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zUMIs-mapping.R
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#!/usr/bin/env Rscript
suppressMessages(require(yaml))
suppressMessages(require(data.table))
options(datatable.fread.input.cmd.message=FALSE)
Sys.time()
y <- commandArgs(trailingOnly = T)
inp<-yaml::read_yaml(y)
additional_fq <- inp$reference$additional_files
samtools <- inp$samtools_exec
STAR_exec <- inp$STAR_exec
if(is.null(inp$mem_limit)){
inp$mem_limit <- 100
}else if(inp$mem_limit == 0){
inp$mem_limit <- 100
}
# collect filtered bam files ----------------------------------------------
tmpfolder <- paste(inp$out_dir,"/zUMIs_output/.tmpMerge/",sep="")
if(inp$which_Stage == "Filtering"){
filtered_bams <- list.files(path = tmpfolder, pattern=paste(inp$project,".*.filtered.tagged.bam$",sep=""),full.names=T)
#also merge the unmapped bam files:
sammerge_command <- paste(samtools,"cat -o",paste0(inp$out_dir,"/",inp$project,".filtered.tagged.unmapped.bam"),paste0(filtered_bams,collapse=" "))
}else{
filtered_bams <- paste0(inp$out_dir,"/",inp$project,".filtered.tagged.unmapped.bam") # for resuming from mapping state using the merged unmapped bam
}
# check if multiple STAR instances can be run -----------------------------
genome_size <- system(command = paste("du -sh",inp$reference$STAR_index,"| cut -f1"), intern = TRUE)
genome_size <- as.numeric(gsub(pattern = "G",replacement = "", x = genome_size))
if(is.na(genome_size)){
genome_size <- 25 #set average genome size if there was a problem detecting
}
num_star_instances <- floor(inp$mem_limit/genome_size)
if(num_star_instances < 1){
num_star_instances = 1 #set the number of STAR instances to 1 if it is 0
}
if(num_star_instances > inp$num_threads){
num_star_instances = inp$num_threads
}
# GTF file setup ----------------------------------------------------------
#in case of additional sequences, we need to create a custom GTF
if ( is.null(additional_fq[1]) | length(additional_fq)==0 ) {
gtf_to_use <- inp$reference$GTF_file
param_additional_fa <- NULL
system(paste0("cp ",gtf_to_use," ",inp$out_dir,"/",inp$project,".final_annot.gtf"))
}else{
for (i in additional_fq) {
system(paste(samtools,"faidx",i))
assign(paste("fai",i,sep="_"),data.table::fread(input = paste("cut -f1,2 ",i,".fai",sep=""),stringsAsFactors = F,data.table = F))
}
ref_df <- do.call("rbind", mget(ls(pattern = "fai_")))
user_gtf <- data.frame(
V1 = ref_df$V1,
V2 = "User",
V3 = "exon",
V4 = 1,
V5 = ref_df$V2,
V6 = ".",
V7 = "+",
V8 = ".",
V9 = paste('gene_id "',ref_df$V1,'"; transcript_id "',ref_df$V1,'"; exon_number "1"; gene_name "',ref_df$V1,'"; gene_biotype "User"; transcript_name "',ref_df$V1,'"; exon_id "',ref_df$V1,'"',sep = ""),
stringsAsFactors = F
)
write.table(user_gtf,file = paste(inp$out_dir,"/additional_sequence_annot.gtf",sep = ""),sep = "\t",quote = F,row.names = F,col.names = F)
system(command = paste("cat ",inp$reference$GTF_file," ",paste(inp$out_dir,"/additional_sequence_annot.gtf",sep = "")," > ",inp$out_dir,"/",inp$project,".final_annot.gtf",sep=""))
gtf_to_use <- paste(inp$out_dir,"/",inp$project,".final_annot.gtf",sep="")
param_additional_fa <- paste("--genomeFastaFiles",paste(inp$reference$additional_files,collapse = " "))
}
#inp$reference$GTF_file_final <- gtf_to_use
#yaml::write_yaml(inp,file = paste(inp$out_dir,"/",inp$project,".postmap.yaml",sep=""))
# Detect read length ------------------------------------------------------
#check the first 100 reads to detect the read length of the cDNA read
