featureCount_edgeR.r

In sarangian/RNASeqDEA: Differential Expression Analysis based on the read count data generated by either of Salmon, Kallisto, featureCounts and perform statistical analysis to discover quantitative changes in expression levels between two different experimental groups

#!/usr/bin/env Rscript
################################################################################
### R script to compare two different conditions with count files (generated using featureCounts) and edgeR packages
### Aditya Narayan Sarangi
### Designed to be executed with bulkRNASeqPIPE
################################################################################

rm(list=ls())                                        # remove all the objects from the R session
suppressMessages(library(rnaseqdea))
suppressMessages(library(edgeR))
suppressMessages(library(DT))
suppressMessages(library(ggplot2))
suppressMessages(library(gplots))
suppressMessages(library(EnhancedVolcano))
suppressMessages(library(GenomicFeatures))
suppressMessages(library(regionReport))
suppressMessages(library(DEFormats))
suppressMessages(library(RColorBrewer))
suppressMessages(library(pheatmap))
suppressMessages(library(dplyr))
suppressMessages(library(colorspace))
suppressMessages(library(optparse))
suppressMessages(library(scales))
suppressMessages(library(readr))

# options list with associated default value.
option_list <- list( 
make_option(c("-P", "--projectName"),
			default=basename(getwd()),
			dest="projectName",
			help="name of the project used for storing images and tables [default: name of the current directory]."),

make_option(c("-R", "--reportName"),
			default="featureCounts_edgeR_HTML_Report",
			dest="reportName",
			help="name of the project used for the report [default: name of the current directory]."),


make_option(c("-t", "--targetFile"),
			default="target.txt",
			dest="targetFile",
			help="path to the design/target file [default: %default]."),

make_option(c("-T", "--templateFile"),
			dest="templateFile",
			help="path to the R markdown Template file"),


make_option(c("-q", "--featureQuant"),
			dest="featureQuant",
			help="path to the featureCount Quantification file [default: %default]."),

	
make_option(c("-v", "--varInt"),
			default="group",
			dest="varInt", 
			help="factor of interest [default: %default]"),

make_option(c("-c", "--condRef"),
			default="WT",
			dest="condRef",
			help="reference biological condition [default: %default]"),

make_option(c("-b", "--batch"),
			default=NULL,
			dest="batch",
			help="blocking factor [default: %default] or \"batch\" for example"),

make_option(c("-f", "--fitType"),
			default="parametric",
			dest="fitType", 
			help="mean-variance relationship: [default: %default],local or mean"),

make_option(c("-a", "--alpha"),
			default=0.05,
			dest="alpha", 
			help="threshold of statistical significance [default: %default]"),

make_option(c("-p", "--pAdjustMethod"),
			default="BH",
			dest="pAdjustMethod", 
			help="p-value adjustment method: \"BH\" or \"BY\" [default: %default]"),


make_option(c("-l", "--locfunc"),
			default="median",
			dest="locfunc", 
			help="median or shorth to estimate the size factors [default: %default]"),

make_option(c("-m", "--cpmCutoff"),
			default=1,
			dest="cpmCutoff", 
			help="counts-per-million cut-off to filter low counts"),
		
make_option(c("-n", "--normalizationMethod"),
			default="TMM",
			dest="normalizationMethod", 
			help="normalization method in calcNormFactors: \"TMM\", \"RLE\" or \"upperquartile\" [default: %default]")

)

# now parse the command line to check which option is given and get associated values
parser <- OptionParser(usage="usage: %prog [options]",
					   option_list=option_list, 
					   description="Compare two or more biological conditions in a RNA-Seq framework with DESeq2.",
					   epilogue="Computational Genome Biology Lab, CSIR-IICB, Kolkata 700032")
opt <- parse_args(parser, args=commandArgs(trailingOnly=TRUE), positional_arguments=0)$options



#Check mandetory inputs 
if ( is.null(opt$targetFile) ) {
  stop("--sample groupfile file / target file must be provided. See script usage (--help)")
}

if ( is.null(opt$featureQuant) ) {
  stop("--path to the featureCounts Countfile file must be provided. See script usage (--help)")
}


if ( is.null(opt$condRef) ) {
  stop("--reference biological condition name must be provided. See script usage (--help)")
}





