view edgeR_DGE_test.R @ 4:b1aee4b59049 draft

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#!/usr/bin/env Rscript 

# edgeR citation:
#  Robinson MD, McCarthy DJ and Smyth GK (2010). edgeR: a Bioconductor package for differential expression
#  analysis of digital gene expression data. Bioinformatics 26, 139-140
#
#  Robinson MD and Smyth GK (2007). Moderated statistical tests for assessing differences in tag
#  abundance. Bioinformatics 23, 2881-2887

#  Robinson MD and Smyth GK (2008). Small-sample estimation of negative binomial dispersion, with
#  applications to SAGE data. Biostatistics, 9, 321-332

#  McCarthy DJ, Chen Y and Smyth GK (2012). Differential expression analysis of multifactor RNA-Seq
#  experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297

# R script Author:
# - MSc. René Böttcher (Erasmus MC)

# Hooked into Galaxy Server:
# - MSc. Youri Hoogstrate (Erasmus MC)


setwd("/home/youri/Desktop/galaxy/tools/TraIT/edgeR/DGE")

library(edgeR)
 
# Fetch commandline arguments
args <- commandArgs(trailingOnly = TRUE)
designmatrix        = args[1]
contrast            = args[2]

output_1            = args[3]
output_2            = args[4]
output_3            = args[5]		#FPKM file - to be implemented
output_4            = args[6]

QC                  = nchar(args[7]) > 0

output_5            = args[8]
output_6            = args[9]
output_7            = args[10]

output_8            = args[11]




library(edgeR)
raw_data <- read.delim(designmatrix,header=T,stringsAsFactors=T)

# Obtain read-counts
read_counts 		= read.delim(as.character(raw_data[1,1]),header=F,stringsAsFactors=F,row.names=1)
for(i in 2:length(raw_data[,1])) {
  print("parsing counts from:")
  print(raw_data[i,1])
  read_counts = cbind(read_counts,read.delim(as.character(raw_data[i,1]),header=F,stringsAsFactors=F,row.names=1))
  print(i)
}



# Filter for HTSeq predifined counts:
exclude_HTSeq = c("no_feature","ambiguous","too_low_aQual","not_aligned","alignment_not_unique")
exclude_DEXSeq = c("_ambiguous","_empty","_lowaqual","_notaligned")

exclude = match(c(exclude_HTSeq, exclude_DEXSeq),rownames(read_counts))
exclude = exclude[is.na(exclude)==0]
if(length(exclude) != 0)  {
  read_counts = read_counts[-exclude,]
}









colnames(read_counts) = raw_data[,2]
dge 					= DGEList(counts=read_counts,genes=rownames(read_counts))

design_tmp <- raw_data[3:length(raw_data)]
rownames(design_tmp)     <- colnames(dge)
formula = paste(c("~0",colnames(design_tmp)),collapse = " + ")
design <- model.matrix(as.formula(formula),design_tmp)

prefixes = colnames(design_tmp)[attr(design,"assign")]
avoid = nchar(prefixes) == nchar(colnames(design))
replacements = substr(colnames(design),nchar(prefixes)+1,nchar(colnames(design)))
replacements[avoid] = colnames(design)[avoid]
colnames(design) = replacements



print("Calculating normalization factors...")
dge		= calcNormFactors(dge)
print("Estimating common dispersion...")
dge 	= estimateGLMCommonDisp(dge,design)
print("Estimating trended dispersion...")
dge 	= estimateGLMTrendedDisp(dge,design)
print("Estimating tagwise dispersion...")
dge 	= estimateGLMTagwiseDisp(dge,design)




if (QC == TRUE) {
  print("Creating QC plots...")
  ## MDS Plot
  pdf(output_5)
  plotMDS(dge, main="edgeR MDS Plot")
  dev.off()
  ## Biological coefficient of variation plot
  pdf(output_6)
  plotBCV(dge, cex=0.4, main="edgeR: Biological coefficient of variation (BCV) vs abundance")
  dev.off()
}



print("Fitting GLM...")
fit 	= glmFit(dge,design)

print(paste("Performing likelihood ratio test: ",contrast,sep=""))
cont <- c(contrast)
cont <- makeContrasts(contrasts=cont, levels=design)

lrt <- glmLRT(fit, contrast=cont[,1])
print(paste("Exporting to file: ",output_1,sep=""))
write.table(file=output_1,topTags(lrt,n=nrow(read_counts))$table,sep="\t",row.names=T)
write.table(file=output_2,cpm(dge,normalized.lib.sizes=TRUE),sep="\t")
# todo EXPORT FPKM
write.table(file=output_4,dge$counts,sep="\t")



if (QC == TRUE) {
  print("Creating MA plots...")
  
  
  etable <- topTags(lrt, n=nrow(dge))$table
  etable <- etable[order(etable$FDR), ]
  pdf(output_7)
  with(etable, plot(logCPM, logFC, pch=20, main="edgeR: Fold change vs abundance"))
  with(subset(etable, FDR<0.05), points(logCPM, logFC, pch=20, col="red"))
  abline(h=c(-1,1), col="blue")
  dev.off()
}
print("Done!")