Mercurial > repos > fubar > edger_test
changeset 13:b2ec6ec6ef74 draft
Uploaded
| author | fubar |
|---|---|
| date | Wed, 12 Jun 2013 06:13:41 -0400 (2013-06-12) |
| parents | 65a97686ca5d |
| children | a4d7ec124c53 |
| files | rgedgeR/rgedgeRpaired.xml rgedgeR/rgedgeRpaired.xml~ rgedgeR/test-data/gentestdata.sh~ rgedgeR/tool_dependencies.xml rgedgeR/tool_dependencies.xml~ |
| diffstat | 5 files changed, 1 insertions(+), 714 deletions(-) [+] |
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--- a/rgedgeR/rgedgeRpaired.xml Wed Jun 12 05:21:25 2013 -0400 +++ b/rgedgeR/rgedgeRpaired.xml Wed Jun 12 06:13:41 2013 -0400 @@ -1,7 +1,6 @@ <tool id="rgedgeRpaired" name="edgeR" version="0.18"> <description>1 or 2 level models for count data</description> <requirements> - <requirement type="package" version="6.2">readline</requirement> <requirement type="package" version="3.0.1">package_R</requirement> <requirement type="package" version="2.12">package_BioCBasics</requirement> </requirements>
--- a/rgedgeR/rgedgeRpaired.xml~ Wed Jun 12 05:21:25 2013 -0400 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,633 +0,0 @@ -<tool id="rgedgeRpaired" name="edgeR" version="0.18"> - <description>1 or 2 level models for count data</description> - <requirements> - <requirement type="package" version="6.2">name=readline</requirement> - <requirement type="package" version="3.0.1">name=package_R</requirement> - <requirement type="package" version="2.12">name=package_BioCBasics</requirement> - </requirements> - - <command interpreter="python"> - rgToolFactory.py --script_path "$runme" --interpreter "Rscript" --tool_name "edgeR" - --output_dir "$html_file.files_path" --output_html "$html_file" --output_tab "$outtab" --make_HTML "yes" - </command> - <inputs> - <param name="input1" type="data" format="tabular" label="Select an input matrix - rows are contigs, columns are counts for each sample" - help="Use the HTSeq based count matrix preparation tool to create these matrices from BAM/SAM files and a GTF file of genomic features"/> - <param name="title" type="text" value="edgeR" size="80" label="Title for job outputs" help="Supply a meaningful name here to remind you what the outputs contain"> - <sanitizer invalid_char=""> - <valid initial="string.letters,string.digits"><add value="_" /> </valid> - </sanitizer> - </param> - <param name="treatment_name" type="text" value="Treatment" size="50" label="Treatment Name"/> - <param name="Treat_cols" label="Select columns containing treatment." type="data_column" data_ref="input1" numerical="True" - multiple="true" use_header_names="true" size="120" display="checkboxes"> - <validator type="no_options" message="Please select at least one column."/> - </param> - <param name="control_name" type="text" value="Control" size="50" label="Control Name"/> - <param name="Control_cols" label="Select columns containing control." type="data_column" data_ref="input1" numerical="True" - multiple="true" use_header_names="true" size="120" display="checkboxes" optional="true"> - </param> - <param name="subjectids" type="text" optional="true" size="120" - label="IF SUBJECTS NOT ALL INDEPENDENT! Enter integers to indicate sample pairing for every column in input" - help="Leave blank if no pairing, but eg if data from sample id A99 is in columns 2,4 and id C21 is in 3,5 then enter '1,2,1,2'"> - <sanitizer> - <valid initial="string.digits"><add value="," /> </valid> - </sanitizer> - </param> - <param name="fQ" type="float" value="0.3" size="5" label="Non-differential contig count quantile threshold - zero to analyze all non-zero read count contigs" - help="May be a good or a bad idea depending on the biology and the question. EG 0.3 = sparsest 30% of contigs with at least one read are removed before analysis"/> - <param name="useNDF" type="boolean" truevalue="T" checked='false' falsevalue="" size="1" label="Non differential filter - remove contigs below a threshold (1 per million) for half or more samples" - help="May be a good or a bad idea depending on the biology and the question. This was the old default. Quantile based is available as an alternative"/> - <param name="priordf" type="integer" value="20" size="3" label="prior.df for tagwise