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chipseq.bds
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chipseq.bds
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#!/usr/bin/env bds
#vim: syntax=java
help == chipseq pipeline settings
// general
type := "TF" help Type of ChIP-Seq pipeline. TF or histone (default: TF).
histone := false help (LEGACY PARAM) Histone ChIP-Seq. Equivalent to '-type histone'
final_stage := "" help Final stage for pipeline (bam, filt_bam, tag, xcor and peak).
// signal track generation
sig_trk_for_pooled_rep_only := false help Generate MACS2 signal tracks for pooled replicate only.
// mapping/filtering/aligning
aligner := "bwa" help Aligner to map raw reads in FASTQs (default: bwa).
subsample_xcor := "15M" help # reads to be subsampled for cross corr. analysis (default: 15M).
subsample_chip := "0" help # reads to subsample exp. replicate. Subsampled tagalign will be used for steps downstream (default: 0; no subsampling).
subsample := "0" help (LEGACY PARAM) # reads to subsample exp. replicate. Subsampled tagalign will be used for steps downstream (default: 0; no subsampling).
subsample_ctl := "0" help # reads to subsample control if non-zero (recommended: 40M or lower).
anon_filt_bam := false help Generate an annomymized filtered bam. This will not affect tasks downsteam.
// peak calling
peak_caller := "spp" help Peak caller for IDR analysis (default: spp for TF ChIP-seq and macs2 for Histone ChIP-seq ).
ctl_depth_ratio := 1.2 help Cut-off ratio of two control tagaligns for pooling (default: 1.2).
fraglen := 0 help (LEGACY PARAM) Manually specify fragment length (default: 0, 0 means parsing fraglen from cross-corr analysis log file).
idr_rank := "" help Scoring column in narrow peak files for IDR. If not specified, signal.value for SPP peaks (TF) and p.value for MACS2 peaks (histone) are used.
idr_thresh := 0.05 help IDR threshold : -log_10(score) (default: 0.05).
use_pooled_ctl := false help Force to use pooled control (ignoring criteria to choose control for each replicate).
true_rep := false help Call peaks on true replicates only.
no_pseudo_rep := false help Do not call peaks on self pseudo replicates.
no_xcor := false help Disable cross-correlation analysis.
no_gpeak_filt := false help Disable gapped peak filtering through narrow peak (for histone ChIP-Seq only).
no_browser_tracks := false help Disable generation of genome browser tracks (workaround for bzip2 shared library issue).
pe_xcor_only := false help (PE ONLY) Align R1 of paired end fastqs only and for cross-correlation analysis. All other analyses and QCs will be disabled.
pe_no_trim_fastq := false help (PE ONLY) No fastq trimming and use PE tagAlign for cross-correlation analysis.
no_jsd := false help Disable JSD plot generation
disable_gb_peak := true // disable gapped/broad peak generation
help() // print help and exit if no parameters are given
include "modules/pipeline_template.bds"
include "modules/input.bds"
include "modules/align_bwa.bds"
include "modules/align_trim_fastq.bds"
include "modules/postalign_bam.bds"
include "modules/postalign_bed.bds"
include "modules/postalign_xcor.bds"
include "modules/callpeak_spp.bds"
include "modules/callpeak_gem.bds"
include "modules/callpeak_peakseq.bds"
include "modules/callpeak_macs2_chipseq.bds"
include "modules/callpeak_naive_overlap.bds"
include "modules/callpeak_idr.bds"
include "modules/callpeak_blacklist_filter.bds"
//include "modules/signal.bds"
// Important file names are stored in global variables (usually a string map string{} with a key with replicate id and peakcaller name)
// e.g. filt_bam{"Replicate 1"} = filtered bam for replicate 1, peak_pr1{"spp,2"} = peak file for pseudo replicate 1 of replicate 2 generated from spp
