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zUMIs-dge2.R
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zUMIs-dge2.R
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#!/usr/bin/env Rscript
library(methods)
library(data.table)
library(yaml)
library(ggplot2)
suppressPackageStartupMessages(library(Rsamtools))
##########################
myYaml <- commandArgs(trailingOnly = T)
opt <-read_yaml(myYaml)
setwd(opt$out_dir)
#try(unixtools::set.tempdir(opt$out_dir))
source(paste0(opt$zUMIs_directory,"/runfeatureCountFUN.R"))
source(paste0(opt$zUMIs_directory,"/misc/featureCounts.R"))
source(paste0(opt$zUMIs_directory,"/UMIstuffFUN.R"))
source(paste0(opt$zUMIs_directory,"/barcodeIDFUN.R"))
options(datatable.fread.input.cmd.message=FALSE)
print(Sys.time())
samtoolsexc <- opt$samtools_exec
data.table::setDTthreads(threads=1)
#Check the version of Rsubread
#checkRsubreadVersion()
fcounts_clib <- paste0(opt$zUMIs_directory,"/misc/fcountsLib2")
opt <- fixMissingOptions(opt)
#######################################################################
########################## double check for non-UMI method
UMIcheck <- check_nonUMIcollapse(opt$sequence_files)
if(UMIcheck == "nonUMI"){
opt$counting_opts$Ham_Dist <- 0
}
#is the data Smart-seq3?
smart3_flag <- ifelse(any(grepl(pattern = "ATTGCGCAATG",x = unlist(opt$sequence_files))), TRUE, FALSE)
#######################################################################
##### Barcode handling & chunking
#read file with barcodecounts
#check if binning of adjacent barcodes should be run
if(opt$barcodes$BarcodeBinning > 0){
bccount <- fread(paste0(opt$out_dir,"/zUMIs_output/",opt$project,"kept_barcodes_binned.txt"))
}else{
bccount <- fread(paste0(opt$out_dir,"/zUMIs_output/",opt$project,"kept_barcodes.txt"))
}
bccount<-splitRG(bccount=bccount, mem= opt$mem_limit, hamdist = opt$counting_opts$Ham_Dist)
##############################################################
##### featureCounts
abamfile<-paste0(opt$out_dir,"/",opt$project,".filtered.tagged.Aligned.out.bam")
outbamfile <-paste0(opt$out_dir,"/",opt$project,".filtered.Aligned.GeneTagged.bam")
## gene annotation
saf<-.makeSAF(gtf = paste0(opt$out_dir,"/",opt$project,".final_annot.gtf"),
extension_var = opt$reference$exon_extension,
exon_extension = opt$reference$extension_length,
buffer_length = (opt$reference$extension_length / 2),
scaff_length = opt$reference$scaffold_length_min,
multi_overlap_var = opt$counting_opts$multi_overlap,
samtoolsexc = samtoolsexc)
try(gene_name_mapping <- .get_gene_names(gtf = paste0(opt$out_dir,"/",opt$project,".final_annot.gtf"), threads = opt$num_threads), silent = TRUE)
try(data.table::fwrite(gene_name_mapping, file = paste0(opt$out_dir,"/zUMIs_output/expression/",opt$project,".gene_names.txt"), sep ="\t", quote = FALSE), silent = TRUE)
##
if(smart3_flag & opt$counting_opts$strand == 1){
#split bam in UMU ends and internal
print("Preparing Smart-seq3 data for stranded gene assignment...")
