlePaired.sParams.sSettingsType + #if $singlePaired.sParams.sSettingsType == "full": + -a $singlePaired.sParams.anchor_length + -m $singlePaired.sParams.splice_mismatches + -i $singlePaired.sParams.min_intron_length + -I $singlePaired.sParams.max_intron_length + -F $singlePaired.sParams.junction_filter + -g $singlePaired.sParams.max_multihits + --min-segment-intron $singlePaired.sParams.min_segment_intron + --max-segment-intron $singlePaired.sParams.max_segment_intron + --initial-read-mismatches=$singlePaired.sParams.initial_read_mismatches + --seg-mismatches=$singlePaired.sParams.seg_mismatches + --seg-length=$singlePaired.sParams.seg_length + --library-type=$singlePaired.sParams.library_type + + ## Indel search. + #if $singlePaired.sParams.indel_search.allow_indel_search == "Yes": + ## --allow-indels + --max-insertion-length $singlePaired.sParams.indel_search.max_insertion_length + --max-deletion-length $singlePaired.sParams.indel_search.max_deletion_length + #else: + --no-novel-indels + #end if + + ## Supplying junctions parameters. + #if $singlePaired.sParams.own_junctions.use_junctions == "Yes": + #if $singlePaired.sParams.own_junctions.gene_model_ann.use_annotations == "Yes": + -G $singlePaired.sParams.own_junctions.gene_model_ann.gene_annotation_model + #end if + #if $singlePaired.sParams.own_junctions.raw_juncs.use_juncs == "Yes": + -j $singlePaired.sParams.own_junctions.raw_juncs.raw_juncs + #end if + ## TODO: No idea why a string cast is necessary, but it is: + #if str($singlePaired.sParams.own_junctions.no_novel_juncs) == "Yes": + --no-novel-juncs + #end if + #end if + + #if $singlePaired.sParams.closure_search.use_search == "Yes": + --closure-search + --min-closure-exon $singlePaired.sParams.closure_search.min_closure_exon + --min-closure-intron $singlePaired.sParams.closure_search.min_closure_intron + --max-closure-intron $singlePaired.sParams.closure_search.max_closure_intron + #else: + --no-closure-search + #end if + #if $singlePaired.sParams.coverage_search.use_search == "Yes": + --coverage-search + --min-coverage-intron $singlePaired.sParams.coverage_search.min_coverage_intron + --max-coverage-intron $singlePaired.sParams.coverage_search.max_coverage_intron + #else: + --no-coverage-search + #end if + ## TODO: No idea why the type conversion is necessary, but it seems to be. + #if str($singlePaired.sParams.microexon_search) == "Yes": + --microexon-search + #end if + #end if + #else: + --input2=$singlePaired.input2 + -r $singlePaired.mate_inner_distance + --settings=$singlePaired.pParams.pSettingsType + #if $singlePaired.pParams.pSettingsType == "full": + --mate-std-dev=$singlePaired.pParams.mate_std_dev + -a $singlePaired.pParams.anchor_length + -m $singlePaired.pParams.splice_mismatches + -i $singlePaired.pParams.min_intron_length + -I $singlePaired.pParams.max_intron_length + -F $singlePaired.pParams.junction_filter + -g $singlePaired.pParams.max_multihits + --min-segment-intron $singlePaired.pParams.min_segment_intron + --max-segment-intron $singlePaired.pParams.max_segment_intron + --initial-read-mismatches=$singlePaired.pParams.initial_read_mismatches + --seg-mismatches=$singlePaired.pParams.seg_mismatches + --seg-length=$singlePaired.pParams.seg_length + --library-type=$singlePaired.pParams.library_type + + ## Indel search. + #if $singlePaired.pParams.indel_search.allow_indel_search == "Yes": + ## --allow-indels + --max-insertion-length $singlePaired.pParams.indel_search.max_insertion_length + --max-deletion-length $singlePaired.pParams.indel_search.max_deletion_length + #else: + --no-novel-indels + #end if + + ## Supplying junctions parameters. + #if $singlePaired.pParams.own_junctions.use_junctions == "Yes": + #if $singlePaired.pParams.own_junctions.gene_model_ann.use_annotations == "Yes": + -G $singlePaired.pParams.own_junctions.gene_model_ann.gene_annotation_model + #end if + #if $singlePaired.pParams.own_junctions.raw_juncs.use_juncs == "Yes": + -j $singlePaired.pParams.own_junctions.raw_juncs.raw_juncs + #end if + ## TODO: No idea why type cast is necessary, but it is: + #if str($singlePaired.pParams.own_junctions.no_novel_juncs) == "Yes": + --no-novel-juncs + #end if + #end if + + #if $singlePaired.pParams.closure_search.use_search == "Yes": + --closure-search + --min-closure-exon $singlePaired.pParams.closure_search.min_closure_exon + --min-closure-intron $singlePaired.pParams.closure_search.min_closure_intron + --max-closure-intron $singlePaired.pParams.closure_search.max_closure_intron + #else: + --no-closure-search + #end if + #if $singlePaired.pParams.coverage_search.use_search == "Yes": + --coverage-search + --min-coverage-intron $singlePaired.pParams.coverage_search.min_coverage_intron + --max-coverage-intron $singlePaired.pParams.coverage_search.max_coverage_intron + #else: + --no-coverage-search + #end if + ## TODO: No idea why the type conversion is necessary, but it seems to be. + #if str ($singlePaired.pParams.microexon_search) == "Yes": + --microexon-search + #end if + #end if + #end if + $tar $outputTarFile + </command> + <inputs> + <param format="txt" name="input1" type="data" label="RNA-Seq FASTQ file" help="Please identify a txt input file, which contains a list of fastqsanger files. Nucleotide-space: Must have Sanger-scaled quality values with ASCII offset 33. " /> + <conditional name="refGenomeSource"> + <param name="genomeSource" type="select" label="Will you select a reference genome from your history or use a built-in index?" help="Built-ins were indexed using default options"> + <option value="indexed">Use a built-in index</option> + <option value="history">Use one from the history</option> + </param> + <when value="indexed"> + <param name="index" type="select" label="Select a reference genome" help="If your genome of interest is not listed, contact the Galaxy team"> + <options from_data_table="tophat_indexes"> + <filter type="sort_by" column="2"/> + <validator type="no_options" message="No indexes are available for the selected input dataset"/> + </options> + </param> + </when> + <when value="history"> + <param name="ownFile" type="data" format="fasta" metadata_name="dbkey" label="Select the reference genome" /> + </when> <!-- history --> + </conditional> <!-- refGenomeSource --> + <conditional name="singlePaired"> + <param name="sPaired" type="select" label="Is this library mate-paired?"> + <option value="single">Single-end</option> + <option value="paired">Paired-end</option> + </param> + <when value="single"> + <conditional name="sParams"> + <param name="sSettingsType" type="select" label="TopHat settings to use" help="You can use the default settings or set custom values for any of Tophat's parameters."> + <option value="preSet">Use Defaults</option> + <option value="full">Full parameter list</option> + </param> + <when value="preSet" /> + <!-- Full/advanced params. --> + <when value="full"> + <param name="library_type" type="select" label="Library Type" help="TopHat will treat the reads as strand specific. Every read alignment will have an XS attribute tag. Consider supplying library type options below to select the correct RNA-seq protocol."> + <option value="fr-unstranded">FR Unstranded</option> + <option value="fr-firststrand">FR First Strand</option> + <option value="fr-secondstrand">FR Second Strand</option> + </param> + <param name="anchor_length" type="integer" value="8" label="Anchor length (at least 3)" help="Report junctions spanned by reads with at least this many bases on each side of the junction." /> + <param name="splice_mismatches" type="integer" value="0" label="Maximum number of mismatches that can appear in the anchor region of spliced alignment" /> + <param name="min_intron_length" type="integer" value="70" label="The minimum intron length" help="TopHat will ignore donor/acceptor pairs closer than this many bases apart." /> + <param name="max_intron_length" type="integer" value="500000" label="The maximum intron length" help="When searching for junctions ab initio, TopHat will ignore donor/acceptor pairs farther than this many bases apart, except when such a pair is supported by a split segment alignment of a long read." /> + <conditional name="indel_search"> + <param name="allow_indel_search" type="select" label="Allow indel search"> + <option value="Yes">Yes</option> + <option value="No">No</option> + </param> + <when value="No"/> + <when value="Yes"> + <param name="max_insertion_length" type="integer" value="3" label="Max insertion length." help="The maximum insertion length." /> + <param