string{} flagstat_qc, dup_qc, flagstat_nodup_qc, pbc_qc, xcor_qc, xcor_plot, jsd_qc
string{} bam, filt_bam
string jsd_png
string{} tag, tag_pr1, tag_pr2
string tag_ppr1, tag_ppr2
string{} peak, peak_pr1, peak_pr2, peak_pooled, peak_ppr1, peak_ppr2
string{} gpeak, gpeak_pr1, gpeak_pr2, gpeak_pooled, gpeak_ppr1, gpeak_ppr2
string{} bpeak, bpeak_pr1, bpeak_pr2, bpeak_pooled, bpeak_ppr1, bpeak_ppr2
// string peak_overlap, gpeak_overlap, bpeak_overlap
string{} raw_peak_qc_FRiP
string{} idr_tr, idr_pr, idr_tr_png, idr_pr_png
string idr_ppr, idr_opt, idr_consv, idr_ppr_png
string idr_qc
string{} idr_qc_FRiP
string{} peak_overlap_tr, peak_overlap_pr
string peak_overlap_ppr, peak_overlap_opt, peak_overlap_consv
string peak_overlap_qc
string{} peak_overlap_qc_FRiP
string{} sig_trk // signal tracks from deepTools or bamCoverage: map with key ("$ctl,$rep")
string{} pval_bigwig, fc_bigwig // signal track from macs2
string num_peak_log
main()
void main() { // chipseq pipeline starts here
init_chipseq() // read command line parameters or configruation file
chk_param() // check if parameters are valid
chk_input( true_rep, no_pseudo_rep ) // if inputs are fastq, bam, or tagalign, check if they exist
align() // align and postalign
jsd() // plot fingerprint and compute synthetic JS distance
if ( !pe_xcor_only ) {
pool_tags() // make pooled tagaligns and choose appropriate control for each replicate
call_peaks() // call peaks in parallel (MACS2,SPP)
read_input_peak() // if inputs are peaks, read them
naive_overlap() // get naive overlap peaks
log_number_of_peaks()
do_idr() // IDR
filter_peak() // blacklist-filter peaks
}
report()
}
void init_chipseq() {
read_conf_chipseq()
init_filetable()
}
void read_conf_chipseq() {
type = get_conf_val( type, ["type"] )
histone = get_conf_val_bool( histone, ["histone"] )
if ( histone ) type = "histone" // backward compatibility for LEGACY param
if ( type == "histone" && !cmd_line_arg_has_key("peak_caller") ) peak_caller = "macs2"
final_stage = get_conf_val( final_stage, ["final_stage"] )
sig_trk_for_pooled_rep_only = get_conf_val_bool( sig_trk_for_pooled_rep_only, ["sig_trk_for_pooled_rep_only"] )
aligner = get_conf_val( aligner, ["aligner"] )
subsample_xcor = get_conf_val( subsample_xcor, ["subsample_xcor"] )
subsample_chip = get_conf_val( subsample_chip, ["subsample_chip"] )
subsample = get_conf_val( subsample, ["subsample"] )
subsample_ctl = get_conf_val( subsample_ctl, ["subsample_ctl"] )
anon_filt_bam = get_conf_val_bool( anon_filt_bam, ["anon_filt_bam"] )
peak_caller = get_conf_val( peak_caller, ["peak_caller"] )
ctl_depth_ratio = get_conf_val_real( ctl_depth_ratio, ["ctl_depth_ratio"])
fraglen = get_conf_val_int( fraglen, ["fraglen"] )
idr_rank = get_conf_val( idr_rank, ["idr_rank"] )
idr_thresh = get_conf_val_real( idr_thresh, ["idr_thresh"] )
use_pooled_ctl = get_conf_val_bool( use_pooled_ctl, ["use_pooled_ctl"] )
true_rep = get_conf_val_bool( true_rep, ["true_rep"] )
no_pseudo_rep = get_conf_val_bool( no_pseudo_rep, ["no_pseudo_rep"] )
no_xcor = get_conf_val_bool( no_xcor, ["no_xcor"] )
no_gpeak_filt = get_conf_val_bool( no_gpeak_filt, ["no_gpeak_filt"] )
no_jsd = get_conf_val_bool( no_jsd, ["no_jsd"] )
no_browser_tracks = get_conf_val_bool( no_browser_tracks, ["no_browser_tracks"] )
pe_xcor_only = get_conf_val_bool( pe_xcor_only, ["pe_xcor_only"] )
pe_no_trim_fastq = get_conf_val_bool( pe_no_trim_fastq, ["pe_no_trim_fastq"] )
check_peak_caller_valid()
check_aligner_valid()
if ( pe_xcor_only ) final_stage = "xcor"
if ( idr_rank == "" ) {
if ( peak_caller == "spp" || peak_caller == "gem" ) {
idr_rank = "signal.value"
}
else if ( peak_caller == "macs2" ) {
idr_rank = "p.value"
}
else if ( peak_caller == "peakseq" ) {
idr_rank = "q.value"
}
else error("Unsupported peak_caller ($peak_caller)!")