print(Sys.time())
tmp_bams <- split_bam(bam = abamfile, cpu = opt$num_threads, samtoolsexc=samtoolsexc)
#assign features with appropriate strand
fnex_int<-.runFeatureCount(tmp_bams[1], saf=saf$exons, strand=0, type="ex", primaryOnly = opt$counting_opts$primaryHit, cpu = opt$num_threads, mem = opt$mem_limit, fcounts_clib = fcounts_clib, multi_overlap_var = opt$counting_opts$multi_overlap, fraction_overlap = opt$counting_opts$fraction_overlap)
fnex_umi<-.runFeatureCount(tmp_bams[2], saf=saf$exons, strand=1, type="ex", primaryOnly = opt$counting_opts$primaryHit, cpu = opt$num_threads, mem = opt$mem_limit, fcounts_clib = fcounts_clib, multi_overlap_var = opt$counting_opts$multi_overlap, fraction_overlap = opt$counting_opts$fraction_overlap)
ffiles_int <- paste0(fnex_int,".tmp")
ffiles_umi <- paste0(fnex_umi,".tmp")
if(opt$counting_opts$introns){
fnin_int<-.runFeatureCount(ffiles_int, saf=saf$introns, strand=0, type="in", primaryOnly = opt$counting_opts$primaryHit, cpu = opt$num_threads, mem = opt$mem_limit, fcounts_clib = fcounts_clib, multi_overlap_var = opt$counting_opts$multi_overlap, fraction_overlap = opt$counting_opts$fraction_overlap)
fnin_umi<-.runFeatureCount(ffiles_umi, saf=saf$introns, strand=1, type="in", primaryOnly = opt$counting_opts$primaryHit, cpu = opt$num_threads, mem = opt$mem_limit, fcounts_clib = fcounts_clib, multi_overlap_var = opt$counting_opts$multi_overlap, fraction_overlap = opt$counting_opts$fraction_overlap)
ffiles_int <- paste0(fnin_int,".tmp")
ffiles_umi <- paste0(fnin_umi,".tmp")
}
join_bam_cmd <- paste(samtoolsexc, "cat -o", outbamfile, ffiles_int, ffiles_umi)
system(join_bam_cmd)
system(paste0("rm ",tmp_bams[1],"* ",tmp_bams[2],"*"))
}else{
fnex<-.runFeatureCount(abamfile,
saf=saf$exons,
strand=opt$counting_opts$strand,
type="ex",
primaryOnly = opt$counting_opts$primaryHit,
cpu = opt$num_threads,
mem = opt$mem_limit,
fcounts_clib = fcounts_clib,
multi_overlap_var = opt$counting_opts$multi_overlap,
fraction_overlap = opt$counting_opts$fraction_overlap)
ffiles<-paste0(fnex,".tmp")
if(opt$counting_opts$introns){
fnin <-.runFeatureCount(ffiles,
saf=saf$introns,
strand=opt$counting_opts$strand,
type="in",
primaryOnly = opt$counting_opts$primaryHit,
cpu = opt$num_threads,
mem = opt$mem_limit,
fcounts_clib = fcounts_clib,
multi_overlap_var = opt$counting_opts$multi_overlap,
fraction_overlap = opt$counting_opts$fraction_overlap)
system(paste0("rm ",fnex,".tmp"))
ffiles<-paste0(fnin,".tmp")
}
system(paste("mv",ffiles,outbamfile))
}
if(is.null(opt$mem_limit)){
mempercpu <- max(round(100/opt$num_threads,0),1)
}else{
mempercpu <- max(round(opt$mem_limit/opt$num_threads,0),1)
}
if(opt$counting_opts$Ham_Dist == 0){
sortbamfile <-paste0(opt$out_dir,"/",opt$project,".filtered.Aligned.GeneTagged.sorted.bam")
print(Sys.time())
print("Coordinate sorting final bam file...")
sort_cmd <- paste0(samtoolsexc," sort -O 'BAM' -@ ",opt$num_threads," -m ",mempercpu,"G -o ",sortbamfile," ",outbamfile)
system(sort_cmd)
system(paste0("rm ",outbamfile))
}else{
#run hamming distance collapsing here and write output into bam file
if(!dir.exists( paste0(opt$out_dir,"/zUMIs_output/molecule_mapping/") )){
dir.create( paste0(opt$out_dir,"/zUMIs_output/molecule_mapping/") )
}
tmpbamfile <- outbamfile
outbamfile <- paste0(opt$out_dir,"/",opt$project,".filtered.Aligned.GeneTagged.sorted.bam")
print(Sys.time())
print("Coordinate sorting intermediate bam file...")
sort_cmd <- paste0(samtoolsexc," sort -O 'BAM' -@ ",opt$num_threads," -m ",mempercpu,"G -o ",outbamfile," ",tmpbamfile)
system(sort_cmd)
index_cmd <- paste(samtoolsexc,"index -@",opt$num_threads,outbamfile)
system(index_cmd)
system(paste0("rm ",tmpbamfile))
print(Sys.time())
#check if PE / SE flag is set correctly
if(is.null(opt$read_layout)){
opt$read_layout <- check_read_layout(outbamfile)
}
for(i in unique(bccount$chunkID)){
print( paste( "Hamming distance collapse in barcode chunk", i, "out of",length(unique(bccount$chunkID)) ))
reads <- reads2genes_new(featfile = outbamfile,
bccount = bccount,
inex = opt$counting_opts$introns,
chunk = i,
cores = opt$num_threads)
reads <- reads[!UB==""] #make sure only UMI-containing reads go further
u <- umiCollapseHam(reads,bccount, HamDist=opt$counting_opts$Ham_Dist)
}
#print("Demultiplexing output bam file by cell barcode...")
