#!/usr/bin/env Rscript # parse parameter --------------------------------------------------------- library(argparser, quietly=TRUE) # Create a parser p <- arg_parser("run featureCounts and calculate FPKM/TPM") # Add command line arguments p <- add_argument(p, "--bam", help="input: bam file", type="character") p <- add_argument(p, "--gtf", help="input: gtf file", type="character") p <- add_argument(p, "--featureType", help="a character string or a vector of character strings giving the feature type or types used to select rows in the GTF annotation which will be used for read summarization", type="character", default="exon") p <- add_argument(p, "--attrType", help="a character string giving the attribute type in the GTF annotation which will be used to group features (eg. exons) into meta-features", type="character", default="gene_id") p <- add_argument(p, "--isPairedEnd", help="indicating whether libraries contain paired-end reads or not", type="logical", default=TRUE) p <- add_argument(p, "--strandSpecific", help="0 (unstranded), 1 (stranded) and 2 (reversely stranded)", type="numeric", default=0) p <- add_argument(p, "--output", help="output prefix", type="character") # Parse the command line arguments argv <- parse_args(p) library(Rsubread) library(limma) library(edgeR) bamFile <- argv$bam gtfFile <- argv$gtf nthreads <- 1 outFilePref <- argv$output outStatsFilePath <- paste(outFilePref, '.log', sep = ''); outCountsFilePath <- paste(outFilePref, '.count', sep = ''); fCountsList = featureCounts(bamFile, annot.ext=gtfFile, isGTFAnnotationFile=TRUE, nthreads=nthreads, GTF.featureType=argv$featureType, GTF.attrType=argv$attrType, isPairedEnd=argv$isPairedEnd, strandSpecific=argv$strandSpecific) dgeList = DGEList(counts=fCountsList$counts, genes=fCountsList$annotation) fpkm = rpkm(dgeList, dgeList$genes$Length) tpm = exp(log(fpkm) - log(sum(fpkm)) + log(1e6)) write.table(fCountsList$stat, outStatsFilePath, sep="\t", col.names=FALSE, row.names=FALSE, quote=FALSE) featureCounts = cbind(fCountsList$annotation[,1], fCountsList$counts, fpkm, tpm) colnames(featureCounts) = c('gene_id', 'counts', 'fpkm','tpm') write.table(featureCounts, outCountsFilePath, sep="\t", col.names=TRUE, row.names=FALSE, quote=FALSE)