RNAseqDownstream2report/RNAseq_5_pwGSEA.R

#!/usr/bin/env Rscript
###Copyright 2019 Ying Yu from Fudan-PGx group 
# example:
# Rscript RNAseq_5_pwGSEA.R -o /home/yuying/rnaseqreport_test -i example_geneexp_log2fpkm_floor0p01_c13r58395_2019-04-30.txt  -g group13_2.txt 

suppressPackageStartupMessages(library("optparse"))
suppressPackageStartupMessages(library("fgsea"))

# specify our desired options in a list
# by default OptionParser will add an help option equivalent to 
# make_option(c("-h", "--help"), action="store_true", default=FALSE, 
#               help="Show this help message and exit")

# input input list , rds, from * to *

option_list <- list( 
    make_option(c("-o", "--out_dir"), type="character",default="./",
        help="The output directory [default ./]"),
    make_option(c("-i", "--input"),type="character", default=NULL,
        help="The input expression files. Required!"),
	make_option(c("-e", "--type_gene_id"),type="character", default="EnsemblGID",
        help="The type of gene symbol. Could be either of EnsemblGID/EntrezID/GeneSymbol [default: EnsemblGID]"),
	make_option(c("-g", "--sample_group"),type="character",  default=NULL,
        help="File in tab-delimited format for sample group infomation. The input file containing sample name and group infomation. note colname must be like: sample	group1	group2... Required! "),
	make_option(c("-q", "--padjvalueCutoff"), type="double",default=0.2,metavar="number",
		help="Cutoff value of adjusted p value. [default: 0.2]"),
   	make_option(c("-p", "--project_code"), type="character",default="rnaseq",
        help="Project code, which is used as prefix of output file. [default: rnaseq]"),
	make_option(c("-d", "--ref_rdata_dir"), type="character",default="./",
        help="The directory of reference files: human_c2_v5p2.rdata, human_c5_v5p2.rdata and ID_convert_table.rds. [default: ./]")
		)

# get command line options, if help option encountered print help and exit,
# otherwise if options not found on command line then set defaults, 
opt <- parse_args(OptionParser(option_list=option_list))

if (is.null(opt$input)){
  print_help(opt_parser)
  stop("At least one argument must be supplied (input file).", call.=FALSE)
}

if (is.null(opt$sample_group)){
  stop("At least one argument must be supplied (input group infomation for DEG analysis).", call.=FALSE)
}

##import file
out_dir<-paste(gsub("/$","",opt$out_dir),"/",sep="")
logexpr<-read.table(opt$input,header=T,stringsAsFactors=F,row.names=1,check.names=F)

#check exp file is log scale
if(max(logexpr[,1])-min(logexpr[,1])>100){
  stop("DEG anlaysis should be conducted based on expression profile on log scale. Please run log2 first", call.=FALSE)
}

##import sample group file and check 
sample_group<-read.table(opt$sample_group,sep="\t",header=T)

if(length(grep("group",colnames(sample_group)))==0){
stop("No group is identified in sample_group file. Make sure the head of sample_group file is like sample, group1, group2.")
}

#refdir
refdir<-paste(gsub("/$","",opt$ref_rdata_dir),"/",sep="")
#c2: curated gene sets (rdata file)
#c5: GO gene sets (rdata file)

if(length(grep("human_c2_v5p2.rdata",dir(refdir)))>0){
load(paste(refdir,"human_c2_v5p2.rdata",sep=""))
}else{
 stop("Cannot find human_c2_v5p2.rds in the ref_rdata_dir. Exit!", call.=FALSE)
}

if(length(grep("human_c5_v5p2.rdata",dir(refdir)))>0){
load(paste(refdir,"human_c5_v5p2.rdata",sep=""))
}else{
 stop("Cannot find human_c5_v5p2.rds in the ref_rdata_dir. Exit!", call.=FALSE)
}

##########################
#########ID convert#######
##########################

message("Begin ID conversion.")