name="max_deletion_length" type="integer" value="3" label="Max deletion length." help="The maximum deletion length." /> + </when> + </conditional> + <param name="junction_filter" type="float" value="0.15" label="Minimum isoform fraction: filter out junctions supported by too few alignments (number of reads divided by average depth of coverage)" help="0.0 to 1.0 (0 to turn off)" /> + <param name="max_multihits" type="integer" value="40" label="Maximum number of alignments to be allowed" /> + <param name="min_segment_intron" type="integer" value="50" label="Minimum intron length that may be found during split-segment (default) search" /> + <param name="max_segment_intron" type="integer" value="500000" label="Maximum intron length that may be found during split-segment (default) search" /> + <param name="initial_read_mismatches" type="integer" min="0" max="3" value="2" label="Number of mismatches allowed in the initial read mapping" /> + <param name="seg_mismatches" type="integer" min="0" max="3" value="2" label="Number of mismatches allowed in each segment alignment for reads mapped independently" /> + <param name="seg_length" type="integer" value="25" label="Minimum length of read segments" /> + + <!-- Options for supplying own junctions. --> + <conditional name="own_junctions"> + <param name="use_junctions" type="select" label="Use Own Junctions"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="Yes"> + <conditional name="gene_model_ann"> + <param name="use_annotations" type="select" label="Use Gene Annotation Model"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="No" /> + <when value="Yes"> + <param format="gtf" name="gene_annotation_model" type="data" label="Gene Model Annotations" help="TopHat will use the exon records in this file to build a set of known splice junctions for each gene, and will attempt to align reads to these junctions even if they would not normally be covered by the initial mapping."/> + </when> + </conditional> + <conditional name="raw_juncs"> + <param name="use_juncs" type="select" label="Use Raw Junctions"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="No" /> + <when value="Yes"> + <param format="interval" name="raw_juncs" type="data" label="Raw Junctions" help="Supply TopHat with a list of raw junctions. Junctions are specified one per line, in a tab-delimited format. Records look like: [chrom] [left] [right] [+/-] left and right are zero-based coordinates, and specify the last character of the left sequenced to be spliced to the first character of the right sequence, inclusive."/> + </when> + </conditional> + <param name="no_novel_juncs" type="select" label="Only look for supplied junctions"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + </when> + <when value="No" /> + </conditional> <!-- /own_junctions --> + + <!-- Closure search. --> + <conditional name="closure_search"> + <param name="use_search" type="select" label="Use Closure Search"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="Yes"> + <param name="min_closure_exon" type="integer" value="50" label="During closure search for paired end reads, exonic hops in the potential splice graph must be at least this long. The default is 50." /> + <param name="min_closure_intron" type="integer" value="50" label="Minimum intron length that may be found during closure search" /> + <param name="max_closure_intron" type="integer" value="5000" label="Maximum intron length that may be found during closure search" /> + </when> + <when value="No" /> + </conditional> + <!-- Coverage search. --> + <conditional name="coverage_search"> + <param name="use_search" type="select" label="Use Coverage Search"> + <option selected="true" value="Yes">Yes</option> + <option value="No">No</option> + </param> + <when value="Yes"> + <param name="min_coverage_intron" type="integer" value="50" label="Minimum intron length that may be found during coverage search" /> + <param name="max_coverage_intron" type="integer" value="20000" label="Maximum intron length that may be found during coverage search" /> + </when> + <when value="No" /> + </conditional> + <param name="microexon_search" type="select" label="Use Microexon Search" help="With this option, the pipeline will attempt to find alignments incident to microexons. Works only for reads 50bp or longer."> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + </when> <!