dispersion - lower value = more emphasis on each tag's variance. Replaces prior.n and prior.df = prior.n * residual.df" - help="Zero = Use edgeR default. Use a small value to 'smooth' small samples. See edgeR docs and note below"/> - <param name="fdrthresh" type="float" value="0.05" size="5" label="P value threshold for FDR filtering for amily wise error rate control" - help="Conventional default value of 0.05 recommended"/> - <param name="fdrtype" type="select" label="FDR (Type II error) control method" - help="Use fdr or bh typically to control for the number of tests in a reliable way"> - <option value="fdr" selected="true">fdr</option> - <option value="BH">Benjamini Hochberg</option> - <option value="BY">Benjamini Yukateli</option> - <option value="bonferroni">Bonferroni</option> - <option value="hochberg">Hochberg</option> - <option value="holm">Holm</option> - <option value="hommel">Hommel</option> - <option value="none">no control for multiple tests</option> - </param> - </inputs> - <outputs> - <data format="tabular" name="outtab" label="${title}.xls"/> - <data format="html" name="html_file" label="${title}.html"/> - </outputs> - <stdio> - <exit_code range="4" level="fatal" description="Number of subject ids must match total number of samples in the input matrix" /> - </stdio> - <tests> -<test> -<param name='input1' value='test_bams2mx.xls' ftype='tabular' /> - <param name='treatment_name' value='case' /> - <param name='title' value='edgeRtest' /> - <param name='fdrtype' value='fdr' /> - <param name='priordf' value="0" /> - <param name='fdrthresh' value="0.05" /> - <param name='control_name' value='control' /> - <param name='Treat_cols' value='3,4,5,9' /> - <param name='Control_cols' value='2,6,7,8' /> - <output name='outtab' file='edgeRtest1out.xls' ftype='tabular' compare='diff' /> - <output name='html_file' file='edgeRtest1out.html' ftype='html' compare='diff' lines_diff='20' /> -</test> -</tests> - -<configfiles> -<configfile name="runme"> -<![CDATA[ -# -# edgeR.Rscript -# updated npv 2011 for R 2.14.0 and edgeR 2.4.0 by ross -# Performs DGE on a count table containing n replicates of two conditions -# -# Parameters -# -# 1 - Output Dir - -# Original edgeR code by: S.Lunke and A.Kaspi -reallybig = log10(.Machine\$double.xmax) -reallysmall = log10(.Machine\$double.xmin) -library('stringr') -library('gplots') -library('DESeq') -library('edgeR') -hmap2 = function(cmat,nsamp=100,outpdfname='heatmap2.pdf', TName='Treatment',group=NA,myTitle='title goes here') -{ -# Perform clustering for significant pvalues after controlling FWER - samples = colnames(cmat) - gu = unique(group) - if (length(gu) == 2) { - col.map = function(g) {if (g==gu[1]) "#FF0000" else "#0000FF"} - pcols = unlist(lapply(group,col.map)) - } else { - colours = rainbow(length(gu),start=0,end=4/6) - pcols = colours[match(group,gu)] } - gn = rownames(cmat) - dm = cmat[(! is.na(gn)),] - # remove unlabelled hm rows - nprobes = nrow(dm) - # sub = paste('Showing',nprobes,'contigs ranked for evidence of differential abundance') - if (nprobes > nsamp) { - dm =dm[1:nsamp,] - #sub = paste('Showing',nsamp,'contigs ranked for evidence for differential abundance out of',nprobes,'total') - } - newcolnames = substr(colnames(dm),1,20) - colnames(dm) = newcolnames - pdf(outpdfname) - heatmap.2(dm,main=myTitle,ColSideColors=pcols,col=topo.colors(100),dendrogram="col",key=T,density.info='none', - Rowv=F,scale='row',trace='none',margins=c(8,8),cexRow=0.4,cexCol=0.5) - dev.off() -} - -hmap = function(cmat,nmeans=4,outpdfname="heatMap.pdf",nsamp=250,TName='Treatment',group=NA,myTitle="Title goes here") -{ - # for 2 groups only was - #col.map = function(g) {if (g==TName) "#FF0000" else "#0000FF"} - #pcols = unlist(lapply(group,col.map)) - gu = unique(group) - colours = rainbow(length(gu),start=0.3,end=0.6) - pcols = colours[match(group,gu)] - nrows = nrow(cmat) - mtitle = paste(myTitle,'Heatmap: n contigs =',nrows) - if (nrows > nsamp) { - cmat = cmat[c(1:nsamp),] - mtitle = paste('Heatmap: Top ',nsamp,' DE contigs (of ',nrows,')',sep='') - } - newcolnames = substr(colnames(cmat),1,20) - colnames(cmat) = newcolnames - pdf(outpdfname) - heatmap(cmat,scale='row',main=mtitle,cexRow=0.3,cexCol=0.4,Rowv=NA,ColSideColors=pcols) - dev.off() -} - -qqPlot = function(descr='Title',pvector, ...) -# stolen from https://gist.github.com/703512 -{ - o = -log10(sort(pvector,decreasing=F)) - e = -log10( 1:length(o)/length(o) ) - o[o==-Inf] = reallysmall - o[o==Inf] = reallybig - pdfname = paste(gsub(" ","", descr , fixed=TRUE),'pval_qq.pdf',sep='_') - maint = paste(descr,'QQ Plot') - pdf(pdfname) - plot(e,o,pch=19,cex=1, main=maint, ..., - xlab=expression(Expected~~-log[10](italic(p))), - ylab=expression(Observed~~-log[10](italic(p))), - xlim=c(0,max(e)), ylim=c(0,max(o))) - lines(e,e,col="red") - grid(col = "lightgray", lty = "dotted") - dev.off() -} - -smearPlot = function(DGEList,deTags, outSmear, outMain) - { - pdf(outSmear) - plotSmear(DGEList,de.tags=deTags,main=outMain) - grid(col="blue") - dev.off() - } - -boxPlot = function(rawrs,cleanrs,maint,myTitle) -{ # - nc = ncol(rawrs) - for (i in c(1:nc)) {rawrs[(rawrs[,i] < 0),i] = NA} - fullnames = colnames(rawrs) - newcolnames = substr(colnames(rawrs),1,20) - colnames(rawrs) = newcolnames - newcolnames = substr(colnames(cleanrs),1,20) - colnames(cleanrs) = newcolnames - pdfname = paste(gsub(" ","", myTitle , fixed=TRUE),"sampleBoxplot.pdf",sep='_') - defpar = par(no.readonly=T) - pdf(pdfname,height=6,width=8) - #par(mfrow=c(1,2)) # 1 rows 2 col - l = layout(matrix(c(1,2),1,2,byrow=T)) - print.noquote('raw contig counts by sample:') - print.noquote(summary(rawrs)) - print.noquote('normalised contig counts by sample:') - print.noquote(summary(cleanrs)) - boxplot(rawrs,varwidth=T,notch=T,ylab='log contig count',col="maroon",las=3,cex.axis=0.35,main=paste('Raw:',maint)) - grid(col="blue") - boxplot(cleanrs,varwidth=T,notch=T,ylab='log contig count',col="maroon",las=3,cex.axis=0.35,main=paste('After ',maint)) - grid(col="blue") - dev.off() - pdfname = paste(gsub(" ","", myTitle , fixed=TRUE),"samplehistplot.pdf",sep='_') - nc = ncol(rawrs) - print.noquote(paste('Using ncol rawrs=',nc)) - ncroot = round(sqrt(nc)) - if (ncroot*ncroot < nc) { ncroot = ncroot + 1 } - m = c() - for (i in c(1:nc)) { - rhist = hist(rawrs[,i],breaks=100,plot=F) - m = append(m,max(rhist\$counts)) - } - ymax = max(m) - pdf(pdfname) - par(mfrow=c(ncroot,ncroot)) - for (i in c(1:nc)) { - hist(rawrs[,i], main=paste("Contig logcount",i), xlab='log raw count', col="maroon", - breaks=100,sub=fullnames[i],cex=0.8,ylim=c(0,ymax)) - } - dev.off() - par(defpar) - -} - -cumPlot = function(rawrs,cleanrs,maint,myTitle) -{ # updated to use ecdf - pdfname = paste(gsub(" ","", myTitle , fixed=TRUE),"RowsumCum.pdf",sep='_') - defpar = par(no.readonly=T) - pdf(pdfname) - par(mfrow=c(2,1)) - lrs = log(rawrs,10) - lim = max(lrs) - hist(lrs,breaks=100,main=paste('Before:',maint),xlab="# Reads (log)", - ylab="Count",col="maroon",sub=myTitle, xlim=c(0,lim),las=1) - grid(col="blue") - lrs = log(cleanrs,10) - hist(lrs,breaks=100,main=paste('After:',maint),xlab="# Reads (log)", - ylab="Count",col="maroon",sub=myTitle,xlim=c(0,lim),las=1) - grid(col="blue") - dev.off() - par(defpar) -} - -cumPlot1 = function(rawrs,cleanrs,maint,myTitle) -{ # updated to use ecdf - pdfname = paste(gsub(" ","", myTitle , fixed=TRUE),"RowsumCum.pdf",sep='_') - pdf(pdfname) - par(mfrow=c(2,1)) - lastx = max(rawrs) - rawe = knots(ecdf(rawrs)) - cleane = knots(ecdf(cleanrs)) - cy = 1:length(cleane)/length(cleane) - ry = 1:length(rawe)/length(rawe) - plot(rawe,ry,type='l',main=paste('Before',maint),xlab="Log Contig Total Reads", - ylab="Cumulative proportion",col="maroon",log='x',xlim=c(1,lastx),sub=myTitle) - grid(col="blue") - plot(cleane,cy,type='l',main=paste('After',maint),xlab="Log Contig Total Reads", - ylab="Cumulative proportion",col="maroon",log='x',xlim=c(1,lastx),sub=myTitle) - grid(col="blue") - dev.off() -} - - - -edgeIt = function (Count_Matrix,group,outputfilename,fdrtype='fdr',priordf=5,fdrthresh=0.05,outputdir='.', - myTitle='edgeR',libSize=c(),useNDF="T",filterquantile=0.2,subjects=c()) { - - # Error handling - if (length(unique(group))!