# get options and arguments
workDir <- getwd()
projectName <- opt$projectName 
reportName <-opt$reportName                      # name of the project
targetFile <- opt$targetFile   
templateFile <- opt$templateFile               # path to the design/target file   
featureQuant <- opt$featureQuant                            # path to the directory containing salmon quantification files
varInt <- opt$varInt                                 # factor of interest
condRef <- opt$condRef                               # reference biological condition
batch <- opt$batch                                   # blocking factor: NULL (default) or "batch" for example
fitType <- opt$fitType                               # mean-variance relationship: "parametric" (default), "local" or "mean"
alpha <- as.numeric(opt$alpha)                       # threshold of statistical significance
pAdjustMethod <- opt$pAdjustMethod                   # p-value adjustment method: "BH" (default) or "BY"
locfunc <- opt$locfunc  
normalizationMethod <- opt$normalizationMethod       # normalization method in calcNormFactors
cpmCutoff <- opt$cpmCutoff                             # "median" (default) or "shorth" to estimate the size factors
				

 #print(paste("workDir", workDir))
 print(paste("projectName", projectName))
 print(paste("reportName", reportName))
 print(paste("targetFile", targetFile))
 print(paste("featureQuant", featureQuant))
 print(paste("varInt", varInt))
 print(paste("condRef", condRef))
 print(paste("batch", batch))
 print(paste("fitType", fitType))
 print(paste("alpha", alpha))
 print(paste("pAdjustMethod", pAdjustMethod))
 print(paste("locfunc", locfunc))

################################################################################
###                             running script                               ###
################################################################################
# setwd(workDir)
#dir.create(projectName, showWarnings = FALSE, recursive = TRUE)

#imageFolder <- paste0(projectName,"/figures/")
#tableFolder <- paste0(projectName,"/tables/")

#dir.create(imageFolder, showWarnings = FALSE, recursive = TRUE)
#dir.create(tableFolder, showWarnings = FALSE, recursive = TRUE)
dir.create("tables", showWarnings = FALSE, recursive = TRUE)


#source ("/opt/RNASeqPIPE/tools/utility/load.TargetFile.R", chdir=T)
#source ("/opt/RNASeqPIPE/tools/utility/run.edgeR.r", chdir=T)
#source ("/opt/RNASeqPIPE/tools/utility/exportResults.edgeR.R", chdir=T)

#plots
					   
# loading target file
target <- loadTargetFile(targetFile=targetFile, varInt=varInt, condRef=condRef, batch=batch)
rownames(target) <- as.character(target[,1])

countData <- read.table(featureQuant, header=TRUE, row.names=1)
countData <- countData[ ,6:ncol(countData)]
colnames(countData) <- gsub("\\.[sb]am$", "", colnames(countData))
colnames(countData) <- lapply(colnames(countData), function(x) sapply(strsplit(x, "\\."), tail, 1))
colnames(countData)
countData <- as.matrix(countData)

head (countData)

all(rownames(target) %in% colnames(countData))

countData <- countData[, rownames(target)]
all(rownames(target) == colnames(countData))

out.edgeR <- run.edgeR(counts=countData, target=target, varInt=varInt, condRef=condRef,
                       batch=batch, normalizationMethod=normalizationMethod,
                       pAdjustMethod=pAdjustMethod)



dge <- out.edgeR$dge
res <- out.edgeR$results
lrt <- out.edgeR$lrt


dds = as.DESeqDataSet(dge)
#res <- results(dds)

coldata <- colData(dds)

samples <- as.factor(row.names(coldata))
samples


colData(dds) <- cbind(colData(dds), samples)

coldata <- colData(dds)
intgroup <- colnames(coldata[c(1)])
intgroup

#summaryResults <- summarizeResults.edgeR(out.edgeR, group=target[,varInt], counts=countdata, alpha=alpha)
exportResults.edgeR(out.edgeR, group=target[,varInt], counts=countData, alpha=alpha, export=TRUE)

save.image(file=paste0(reportName, ".RData"))


report <- edgeReport(dge, lrt, projectName, intgroup, outdir = reportName,  output = 'index', nBest = 50000, nBestFeatures = 20, template = templateFile)


if(interactive()) {
    browseURL(report)
}