salmon_edgeR.r

In sarangian/deaRscripts: 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 Salmon and DESeq2 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)) 
suppressMessages(library(rhdf5))
suppressMessages(library(tximport))
suppressMessages(library(DelayedArray))

                                          # to run the script in command lines

# 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="Salmon_edgeR_HTML_Report",
			dest="reportName",
			help="name of the project used for the report [default: name of the current directory]."),

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

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

make_option(c("-q", "--quantDir"),
			dest="quantDir",
			help="path to the directory containing the Salmon Quantification Folder"),		

make_option(c("-G", "--tx2geneDirectory"),
			default="NULL",
			dest="tx2geneDirectory",
			help="path to the tx2gene Directory [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("-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("-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 biological conditions using salmon with edgeR.",
					   epilogue="Computational Genome Biology Lab, CSIR-IICB, Kolkata 700032")
opt <- parse_args(parser, args=commandArgs(trailingOnly=TRUE), positional_arguments=0)$options

# 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
quantDir <- opt$quantDir							 # path to the directory containing raw counts files
tx2geneDirectory <- opt$tx2geneDirectory			 
varInt <- opt$varInt                                 # factor of interest
condRef <- opt$condRef                               # reference biological condition
batch <- opt$batch                                   # blocking factor: NULL (default) or "batch" for example
alpha <- as.numeric(opt$alpha)                       # threshold of statistical significance
pAdjustMethod <- opt$pAdjustMethod                   # p-value adjustment method: "BH" (default) or "BY"
normalizationMethod <- opt$normalizationMethod       # normalization method in calcNormFactors
cpmCutoff <- opt$cpmCutoff                           # counts-per-million cut-off to filter low counts

print(paste("projectName", projectName))
print(paste("targetFile", targetFile))
print(paste("varInt", varInt))
print(paste("condRef", condRef))
print(paste("batch", batch))
print(paste("alpha", alpha))
print(paste("pAdjustMethod", pAdjustMethod))
print(paste("normalizationMethod", normalizationMethod))
print(paste("templateFile", templateFile))

################################################################################
###                             running script                               ###
################################################################################

#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$quantDir) ) {
  stop("--path to the directory containing the Kallisto Quantification files must be provided. See script usage (--help)")
}

if ( is.null(opt$tx2geneDirectory) ) {
  stop("--tx2geneDirectory folder path must be provided. See script usage (--help)")
}
if ( is.null(opt$condRef) ) {
  stop("--reference biological condition name must be provided. See script usage (--help)")
}

################################################################################
# setwd(workDir)



#source ("/opt/RNASeqPIPE/tools/utility/load.TargetFile.R")
#source ("/opt/RNASeqPIPE/tools/utility/run.edgeR_trans.r")
#source ("/opt/RNASeqPIPE/tools/utility/exportResults.edgeR.R")
#source ("/opt/RNASeqPIPE/tools/utility/summarizeResults.edgeR.r")
#source ("/opt/RNASeqPIPE/tools/utility/nDiffTotal.r")


#plots

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

				   
# loading target file
target <- loadTargetFile(targetFile=targetFile, varInt=varInt, condRef=condRef, batch=batch)
#names(target$files)
print (target)
group=unique(target[,varInt])


files <- file.path(quantDir, 'salmon_quant', target$samples, "quant.sf")
#print (files)

salmon_dname <- dirname(files)
salmon_file_names <- basename(salmon_dname)
cat (salmon_file_names)
names(files) <- salmon_file_names
#print (files)



tx2gene <- read_csv(file.path(tx2geneDirectory, "tx2gene.csv"))
txi.salmon <- tximport(files, type="salmon", tx2gene=tx2gene)
counts <- txi.salmon$counts 

#head(counts) 


rownames_countMat <- rownames(counts)

#print (rownames_countMat)

#str(counts)
#countdata = as.data.frame(lapply(counts, as.integer))
#str(countdata)

write.table(counts, file=paste0(quantDir,"counts.txt"), row.names=FALSE, sep="\t", quote=FALSE)

DF <- read.table(file=paste0(quantDir,"counts.txt"), sep = "\t", header=TRUE, quote="", fill=FALSE)

head (DF)
countdata = as.data.frame(lapply(DF, as.integer))

row.names(countdata) <- rownames_countMat

#str(countdata)
#head(countdata)


out.edgeR <- run.edgeR_trans(counts=countdata, target=target, varInt=varInt, condRef=condRef,
                       batch=batch, cpmCutoff=cpmCutoff, 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=group, 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)
}