=2){ - print("Number of conditions identified in experiment does not equal 2") - q() - } - require(edgeR) - mt = paste(unlist(strsplit(myTitle,'_')),collapse=" ") - allN = nrow(Count_Matrix) - nscut = round(ncol(Count_Matrix)/2) - colTotmillionreads = colSums(Count_Matrix)/1e6 - rawrs = rowSums(Count_Matrix) - nonzerod = Count_Matrix[(rawrs > 0),] # remove all zero count genes - nzN = nrow(nonzerod) - nzrs = rowSums(nonzerod) - zN = allN - nzN - print('# Quantiles for non-zero row counts:',quote=F) - print(quantile(nzrs,probs=seq(0,1,0.1)),quote=F) - if (useNDF == "T") - { - gt1rpin3 = rowSums(Count_Matrix/expandAsMatrix(colTotmillionreads,dim(Count_Matrix)) >= 1) >= nscut - lo = colSums(Count_Matrix[!gt1rpin3,]) - workCM = Count_Matrix[gt1rpin3,] - cleanrs = rowSums(workCM) - cleanN = length(cleanrs) - meth = paste( "After removing",length(lo),"contigs with fewer than ",nscut," sample read counts >= 1 per million, there are",sep="") - print(paste("Read",allN,"contigs. Removed",zN,"contigs with no reads.",meth,cleanN,"contigs"),quote=F) - maint = paste('Filter >=1/million reads in >=',nscut,'samples') - } - else { - useme = (nzrs > quantile(nzrs,filterquantile)) - workCM = nonzerod[useme,] - lo = colSums(nonzerod[!useme,]) - cleanrs = rowSums(workCM) - cleanN = length(cleanrs) - meth = paste("After filtering at count quantile =",filterquantile,", there are",sep="") - print(paste('Read',allN,"contigs. Removed",zN,"with no reads.",meth,cleanN,"contigs"),quote=F) - maint = paste('Filter below',filterquantile,'quantile') - } - cumPlot(rawrs=rawrs,cleanrs=cleanrs,maint=maint,myTitle=myTitle) - allgenes <- rownames(workCM) - print(paste("# Total low count contigs per sample = ",paste(lo,collapse=',')),quote=F) - rsums = rowSums(workCM) - TName=unique(group)[1] - CName=unique(group)[2] - # Setup DGEList object - DGEList = DGEList(counts=workCM, group = group) - if (length(subjects) == 0) - { - doDESEQ = T - mydesign = model.matrix(~group) - } - else { - doDESEQ = F - subjf = factor(subjects) - mydesign = model.matrix(~subjf+group) # we block on subject so make group last to simplify finding it - } - print.noquote(paste('Using samples:',paste(colnames(workCM),collapse=','))) - print.noquote('Using design matrix:') - print.noquote(mydesign) - DGEList = estimateGLMCommonDisp(DGEList,mydesign) - comdisp = DGEList\$common.dispersion - DGEList = estimateGLMTrendedDisp(DGEList,mydesign) - if (priordf > 0) { - print.noquote(paste("prior.df =",priordf)) - DGEList = estimateGLMTagwiseDisp(DGEList,mydesign,prior.df = priordf) - } else { - DGEList = estimateGLMTagwiseDisp(DGEList,mydesign) - } - DGLM = glmFit(DGEList,design=mydesign) - efflib = DGEList\$samples\$lib.size*DGEList\$samples\$norm.factors - normData = (1e+06*DGEList\$counts/efflib) - co = length(colnames(mydesign)) - DE = glmLRT(DGLM,coef=co) # always last one - subject is first if needed - uoutput = cbind( - Name=as.character(rownames(DGEList\$counts)), - DE\$table, - adj.p.value=p.adjust(DE\$table\$PValue, method=fdrtype), - Dispersion=DGEList\$tagwise.dispersion,totreads=rsums,normData, - DGEList\$counts - ) - soutput = uoutput[order(DE\$table\$PValue),] # sorted into p value order - for quick toptable - goodness = gof(DGLM, pcutoff=fdrthresh) - if (sum(goodness\$outlier) > 0) { - print.noquote('GLM outliers:') - print(paste(rownames(DGLM)[(goodness\$outlier != 0)],collapse=','),quote=F) - z = limma::zscoreGamma(goodness\$gof.statistic, shape=goodness\$df/2, scale=2) - pdf(paste(mt,"GoodnessofFit.pdf",sep='_')) - qq = qqnorm(z, panel.first=grid(), main="tagwise dispersion") - abline(0,1,lwd=3) - points(qq\$x[goodness\$outlier],qq\$y[goodness\$outlier], pch=16, col="dodgerblue") - dev.off() - } else { print('No GLM fit outlier genes found\n')} - estpriorn = getPriorN(DGEList) - print(paste("Common Dispersion =",comdisp,"CV = ",sqrt(comdisp),"getPriorN = ",estpriorn),quote=F) - efflib = DGEList\$samples\$lib.size*DGEList\$samples\$norm.factors - normData = (1e+06*DGEList\$counts/efflib) - uniqueg = unique(group) - # Plot MDS - sample_colors = match(group,levels(group)) - pdf(paste(mt,"MDSplot.pdf",sep='_')) - sampleTypes = levels(group) - plotMDS.DGEList(DGEList,main=paste("MDS Plot for",myTitle),cex=0.5,col=sample_colors,pch=sample_colors) - legend(x="topleft", legend = sampleTypes,col=c(1:length(sampleTypes)), pch=19) - grid(col="blue") - dev.off() - colnames(normData) = paste( colnames(normData),'N',sep="_") - print(paste('Raw