-- full --> + </conditional> <!-- sParams --> + </when> <!-- single --> + <when value="paired"> + <param format="txt" name="input2" type="data" label="list of RNA-Seq paired end FASTQ file" help="Nucleotide-space: Must have Sanger-scaled quality values with ASCII offset 33" /> + <param name="mate_inner_distance" type="integer" value="20" label="Mean Inner Distance between Mate Pairs" /> + <conditional name="pParams"> + <param name="pSettingsType" type="select" label="TopHat settings to use" help="For most mapping needs use Commonly used settings. If you want full control use Full parameter list"> + <option value="preSet">Commonly used</option> + <option value="full">Full parameter list</option> + </param> + <when value="preSet" /> + <!-- Full/advanced params. --> + <when value="full"> + <param name="library_type" type="select" label="Library Type" help="TopHat will treat the reads as strand specific. Every read alignment will have an XS attribute tag. Consider supplying library type options below to select the correct RNA-seq protocol."> + <option value="fr-unstranded">FR Unstranded</option> + <option value="fr-firststrand">FR First Strand</option> + <option value="fr-secondstrand">FR Second Strand</option> + </param> + <param name="mate_std_dev" type="integer" value="20" label="Std. Dev for Distance between Mate Pairs" help="The standard deviation for the distribution on inner distances between mate pairs."/> + <param name="anchor_length" type="integer" value="8" label="Anchor length (at least 3)" help="Report junctions spanned by reads with at least this many bases on each side of the junction." /> + <param name="splice_mismatches" type="integer" value="0" label="Maximum number of mismatches that can appear in the anchor region of spliced alignment" /> + <param name="min_intron_length" type="integer" value="70" label="The minimum intron length" help="TopHat will ignore donor/acceptor pairs closer than this many bases apart." /> + <param name="max_intron_length" type="integer" value="500000" label="The maximum intron length" help="When searching for junctions ab initio, TopHat will ignore donor/acceptor pairs farther than this many bases apart, except when such a pair is supported by a split segment alignment of a long read." /> + <conditional name="indel_search"> + <param name="allow_indel_search" type="select" label="Allow indel search"> + <option value="Yes">Yes</option> + <option value="No">No</option> + </param> + <when value="No"/> + <when value="Yes"> + <param name="max_insertion_length" type="integer" value="3" label="Max insertion length." help="The maximum insertion length." /> + <param name="max_deletion_length" type="integer" value="3" label="Max deletion length." help="The maximum deletion length." /> + </when> + </conditional> + <param name="junction_filter" type="float" value="0.15" label="Minimum isoform fraction: filter out junctions supported by too few alignments (number of reads divided by average depth of coverage)" help="0.0 to 1.0 (0 to turn off)" /> + <param name="max_multihits" type="integer" value="40" label="Maximum number of alignments to be allowed" /> + <param name="min_segment_intron" type="integer" value="50" label="Minimum intron length that may be found during split-segment (default) search" /> + <param name="max_segment_intron" type="integer" value="500000" label="Maximum intron length that may be found during split-segment (default) search" /> + <param name="initial_read_mismatches" type="integer" min="0" max="3" value="2" label="Number of mismatches allowed in the initial read mapping" /> + <param name="seg_mismatches" type="integer" min="0" max="3" value="2" label="Number of mismatches allowed in each segment alignment for reads mapped independently" /> + <param name="seg_length" type="integer" value="25" label="Minimum length of read segments" /> + <!