idr_rank = (peak_caller == "spp" ? "signal.value" : "p.value")
}
// for backward compatibility for legacy params
if ( subsample_chip == "0" && subsample != "0" ) subsample_chip = subsample
if ( extsize_macs2 == -1 && fraglen > 0 ) extsize_macs2 = fraglen
if ( speak_spp == -1 && fraglen > 0 ) speak_spp = fraglen
if ( is_final_stage_after_xcor() && no_xcor ) {
if ( speak_spp == -1 ) error("For the case of no cross-corr. analysis (-no_xcor). You need to specify -speak_spp.")
if ( extsize_macs2 == -1 ) error("For the case of no cross-corr. analysis (-no_xcor). You need to specify -extsize_macs2.")
}
peak_caller = peak_caller.toLower()
print( "\n\n== chipseq pipeline settings\n")
print( "# replicates \t\t\t\t: "+get_num_rep()+"\n" )
print( "Type of ChIP-Seq pipeline\t\t: $type\n")
print( "Final stage for ChIP-Seq\t\t: $final_stage\n" )
print( "Signal tracks for pooled rep. only\t: $sig_trk_for_pooled_rep_only\n")
print( "Aligner to map raw reads\t\t\t: $aligner\n")
print( "# reads to subsample for cross-corr. analysis\t\t: "+parse_number( subsample_xcor)+"\n")
print( "# reads to subsample exp. replicates (0: no subsampling): "+parse_number( subsample_chip )+"\n")
print( "# reads to subsample controls (0: no subsampling)\t: "+parse_number( subsample_ctl)+"\n")
print( "Generate anonymized filt. bam\t\t: $anon_filt_bam\n")
print( "Peak caller for IDR analysis\t\t: $peak_caller\n")
print( "Control rep. depth ratio\t\t: $ctl_depth_ratio\n" )
// print( "Specified fraglen (0:auto)\t\t: $fraglen\n")
print( "Scoring column for IDR\t\t\t: $idr_rank\n")
print( "IDR threshold\t\t\t\t: $idr_thresh\n" )
print( "Force to use pooled ctl\t\t\t: $use_pooled_ctl\n")
print( "Peak calling for true reps only\t\t: $true_rep\n" )
print( "No peak calling for self pseudo reps\t: $no_pseudo_rep\n" )
print( "Disable cross-correlation analysis\t: $no_xcor\n" )
print( "Disable g. peak filt. thru. n. peak\t: $no_gpeak_filt\n" )
print( "Disable genome browser tracks\t: $no_browser_tracks\n" )
print( "Disable all QCs except cross-corr. (PE only)\t: $pe_xcor_only\n" )
print( "No fastq trimming and use PE tagAlign for cross-corr. (PE only)\t: $pe_no_trim_fastq\n" )
print( "Disable JSD plot generation\t\t: $no_jsd\n" )
}
void init_filetable() { // init file table labels in HTML report
// add label to graphviz
// : Items in filetable will be sorted in the ascending order of rank
// : Items added later will have higher rank
// Level 1
add_label_to_table("Raw reads")
add_label_to_table("Alignment")
add_label_to_table("Signal tracks")
add_label_to_table("Peaks")
add_label_to_table("QC and logs")
// Level 2
for (int i=1; i<=100; i++) \
add_label_to_table("Replicate $i")
for (int i=1; i<=100; i++) \
add_label_to_table("Control $i")
add_label_to_table("True replicates")
add_label_to_table("Pooled replicate")
add_label_to_table("Pooled control")
add_label_to_table("Pseudo-replicates")
add_label_to_table("Pooled pseudo-replicate")
add_label_to_table("Pooled pseudo-replicates")