sample read totals',paste(colSums(nonzerod,na.rm=T),collapse=','))) - nzd = data.frame(log(nonzerod + 1e-2,10)) - boxPlot(rawrs=nzd,cleanrs=log(normData,10),maint='TMM Normalisation',myTitle=myTitle) - if (doDESEQ) - { - # DESeq - deSeqDatcount <- newCountDataSet(workCM, group) - deSeqDatsizefac <- estimateSizeFactors(deSeqDatcount) - deSeqDatdisp <- estimateDispersions(deSeqDatsizefac) - rDESeq <- nbinomTest(deSeqDatdisp, levels(group)[1], levels(group)[2]) - rDESeq <- rDESeq[order(rDESeq\$pval), ] - write.table(rDESeq,paste(mt,'DESeq_TopTable.xls',sep='_'), quote=FALSE, sep="\t",row.names=F) - topresults.DESeq <- rDESeq[which(rDESeq\$padj < fdrthresh), ] - DESeqcountsindex <- which(allgenes %in% topresults.DESeq\$id) - DESeqcounts <- rep(0, length(allgenes)) - DESeqcounts[DESeqcountsindex] <- 1 - } - DGEList = calcNormFactors(DGEList) - norm.factor = DGEList\$samples\$norm.factors - pdf(paste(mt,"voomplot.pdf",sep='_')) - dat.voomed <- voom(DGEList, mydesign, plot = TRUE, lib.size = colSums(workCM) * norm.factor) - dev.off() - # Use limma to fit data - fit <- lmFit(dat.voomed, mydesign) - fit <- eBayes(fit) - rvoom <- topTable(fit, coef = length(colnames(mydesign)), adj = "BH", n = Inf) - write.table(rvoom,paste(mt,'VOOM_topTable.xls',sep='_'), quote=FALSE, sep="\t",row.names=F) - # Use an FDR cutoff to find interesting samples for edgeR, DESeq and voom/limma - topresults.voom <- rvoom[which(rvoom\$adj.P.Val < fdrthresh), ] - topresults.edgeR <- soutput[which(soutput\$adj.p.value < fdrthresh), ] - # Create venn diagram of hits - edgeRcountsindex <- which(allgenes %in% rownames(topresults.edgeR)) - voomcountsindex <- which(allgenes %in% topresults.voom\$ID) - edgeRcounts <- rep(0, length(allgenes)) - edgeRcounts[edgeRcountsindex] <- 1 - voomcounts <- rep(0, length(allgenes)) - voomcounts[voomcountsindex] <- 1 - if (doDESEQ) { - vennmain = paste(mt,'Voom,edgeR and DESeq overlap at FDR=',fdrthresh) - counts.dataframe <- data.frame(edgeRcounts = edgeRcounts, DESeqcounts = DESeqcounts, - voomcounts = voomcounts, row.names = allgenes) - } else { - vennmain = paste(mt,'Voom and edgeR overlap at FDR=',fdrthresh) - counts.dataframe <- data.frame(edgeRcounts = edgeRcounts, voomcounts = voomcounts, row.names = allgenes) - } - counts.venn <- vennCounts(counts.dataframe) - vennf = paste(mt,'venn.pdf',sep='_') - pdf(vennf) - vennDiagram(counts.venn,main=vennmain,col="maroon") - dev.off() - #Prepare our output file - nreads = soutput\$totreads # ordered same way - print('# writing output',quote=F) - write.table(soutput,outputfilename, quote=FALSE, sep="\t",row.names=F) - rn = uoutput\$Name - reg = "^chr([0-9]+):([0-9]+)-([0-9]+)" - org="hg19" - genecards="<a href='http://www.genecards.org/index.php?path=/Search/keyword/" - ucsc = paste("<a href='http://genome.ucsc.edu/cgi-bin/hgTracks?db=",org,sep='') - testreg = str_match(rn,reg) - nreads = uoutput\$totreads # ordered same way - if (sum(!is.na(testreg[,1]))/length(testreg[,1]) > 0.8) # is ucsc style string - { - urls = paste(ucsc,'&position=chr',testreg[,2],':',testreg[,3],"-",testreg[,4],"'>",rn,'</a>',sep='') - } else { - urls = paste(genecards,rn,"'></a>",rn,'</a>',sep="") - } - print.noquote('# urls') - cat(head(urls)) - tt = uoutput - cat("# Top tags\n") - tt = cbind(tt,ntotreads=nreads,URL=urls) # add to end so table isn't laid out strangely - tt = tt[order(DE\$table\$PValue),] - options(width = 500) - print.noquote(tt[1:50,]) - pdf(paste(mt,"BCV_vs_abundance.pdf",sep='_')) - plotBCV(DGEList, cex=0.3, main="Biological CV vs abundance") - dev.off() - # Plot MAplot - deTags = rownames(uoutput[uoutput\$adj.p.value < fdrthresh,]) - nsig = length(deTags) - print(paste('#',nsig,'tags significant at adj p=',fdrthresh),quote=F) - print('# deTags',quote=F) - print(head(deTags)) - dg = DGEList[order(DE\$table\$PValue),] - #normData = (1e+06 * dg\$counts/expandAsMatrix(dg\$samples\$lib.size, dim(dg))) - efflib = dg\$samples\$lib.size*dg\$samples\$norm.factors - normData = (1e+06*dg\$counts/efflib) - outpdfname=paste(mt,"heatmap.pdf",sep='_') - hmap2(normData,nsamp=100,TName=TName,group=group,outpdfname=outpdfname,myTitle=myTitle) - outSmear = paste(mt,"Smearplot.pdf",sep='_') - outMain = paste("Smear Plot for ",TName,' Vs ',CName,' (FDR@',fdrthresh,' N = ',nsig,')',sep='') - smearPlot(DGEList=DGEList,deTags=deTags, outSmear=outSmear, outMain = outMain) - qqPlot(descr=myTitle,pvector=DE\$table\$PValue) - if (doDESEQ) { - cat("# DESeq top 50\n") - print(rDESeq[1:50,]) - } - cat("# VOOM top 50\n") - print(rvoom[1:50,]) - # need a design matrix and glm to use this - goodness = gof(DGLM, pcutoff=fdrthresh) - nout = sum(goodness\$outlier) - if (nout > 0) { - print.noquote(paste('Found',nout,'Goodness of fit outliers')) - rownames(DGLM)[goodness\$outlier] - z = limma::zscoreGamma(goodness\$gof.statistic, shape=goodness\$df/2, scale=2) - pdf(paste(mt,"GoodnessofFit.pdf",sep='_')) - qq = qqnorm(z, panel.first=grid(), main="tagwise dispersion") - abline(0,1,lwd=3) - points(qq\$x[goodness\$outlier],qq\$y[goodness\$outlier], pch=16, col="dodgerblue") - dev.off() - } - #Return our main table - uoutput - -} #Done -sink(stdout(),append=T,type="message") -options(width=512) -Out_Dir = "$html_file.files_path" -Input = "$input1" -TreatmentName = "$treatment_name" -TreatmentCols = "$Treat_cols" -ControlName = "$control_name" -ControlCols= "$Control_cols" -outputfilename = "$outtab" -fdrtype = "$fdrtype" -priordf = $priordf -fdrthresh = $fdrthresh -useNDF = "$useNDF" -fQ = $fQ # non-differential centile cutoff -myTitle = "$title" -subjects = c($subjectids) -nsubj = length(subjects) -#Set our columns -TCols = as.numeric(strsplit(TreatmentCols,",")[[1]])-1 -CCols = as.numeric(strsplit(ControlCols,",")[[1]])-1 -cat('# got TCols=') -cat(TCols) -cat('; CCols=') -cat(CCols) -cat('\n') -useCols = c(TCols,CCols) -# Create output dir if non existent - if (file.exists(Out_Dir) == F) dir.create(Out_Dir) - -Count_Matrix = read.table(Input,header=T,row.names=1,sep='\t') #Load tab file assume header -snames = colnames(Count_Matrix) -nsamples = length(snames) -if (nsubj > 0 & nsubj != nsamples) { -options("show.error.messages"=T) -mess = paste('Fatal error: Supplied subject id list',paste(subjects,collapse=','),'has length',nsubj,'but there are',nsamples,'samples',paste(snames,collapse=',')) -write(mess, stderr()) -#print(mess) -quit(save="no",status=4) -} - -Count_Matrix = Count_Matrix[,useCols] # reorder columns -if (length(subjects) != 0) {subjects = subjects[useCols]} -rn = rownames(Count_Matrix) -islib = rn %in% c('librarySize','NotInBedRegions') -LibSizes = Count_Matrix[subset(rn,islib),][1] # take first -Count_Matrix = Count_Matrix[subset(rn,! islib),] -group = c(rep(TreatmentName,length(TCols)), rep(ControlName,length(CCols)) ) #Build a group descriptor -group = factor(group, levels=c(ControlName,TreatmentName)) -colnames(Count_Matrix) = paste(group,colnames(Count_Matrix),sep="_") #Relable columns -results = edgeIt(Count_Matrix=Count_Matrix,group=group,outputfilename=outputfilename,fdrtype=fdrtype,priordf=priordf,fdrthresh=fdrthresh, - outputdir=Out_Dir,myTitle=myTitle,libSize=c(),useNDF=useNDF,filterquantile=fQ,subjects=subjects) -#Run the main function -# for the log -sessionInfo() -]]> -</configfile> -</configfiles> -<help> -**What it does** - -Performs digital gene expression analysis between a treatment and control on a count matrix. -Optionally adds a term for subject if not all samples are independent or if some other factor needs to be blocked in the design. - -**Input** - -A matrix consisting of non-negative integers. The matrix must have a unique header row identifiying the samples, and a unique set of row names -as the first column. Typically the row names are gene symbols or probe id's for downstream use in GSEA and other methods. - -If you have (eg) paired samples and wish to include a term in the GLM to account for some other factor (subject in the case of paired samples), -put a comma separated list of indicators for every sample (whether modelled or not!) indicating (eg) the subject number or -A list of integers, one for each subject or an empty string if samples are all independent. -If not empty, there must be exactly as many integers in the supplied integer list as there are columns (samples) in the count matrix. -Integers for samples that are not in the analysis *must* be present in the string as filler even if not used. - -So if