-- Options for supplying own junctions. --> + <conditional name="own_junctions"> + <param name="use_junctions" type="select" label="Use Own Junctions"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="Yes"> + <conditional name="gene_model_ann"> + <param name="use_annotations" type="select" label="Use Gene Annotation Model"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="No" /> + <when value="Yes"> + <param format="gtf" name="gene_annotation_model" type="data" label="Gene Model Annotations" help="TopHat will use the exon records in this file to build a set of known splice junctions for each gene, and will attempt to align reads to these junctions even if they would not normally be covered by the initial mapping."/> + </when> + </conditional> + <conditional name="raw_juncs"> + <param name="use_juncs" type="select" label="Use Raw Junctions"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="No" /> + <when value="Yes"> + <param format="interval" name="raw_juncs" type="data" label="Raw Junctions" help="Supply TopHat with a list of raw junctions. Junctions are specified one per line, in a tab-delimited format. Records look like: [chrom] [left] [right] [+/-] left and right are zero-based coordinates, and specify the last character of the left sequenced to be spliced to the first character of the right sequence, inclusive."/> + </when> + </conditional> + <param name="no_novel_juncs" type="select" label="Only look for supplied junctions"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + </when> + <when value="No" /> + </conditional> <!-- /own_junctions --> + + <!-- Closure search. --> + <conditional name="closure_search"> + <param name="use_search" type="select" label="Use Closure Search"> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + <when value="Yes"> + <param name="min_closure_exon" type="integer" value="50" label="During closure search for paired end reads, exonic hops in the potential splice graph must be at least this long. The default is 50." /> + <param name="min_closure_intron" type="integer" value="50" label="Minimum intron length that may be found during closure search" /> + <param name="max_closure_intron" type="integer" value="5000" label="Maximum intron length that may be found during closure search" /> + </when> + <when value="No" /> + </conditional> + <!-- Coverage search. --> + <conditional name="coverage_search"> + <param name="use_search" type="select" label="Use Coverage Search"> + <option selected="true" value="Yes">Yes</option> + <option value="No">No</option> + </param> + <when value="Yes"> + <param name="min_coverage_intron" type="integer" value="50" label="Minimum intron length that may be found during coverage search" /> + <param name="max_coverage_intron" type="integer" value="20000" label="Maximum intron length that may be found during coverage search" /> + </when> + <when value="No" /> + </conditional> + <param name="microexon_search" type="select" label="Use Microexon Search" help="With this option, the pipeline will attempt to find alignments incident to microexons. Works only for reads 50bp or longer."> + <option value="No">No</option> + <option value="Yes">Yes</option> + </param> + </when> <!-- full --> + </conditional> <!-- pParams --> + </when> <!-- paired --> + </conditional> + <param name="tar" type="boolean" truevalue="-t" falsevalue="" checked="false" label="tar option" help="This option creates a tar file for all out results."/> + </inputs> + + <outputs> + <!-- <data format="bed" name="insertions" label="${tool.name} on ${on_string}: insertions" from_work_dir="tophat_out/insertions.bed"> + <filter> + ( + ( ( 'sParams' in singlePaired ) and ( 'indel_search' in singlePaired['sParams'] ) and + ( singlePaired['sParams']['indel_search']['allow_indel_search'] == 'Yes' ) ) or + ( ( 'pParams' in singlePaired ) and ( 'indel_search' in singlePaired['pParams'] ) and + ( singlePaired['pParams']['indel_search']['allow_indel_search'] == 'Yes' ) ) + ) + </filter> + <actions> + <conditional name="refGenomeSource.genomeSource"> + <when value="indexed"> + <action type="metadata" name="dbkey"> + <option type="from_data_table" name="tophat_indexes" column="1" offset="0"> + <filter type="param_value" column="0" value="#" compare="startswith" keep="False"/> + <filter type="param_value" ref="refGenomeSource.index" column="0"/> + </option> + </action> + </when> + <when value="history"> + <action type="metadata" name="dbkey"> + <option type="from_param" name="refGenomeSource.ownFile" param_attribute="dbkey" /> + </action> + </when> + </conditional> + </actions> + </data> + <data