if(length(grep("ID_convert_table.rds",dir(refdir)))>0){
idconvert<-readRDS(paste(refdir,"ID_convert_table.rds",sep=""))
}else{
 stop("Cannot find ID_convert_table.rds in the working folder. Exit!", call.=FALSE)
}

if(opt$type_gene_id=="EnsemblGID"){
gene_entrez<-idconvert$EntrezID[match(rownames(logexpr),idconvert$EnsemblID)]
if(length(which(is.na(gene_entrez)))==nrow(logexpr)){
 stop("Cannot convert Ensembl gene ID to Entrez gene ID. Exit!", call.=FALSE)
}else{
logexpr1<-logexpr[!gene_entrez=="",]
gene_entrez1<-gene_entrez[!gene_entrez==""]
logexpr.entrez<-apply(logexpr1,2,function(x){unlist(tapply(x,as.factor(gene_entrez1),mean))})
}
}

if(opt$type_gene_id=="GeneSymbol"){
gene_entrez<-idconvert$EntrezID[match(rownames(logexpr),idconvert$GeneSymbol)]
if(length(which(is.na(gene_entrez)))==nrow(logexpr)){
 stop("Cannot convert Ensembl gene ID to Entrez gene ID. Exit!", call.=FALSE)
}else{
logexpr1<-logexpr[!gene_entrez=="",]
gene_entrez1<-gene_entrez[!gene_entrez==""]
logexpr.entrez<-apply(logexpr1,2,function(x){unlist(tapply(x,as.factor(gene_entrez1),mean))})
}
}

if(opt$type_gene_id=="EntrezID"){
logexpr.entrez<-logexpr
}
message("Finish ID conversion.")

######################
######## GSEA ########
######################

groupn<-grep("group",colnames(sample_group))

c5sigall<-c()
c2sigall<-c()
for ( i in groupn){
compgroup<-combn(unique(sample_group[,i]), 2)
for ( j in 1:ncol(compgroup)){
nam<-paste(compgroup[1,j],"vs",compgroup[2,j],sep="")
versus<-paste(compgroup[1,j],"vs",compgroup[2,j],sep=" ")
groupA<-logexpr.entrez[,as.character(sample_group$sample[sample_group[,i] %in% compgroup[1,j]])]
groupB<-logexpr.entrez[,as.character(sample_group$sample[sample_group[,i] %in% compgroup[2,j]])]

logfc<-rowMeans(groupA)-rowMeans(groupB)
logfc<-logfc[order(-logfc)]

#GSEA in GO term
fgseaRes.c5 <- fgsea(Hs.c5, logfc, minSize=15, maxSize = 500, nperm=1000)
c5sig<-fgseaRes.c5[fgseaRes.c5$padj<opt$padjvalueCutoff,]

if(nrow(c5sig)==0){
message(paste("No significant GO term is identified in group ",nam,".",sep=""))
}else{
message(paste(nrow(c5sig)," significant GO term(s) is(are) identified in group ",nam,".",sep=""))

c5sig<-c5sig[order(c5sig$pval),]
c5sig<-data.frame(c5sig)
c5sig$leadingEdge<-sapply(c5sig$leadingEdge,function(x){paste0(unlist(x),collapse=", ")})

c5sigall<-rbind(c5sigall,cbind(versus,c5sig))
}
#GSEA in curated gene sets

fgseaRes.c2 <- fgsea(Hs.c2, logfc, minSize=15, maxSize = 500, nperm=1000)
c2sig<-fgseaRes.c2[fgseaRes.c2$padj<opt$padjvalueCutoff,]
if(nrow(c2sig)==0){
message(paste("No significant curated gene sets is identified in group ",nam,".",sep=""))
}else{
message(paste(nrow(c2sig)," significant curated gene sets are identified in group ",nam,".",sep=""))

c2sig<-c2sig[order(c2sig$pval),]
c2sig<-data.frame(c2sig)
c2sig$leadingEdge<-sapply(c2sig$leadingEdge,function(x){paste0(unlist(x),collapse=", ")})
c2sigall<-rbind(c2sigall,cbind(versus,c2sig))
}
}
}

if(length(c5sigall)==0){
message("No significant GO term is identified.")
}else{
c5sigall$pval<-signif(c5sigall$pval,4)
c5sigall$padj<-signif(c5sigall$padj,4)
c5sigall$ES<-signif(c5sigall$ES,4)
c5sigall$NES<-signif(c5sigall$NES,4)
rownames(c5sigall)<-c(1:nrow(c5sigall))
write.csv(c5sigall,paste(out_dir,opt$project_code,"_gsea_go.csv",sep=""))
}

if(length(c2sigall)==0){
message("No significant GO term is identified.")
}else{
c2sigall$pval<-signif(c2sigall$pval,4)
c2sigall$padj<-signif(c2sigall$padj,4)
c2sigall$ES<-signif(c2sigall$ES,4)
c2sigall$NES<-signif(c2sigall$NES,4)
rownames(c2sigall)<-c(1:nrow(c2sigall))
write.csv(c2sigall,paste(out_dir,opt$project_code,"_gsea_curatedgenesets.csv",sep=""))
}