you have 2 pairs out of 6 samples, you need to put in unique integers for the unpaired ones -eg if you had 6 samples with the first two independent but the second and third pairs each being from independent subjects. you might use -8,9,1,1,2,2 -as subject IDs to indicate two paired samples from the same subject in columns 3/4 and 5/6 - -**Output** - -A matrix which consists the original data and relative expression levels and some helpful plots - -**Note on edgeR versions** - -The edgeR authors made a small cosmetic change in the name of one important variable (from p.value to PValue) -breaking this and all other code that assumed the old name for this variable, -between edgeR2.4.4 and 2.4.6 (the version for R 2.14 as at the time of writing). -This means that all code using edgeR is sensitive to the version. I think this was a very unwise thing -to do because it wasted hours of my time to track down and will similarly cost other edgeR users dearly -when their old scripts break. This tool currently now works with 2.4.6. - -**Note on prior.N** - -http://seqanswers.com/forums/showthread.php?t=5591 says: - -*prior.n* - -The value for prior.n determines the amount of smoothing of tagwise dispersions towards the common dispersion. -You can think of it as like a "weight" for the common value. (It is actually the weight for the common likelihood -in the weighted likelihood equation). The larger the value for prior.n, the more smoothing, i.e. the closer your -tagwise dispersion estimates will be to the common dispersion. If you use a prior.n of 1, then that gives the -common likelihood the weight of one observation. - -In answer to your question, it is a good thing to squeeze the tagwise dispersions towards a common value, -or else you will be using very unreliable estimates of the dispersion. I would not recommend using the value that -you obtained from estimateSmoothing()---this is far too small and would result in virtually no moderation -(squeezing) of the tagwise dispersions. How many samples do you have in your experiment? -What is the experimental design? If you have few samples (less than 6) then I would suggest a prior.n of at least 10. -If you have more samples, then the tagwise dispersion estimates will be more reliable, -so you could consider using a smaller prior.n, although I would hesitate to use a prior.n less than 5. - - -From Bioconductor Digest, Vol 118, Issue 5, Gordon writes: - -Dear Dorota, - -The important settings are prior.df and trend. - -prior.n and prior.df are related through prior.df = prior.n * residual.df, -and your experiment has residual.df = 36 - 12 = 24. So the old setting of -prior.n=10 is equivalent for your data to prior.df = 240, a very large -value. Going the other way, the new setting of prior.df=10 is equivalent -to prior.n=10/24. - -To recover old results with the current software you would use - - estimateTagwiseDisp(object, prior.df=240, trend="none") - -To get the new default from old software you would use - - estimateTagwiseDisp(object, prior.n=10/24, trend=TRUE) - -Actually the old trend method is equivalent to trend="loess" in the new -software. You should use plotBCV(object) to see whether a trend is -required. - -Note you could also use - - prior.n = getPriorN(object, prior.df=10) - -to map between prior.df and prior.n. - -</help> - -</tool> - -
--- a/rgedgeR/test-data/gentestdata.sh~ Wed Jun 12 05:21:25 2013 -0400 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,8 +0,0 @@ -#!/bin/bash -# generate test data for rgGSEA -# ross lazarus June 2013 -# adjust gseajar_path ! -GSEAJAR_PATH=/home/rlazarus/galaxy-central/tool_dependency_dir/gsea_jar/2.0.12/fubar/rg_gsea_test/8e291f464aa0/jars/gsea2-2.0.12.jar -python ../rgGSEA.py --input_tab "gsea_test_DGE.xls" --adjpvalcol "5" --signcol "2" --idcol "1" --outhtml "gseatestout.html" --input_name "gsea_test" --setMax "500" --setMin "15" --nPerm "10" --plotTop "20" --gsea_jar "$GSEAJAR_PATH" --output_dir "gseatestout" --mode "Max_probe" --title "GSEA test" --builtin_gmt "gseatestdata.gmt" - -