format="bed" name="deletions" label="${tool.name} on ${on_string}: deletions" from_work_dir="tophat_out/deletions.bed"> + <filter> + ( + ( ( 'sParams' in singlePaired ) and ( 'indel_search' in singlePaired['sParams'] ) and + ( singlePaired['sParams']['indel_search']['allow_indel_search'] == 'Yes' ) ) or + ( ( 'pParams' in singlePaired ) and ( 'indel_search' in singlePaired['pParams'] ) and + ( singlePaired['pParams']['indel_search']['allow_indel_search'] == 'Yes' ) ) + ) + </filter> + <actions> + <conditional name="refGenomeSource.genomeSource"> + <when value="indexed"> + <action type="metadata" name="dbkey"> + <option type="from_data_table" name="tophat_indexes" column="1" offset="0"> + <filter type="param_value" column="0" value="#" compare="startswith" keep="False"/> + <filter type="param_value" ref="refGenomeSource.index" column="0"/> + </option> + </action> + </when> + <when value="history"> + <action type="metadata" name="dbkey"> + <option type="from_param" name="refGenomeSource.ownFile" param_attribute="dbkey" /> + </action> + </when> + </conditional> + </actions> + </data> + <data format="bed" name="junctions" label="${tool.name} on ${on_string}: splice junctions"> + <actions> + <conditional name="refGenomeSource.genomeSource"> + <when value="indexed"> + <action type="metadata" name="dbkey"> + <option type="from_data_table" name="tophat_indexes" column="1" offset="0"> + <filter type="param_value" column="0" value="#" compare="startswith" keep="False"/> + <filter type="param_value" ref="refGenomeSource.index" column="0"/> + </option> + </action> + </when> + <when value="history"> + <action type="metadata" name="dbkey"> + <option type="from_param" name="refGenomeSource.ownFile" param_attribute="dbkey" /> + </action> + </when> + </conditional> + </actions> + </data> + <data format="bam" name="accepted_hits" label="${tool.name} on ${on_string}: accepted_hits"> + <actions> + <conditional name="refGenomeSource.genomeSource"> + <when value="indexed"> + <action type="metadata" name="dbkey"> + <option type="from_data_table" name="tophat_indexes" column="1" offset="0"> + <filter type="param_value" column="0" value="#" compare="startswith" keep="False"/> + <filter type="param_value" ref="refGenomeSource.index" column="0"/> + </option> + </action> + </when> + <when value="history"> + <action type="metadata" name="dbkey"> + <option type="from_param" name="refGenomeSource.ownFile" param_attribute="dbkey" /> + </action> + </when> + </conditional> + </actions> + </data> --> + <data name="outputFileName" format="txt" label="[tophat_parallel] txt File"/> + <data name="outputTarFile" format="tar"> + <filter>tar</filter> + </data> + </outputs> + + + + <help> +**Tophat Overview** + +TopHat_ is a fast splice junction mapper for RNA-Seq reads. It aligns RNA-Seq reads to mammalian-sized genomes using the ultra high-throughput short read aligner Bowtie, and then analyzes the mapping results to identify splice junctions between exons. Please cite: Trapnell, C., Pachter, L. and Salzberg, S.L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111 (2009). + +.. _Tophat: http://tophat.cbcb.umd.edu/ + +------ + +**Know what you are doing** + +.. class:: warningmark + +There is no such thing (yet) as an automated gearshift in splice junction identification. It is all like stick-shift driving in San Francisco. In other words, running this tool with default parameters will probably not give you meaningful results. A way to deal with this is to **understand** the parameters by carefully reading the `documentation`__ and experimenting. Fortunately, Galaxy makes experimenting easy. + +.. __: http://tophat.cbcb.umd.edu/manual.html + +------ + +**Input formats** + +Tophat accepts files in Sanger FASTQ format. Use the FASTQ Groomer to prepare your files. + +------ + +**Outputs** + +Tophat produces two output files: + +- junctions -- A UCSC BED_ track of junctions reported by TopHat. Each junction consists of two connected BED blocks, where each block is as long as the maximal overhang of any read spanning the junction. The score is the number of alignments spanning the junction. +- accepted_hits -- A list of read alignments in BAM_ format. + +.. _BED: http://genome.ucsc.edu/FAQ/FAQformat.html#format1 +.. _BAM: http://samtools.sourceforge.net/ + +Two other possible outputs, depending on the options you choose, are insertions and deletions, both of which are in BED format. + +------- + +**Tophat settings** + +All of the options have a default value. You can change any of them. Some of the options in Tophat have been implemented here. + +------ + +**Tophat parameter list** + +This is a list of implemented Tophat options:: + + -r This is the expected (mean) inner distance between mate pairs. For, example, for paired end runs with fragments + selected at 300bp, where each end is 50bp, you should set -r to be 200. There is no default, and this parameter + is required for paired end runs. + --mate-std-dev INT The standard deviation for the distribution on inner distances between mate pairs. The default is 20bp. + -a/--min-anchor-length INT The "anchor length". TopHat will report junctions spanned by reads with at least this many bases on each side of the junction. Note that individual spliced + alignments may span a junction with fewer than this many bases on one side. However, every junction involved in spliced alignments is supported by at least one + read with this many bases on each side. This must be at least 3 and the default is 8. + -m/--splice-mismatches INT The maximum number of mismatches that may appear in the "anchor" region of a spliced alignment. The default is 0. + -i/--min-intron-length INT The minimum intron length. TopHat will ignore donor/acceptor pairs closer than this many bases apart. The default is 70. + -I/--max-intron-length INT The maximum intron length. When searching for junctions ab initio, TopHat will ignore donor/acceptor pairs farther than this many bases apart, except when such a pair is supported by a split segment alignment of a long read. The default is 500000. + -F/--min-isoform-fraction 0.0-1.0 TopHat filters out junctions supported by too few alignments. Suppose a junction spanning two exons, is supported by S reads. Let the average depth of coverage of + exon A be D, and assume that it is higher than B. If S / D is less than the minimum isoform fraction, the junction is not reported. A value of zero disables the + filter. The default is 0.15. + -g/--max-multihits INT Instructs TopHat to allow up to this many alignments to the reference for a given read, and suppresses all alignments for reads with more than this many + alignments. The default is 40. + -G/--GTF [GTF 2.2 file] Supply TopHat with a list of gene model annotations. TopHat will use the exon records in this file to build a set of known splice junctions for each gene, and will attempt to align reads to these junctions even if they would not normally be covered by the initial mapping. + -j/--raw-juncs [juncs file] Supply TopHat with a list of raw junctions. Junctions are specified one per line, in a tab-delimited format. Records look like: [chrom] [left] [right] [+/-], left and right are zero-based coordinates, and specify the last character of the left sequenced to be spliced to the first character of the right sequence, inclusive. + -no-novel-juncs Only look for junctions indicated in the supplied GFF file. (ignored without -G) + --no-closure-search Disables the mate pair closure-based search for junctions. Currently, has no effect - closure search is off by default. + --closure-search Enables the mate pair closure-based search for junctions. Closure-based search should only be used when the expected inner distance between mates is small (about or less than 50bp) + --no-coverage-search Disables the coverage based search for junctions. + --coverage-search Enables the coverage based search for junctions. Use when coverage search is disabled by default (such as for reads 75bp or longer), for maximum sensitivity. + --microexon-search With this option, the pipeline will attempt to find alignments incident to microexons. Works only for reads 50bp or longer. + --butterfly-search TopHat will use a slower but potentially more sensitive algorithm to find junctions in addition to its standard search. Consider using this if you expect that your experiment produced a lot of reads from pre-mRNA, that fall within the introns of your transcripts. + --segment-mismatches