#filtered_bam <- paste(inp$out_dir,"/",inp$project,".filtered.tagged.bam",sep="")
cDNA_peek <- data.table::fread(cmd = paste(samtools,"view",filtered_bams[1],"| cut -f10 | head -n 1000"),stringsAsFactors = F,data.table = T, header = F)
getmode <- function(v) {
uniqv <- unique(v)
uniqv[which.max(tabulate(match(v, uniqv)))]
}
cDNA_read_length <- getmode(nchar(cDNA_peek$V1))
# Setup STAR mapping ------------------------------------------------------
samtools_load_cores <- ifelse(inp$num_threads>8,2,1)
avail_cores <- inp$num_threads - samtools_load_cores #reserve threads for samtools file opening
if(inp$which_Stage == "Filtering"){
avail_cores <- floor(avail_cores / num_star_instances)
}
if(avail_cores < 2){
avail_cores = 1
}
param_defaults <- paste("--readFilesCommand ",samtools," view -@",samtools_load_cores," --outSAMmultNmax 1 --outFilterMultimapNmax 50 --outSAMunmapped Within --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM")
param_misc <- paste("--genomeDir",inp$reference$STAR_index,
"--sjdbGTFfile",gtf_to_use,
"--runThreadN",avail_cores,
"--sjdbOverhang", cDNA_read_length-1,
"--readFilesType SAM",inp$read_layout)
STAR_command <- paste(STAR_exec,param_defaults,param_misc,inp$reference$additional_STAR_params,param_additional_fa)
if(inp$counting_opts$twoPass==TRUE){
STAR_command <- paste(STAR_command,"--twopassMode Basic")
}
#finally, run STAR
if(num_star_instances>1 & inp$which_Stage == "Filtering"){
map_tmp_dir <- paste0(inp$out_dir,"/zUMIs_output/.tmpMap/")
dir.create(path = map_tmp_dir,showWarnings = FALSE)
input_split <- split(filtered_bams, ceiling(seq_along(filtered_bams) / ceiling(length(filtered_bams) / num_star_instances)))
input_split <- sapply(input_split, paste0, collapse = ",")
STAR_preset <- STAR_command
STAR_command <- lapply(seq(num_star_instances), function(x){
paste(STAR_preset,
"--readFilesIn",input_split[x],
"--outFileNamePrefix",paste0(map_tmp_dir,"/tmp.",inp$project,".",x,"."))
})
STAR_command <- paste(unlist(STAR_command), collapse = " & ")
system(paste(STAR_command,"&",sammerge_command,"& wait"))
#after parallel instance STAR, collect output data in the usual file places
out_logs <- list.files(map_tmp_dir, pattern = paste0("tmp.",inp$project,".*.Log.final.out"), full = TRUE)
merge_logs <- paste("cat",paste(out_logs, collapse = " "),">",paste0(inp$out_dir,"/",inp$project,".filtered.tagged.Log.final.out"))
out_bams <- list.files(map_tmp_dir, pattern = paste0("tmp.",inp$project,".*.Aligned.out.bam"), full = TRUE)
merge_bams <- paste(inp$samtools_exec,"cat -o",paste0(inp$out_dir,"/",inp$project,".filtered.tagged.Aligned.out.bam"),paste(out_bams, collapse = " "))
out_txbams <- list.files(map_tmp_dir, pattern = paste0("tmp.",inp$project,".*.Aligned.toTranscriptome.out.bam"), full = TRUE)
merge_txbams <- paste(inp$samtools_exec,"cat -o",paste0(inp$out_dir,"/",inp$project,".filtered.tagged.Aligned.toTranscriptome.out.bam"),paste(out_txbams, collapse = " "))
system(paste(merge_logs,"&",merge_bams,"&",merge_txbams,"& wait"))
system(paste0("rm -r ", map_tmp_dir, "tmp.", inp$project, ".*"))
}else{
STAR_command <- paste(STAR_command,
"--readFilesIn",paste0(filtered_bams,collapse=","),
"--outFileNamePrefix",paste(inp$out_dir,"/",inp$project,".filtered.tagged.",sep="")
)
if(inp$which_Stage == "Filtering"){
system(paste(STAR_command,"&",sammerge_command,"& wait"))
}else{
system(STAR_command)
}
}
#clean up chunked bam files
if(inp$which_Stage == "Filtering"){
system(paste0("rm ",tmpfolder,"/",inp$project,".*"))
}
q()