--- a/rgedgeR/tool_dependencies.xml Wed Jun 12 05:21:25 2013 -0400 +++ b/rgedgeR/tool_dependencies.xml Wed Jun 12 06:13:41 2013 -0400 @@ -1,37 +1,10 @@ <?xml version="1.0"?> <tool_dependency> - <package name="readline" version="6.2"> - <repository changeset_revision="1301ec7705a8" name="package_readline_6_2" owner="boris" prior_installation_required="True" toolshed="http://testtoolshed.g2.bx.psu.edu/" /> - </package> - <package name="package_R" version="3.0.1"> - <install version="1.0"> - <actions> - <action type="download_by_url">http://cran.ms.unimelb.edu.au/src/base/R-3/R-3.0.1.tar.gz</action> - <action type="set_environment_for_install"> - <repository changeset_revision="1301ec7705a8" name="package_readline_6_2" owner="boris" toolshed="http://testtoolshed.g2.bx.psu.edu/"> - <package name="package_readline_6_2" version="6.2" /> - </repository> - </action> - <action type="make_directory">$INSTALL_DIR</action> - <action type="shell_command">./configure --with-blas --with-lapack --enable-R-shlib --with-x=no --prefix=$INSTALL_DIR && make && make install</action> - <action type="set_environment"> - <environment_variable action="set_to" name="R_HOME">$INSTALL_DIR/lib/R</environment_variable> - <environment_variable action="set_to" name="R_LIBS">$INSTALL_DIR/lib/R/library</environment_variable> - <environment_variable action="prepend_to" name="PATH">$INSTALL_DIR/lib/R/bin</environment_variable> - </action> - </actions> - </install> - <readme>R is a free software environment for statistical computing and graphics - WARNING: See custom compilation options above - Modified from an older version of R by Boris by Ross Lazarus for R 3.0 - Added Bioc basics too - </readme> - </package> <package name="package_BioCBasics" version="2.12"> <install version="1.0"> <actions> <action type="set_environment_for_install"> - <package name="package_R" prior_installation_required="True" toolshed="http://testtoolshed.g2.bx.psu.edu" version="3.0.1" /> + <repository name="package_R" owner="fubar" prior_installation_required="True" toolshed="http://testtoolshed.g2.bx.psu.edu/"/> </action> <action type="shell_command">$R_HOME/bin/R CMD BATCH installBioC.R </action> </actions>
--- a/rgedgeR/tool_dependencies.xml~ Wed Jun 12 05:21:25 2013 -0400 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,44 +0,0 @@ -<?xml version="1.0"?> -<tool_dependency> - <package name="package_readline_6_2" version="6.2"> - <repository name="package_readline_6_2" owner="boris" prior_installation_required="True" - toolshed="http://testtoolshed.g2.bx.psu.edu/" /> - </package> - <package name="package_R" version="3.0.1"> - <install version="1.0"> - <actions> - <action type="download_by_url">http://cran.ms.unimelb.edu.au/src/base/R-3/R-3.0.1.tar.gz</action> - <action type="set_environment_for_install"> - <repository changeset_revision="1301ec7705a8" name="package_readline_6_2" owner="boris" - toolshed="http://testtoolshed.g2.bx.psu.edu/"> - <package name="package_readline_6_2" version="6.2" /> - </repository> - </action> - <action type="make_directory">$INSTALL_DIR</action> - <action type="shell_command">./configure --with-blas --with-lapack --enable-R-shlib --with-readline=no --with-x=no --prefix=$INSTALL_DIR && make && make install</action> - <action type="set_environment"> - <environment_variable action="set_to" name="R_HOME">$INSTALL_DIR/lib/R</environment_variable> - <environment_variable action="set_to" name="R_LIBS">$INSTALL_DIR/lib/R/library</environment_variable> - <environment_variable action="prepend_to" name="PATH">$INSTALL_DIR/lib/R/bin</environment_variable> - </action> - </actions> - </install> - <readme>R is a free software environment for statistical computing and graphics - WARNING: See custom compilation options above - Modified from an older version of R by Boris by Ross Lazarus for R 3.0 - Added Bioc basics too - </readme> - </package> - <package name="package_BioCBasics" version="2.12"> - <install version="1.0"> - <actions> - <action type="shell_command">$INSTALL_DIR/lib/R/bin/R CMD BATCH installBioC.R </action> - </actions> - </install> - <readme>R is a free software environment for statistical computing and graphics - WARNING: See custom compilation options above - Modified from an older version of R by Boris by Ross Lazarus for R 3.0 - Added Bioc basics via this package installBioC.R script - </readme> - </package> -</tool_dependency>