Read segments are mapped independently, allowing up to this many mismatches in each segment alignment. The default is 2. + --segment-length Each read is cut up into segments, each at least this long. These segments are mapped independently. The default is 25. + --min-closure-exon During closure search for paired end reads, exonic hops in the potential splice graph must be at least this long. The default is 50. + --min-closure-intron The minimum intron length that may be found during closure search. The default is 50. + --max-closure-intron The maximum intron length that may be found during closure search. The default is 5000. + --min-coverage-intron The minimum intron length that may be found during coverage search. The default is 50. + --max-coverage-intron The maximum intron length that may be found during coverage search. The default is 20000. + --min-segment-intron The minimum intron length that may be found during split-segment search. The default is 50. + --max-segment-intron The maximum intron length that may be found during split-segment search. The default is 500000. + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/DiffExpAnal/wrappGSNAP.py Tue Jan 22 10:07:03 2013 -0500 @@ -0,0 +1,79 @@ +#! /usr/bin/env python +""" +Yufei LUO +""" + + +import os, sys, subprocess,tempfile +from optparse import OptionParser + +def stop_err(msg): + sys.stderr.write('%s\n' % msg) + sys.exit() + +def __main__(): + #Parse Command Line + description = "GMAP/GSNAP version:2012-12-20." + parser = OptionParser(description = description) + parser.add_option("-d", "--genomeName", dest="genomeName", help="Define the reference genome name.[compulsory]") + parser.add_option("-D", "--workingDir", dest="workingDir", help="Define the directory of writing reference genome index.[compulsory]") + parser.add_option("-k", "--kmer", dest="kmer", defaut=12, help="Choose kmer value (<=16), a big kmer value can take more RAM(4Go).[compulsory]") + parser.add_option("-i", "--inputFasta", dest="inputFastaFile", help="Reference genome file, fasta format.[compulsory]") + parser.add_option("-q", "--inputFastq", dest="inputFastqFile", help="Input fastq file.") + parser.add_option("-p", "--pairedEnd", dest="pairedEndFile", defaut=None, help="Input paired-end fastq file.") + parser.add_option("-A", "--outputFormat", dest="outputFormat", defaut="sam", help="Choose an output format [sam, goby (need to re-compile with appropriate options)].") + (options, args) = parser.parse_args() + + #If workingDir dose not exist, should create before run the job. + dir = options.workingdir + if not os.path.exists(dir): + os.makedirs(dir) + + cmds = [] + cmd_setup = "gmap_setup -d %s -D %s -k %s %s" % (options.genomeName, options.workingdir, options.kmer, options.inputFastaFile) + cmds.append(cmd_setup) + cmd_make_coords = "make Makefile.%s coords" % options.genomeName + cmds.append(cmd_make_coords) + cmd_make_gmapdb = "make Makefile.%s gmapdb" % options.genomeName + cmds.append(cmd_make_gmapdb) + cmd_make_install = "make Makefile.%s install" % options.genomeName + cmds.append(cmd_make_install) + cmd_run = "gsnap -d %s -D %s -A %s %s " % (options.genomeName, options.workingdir, options.outputFormat, options.inputFastqFile) + if options.pairedEndFile != None: + cmd_run += "%s" % options.pairedEndFile + cmds.append(cmd_run) + + tmp_files = [] + for i in range(len(cmds)): + try: + tmp_out = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_out) + tmp_stdout = open(tmp_out, 'wb') + tmp_err = tempfile.NamedTemporaryFile().name + tmp_files.append(tmp_err) + tmp_stderr = open(tmp_err, 'wb') + proc = subprocess.Popen(args=cmds[i], shell=True, cwd=".", stdout=tmp_stdout, stderr=tmp_stderr) + returncode = proc.wait() + tmp_stderr.close() + #get stderr, allowing for case where it's very large + tmp_stderr = open(tmp_err, 'rb') + stderr = '' + buffsize = 1048576 + try: + while True: + stderr += tmp_stderr.read(buffsize) + if not stderr or len(stderr) % buffsize != 0: + break + except OverflowError: + pass + tmp_stdout.close() + tmp_stderr.close() + if returncode != 0: + raise Exception, stderr + except Exception, e: + stop_err('Error in :\n' + str(e)) + + for tmp_file in tmp_files: + os.remove(tmp_file) + +if __name__=="__main__":__main__()