#demultiplex_bam(opt, outbamfile, nBCs = length(unique(bccount$XC)), bccount = bccount, samtoolsexc = samtoolsexc)
print("Correcting UMI barcode tags...")
sortbamfile <- correct_UB_tags_new(outbamfile, opt$project)
file.remove(outbamfile)
#sortbamfile <- correct_UB_tags(bccount, samtoolsexc)
#sortbamfile <-paste0(opt$out_dir,"/",opt$project,".filtered.Aligned.GeneTagged.UBcorrected.sorted.bam")
bccount<-splitRG(bccount=bccount, mem= opt$mem_limit, hamdist = 0) # allow more reads to be in RAM fur subsequent steps
}
index_cmd <- paste(samtoolsexc,"index -@",opt$num_threads,sortbamfile)
system(index_cmd)
print(Sys.time())
#check if PE / SE flag is set correctly
if(is.null(opt$read_layout)){
opt$read_layout <- check_read_layout(sortbamfile)
}
##########################################
#set Downsampling ranges
data.table::setDTthreads(threads=opt$num_threads)
subS<-setDownSamplingOption( opt$counting_opts$downsampling,
bccount= bccount,
filename=paste(opt$out_dir,"/zUMIs_output/stats/",opt$project,
".downsampling_thresholds.pdf",sep=""))
print("Here are the detected subsampling options:")
if(is.null(row.names(subS))){
print("Automatic downsampling")
}else{
print(row.names(subS))
}
if( opt$counting_opts$introns ){
mapList<-list("exon"="exon",
"inex"=c("intron","exon"),
"intron"="intron")
}else{
mapList<-list("exon"="exon")
}
########################## assign reads to UB & GENE
for(i in unique(bccount$chunkID)){
print( paste( "Working on barcode chunk", i, "out of",length(unique(bccount$chunkID)) ))
print( paste( "Processing",length(bccount[chunkID==i]$XC), "barcodes in this chunk..." ))
reads <- reads2genes_new(featfile = sortbamfile,
bccount = bccount,
inex = opt$counting_opts$introns,
chunk = i,
cores = opt$num_threads)
tmp<-collectCounts( reads =reads,
bccount=bccount[chunkID==i],
subsample.splits=subS[which(max(bccount[chunkID==i]$n) >= subS[,1]), , drop = FALSE],
mapList=mapList
)
if(i==1){
allC<-tmp
}else{
allC<-bindList(alldt=allC,newdt=tmp)
}
}
if( UMIcheck == "UMI" ){
if(smart3_flag){
final<-list( umicount = convert2countM(alldt=allC,what="umicount"),
readcount = convert2countM(allC,"readcount"),
readcount_internal = convert2countM(allC,"readcount_internal"))
}else{
final<-list( umicount = convert2countM(alldt=allC,what="umicount"),
readcount = convert2countM(allC,"readcount"))
}
}else{
final<-list(readcount = convert2countM(allC,"readcount"))
}
#Make RPKM if necessary
if(UMIcheck == "nonUMI" | smart3_flag == TRUE ){
tx_len <- .get_tx_lengths( paste0(opt$out_dir,"/",opt$project,".final_annot.gtf") )
rpkms <- if(smart3_flag){
RPKM.calc(final$readcount_internal$exon$all, tx_len)
}else{
RPKM.calc(final$readcount$exon$all, tx_len)
}
final$rpkm <- list(exon = list(all = rpkms))
}
saveRDS(final,file=paste(opt$out_dir,"/zUMIs_output/expression/",opt$project,".dgecounts.rds",sep=""))
################# #Accessory processing scripts
#Intron Probability Score Calculation
if(opt$counting_opts$intronProb == TRUE){
if(opt$counting_opts$introns == FALSE){
print("Intron information is needed to calculate Intron-Probability! Please change yaml settings counting_opts->introns to yes.\n Skipping probability calculation...")
}else{
library(extraDistr)
print("Starting Intron-Probability Calculations...")
# Extractingreads from sam files again, this could be sped up by integrating code further upstream of zUMIs-dge2
genesWithIntronProb<-.intronProbability(bccount=bccount,
featfile=sortbamfile,
inex=opt$counting_opts$introns,
cores=opt$num_threads,
samtoolsexc=samtoolsexc,
allC=allC,
saf=saf)
saveRDS(genesWithIntronProb, file = paste0(opt$out_dir,"/zUMIs_output/stats/",opt$project,".intronProbability.rds"))
}
}
#demultiplexing
if(opt$barcodes$demultiplex){
print("Demultiplexing output bam file by cell barcode...")
demultiplex_bam(opt, sortbamfile, nBCs = length(unique(bccount$XC)), bccount = bccount, samtoolsexc = samtoolsexc)
}
#################
print(Sys.time())
print(paste("I am done!! Look what I produced...",opt$out_dir,"/zUMIs_output/",sep=""))
print(gc())
q()