add_label_to_table("Optimal set")
add_label_to_table("Conservative set")
add_label_to_table("Naive overlap")
add_label_to_table("SPP")
add_label_to_table("MACS2")
add_label_to_table("IDR")
// Level 2 or 3
add_label_to_table("Pseudo-replicate 1")
add_label_to_table("Pseudo-replicate 2")
add_label_to_table("Pooled pseudo-replicate 1")
add_label_to_table("Pooled pseudo-replicate 2")
for (int i=1; i<=20; i++) \
for (int j=i+1; j<=20; j++) \
add_label_to_table("Rep. $i vs Rep. $j")
// Higher levels
add_label_to_table("IDR QC")
add_label_to_table("Fastq")
add_label_to_table("Fastq 1")
add_label_to_table("Fastq 2")
add_label_to_table("BWA map. flagstat log")
add_label_to_table("Bam")
add_label_to_table("Filtered bam")
add_label_to_table("Sorted bam")
add_label_to_table("Dedup. log")
add_label_to_table("Map. flagstat log")
add_label_to_table("PBC log")
add_label_to_table("Bedpe")
add_label_to_table("Subsampled bedpe")
add_label_to_table("Tag-align")
add_label_to_table("Subsampled tag-align")
add_label_to_table("Cross-corr. log")
add_label_to_table("Cross-corr. plot")
add_label_to_table("P-value")
add_label_to_table("Fold enrichment")
add_label_to_table("Narrow peak")
add_label_to_table("Region peak")
add_label_to_table("Broad peak")
add_label_to_table("Gapped peak")
add_label_to_table("IDR peak")
add_label_to_table("Peak")
add_label_to_table("Filtered peak")
add_label_to_table("Filtered gapped peak")
add_label_to_table("Filtered broad peak")
add_label_to_table("IDR plot")
add_label_to_table("Unthresholded IDR peak")
// add label to graphviz (short name, long name)
for (int i=1; i<=50; i++) {
add_label_to_graph("rep$i", "Replicate $i")
add_label_to_graph("rep$i-pr1", "Pseudo-replicate 1 for rep. $i")
add_label_to_graph("rep$i-pr2", "Pseudo-replicate 2 for rep. $i")
add_label_to_graph("rep$i-pr", "Pseudo replicates for rep. $i")
for (int j=1; j<=20; j++) {
add_label_to_graph("rep$i-rep$j", "Rep. $i vs. Rep. $j")
}
}
for (int i=1; i<=100; i++) {
add_label_to_graph("ctl$i", "Control $i")
}
add_label_to_graph("pooled_rep", "Pooled replicate")
add_label_to_graph("pooled_ctl", "Pooled control")
add_label_to_graph("ppr", "Pooled pseudo-replicates")
add_label_to_graph("ppr1", "Pooled pseudo-replicate 1")
add_label_to_graph("ppr2", "Pooled pseudo-replicate 2")
}
void chk_param() {
print( "\n\n== checking chipseq parameters ...\n" )
if ( pe_xcor_only && !is_input_fastq(0,1) ) {
error("-pe_xcor_only is for fastqs only!\n")
}
if ( has_input_fastq() ) chk_align_bwa()
if ( is_final_stage_idr() ) chk_idr()
if ( is_final_stage_idr() || is_final_stage_peak() ) chk_callpeak_macs2()
// errors
if ( peak_caller == "gem" ) chk_callpeak_gem()
if ( peak_caller == "peakseq" ) chk_callpeak_peakseq()
if ( !is_input_peak() && peak_caller == "spp" && !ctl_exists() && !is_final_stage_before_peak() ) \
error("Cannot call peaks (SPP) without controls!\n")
if ( multimapping > 0 ) \
error("Multimapping is not available for chipseq pipeline!\n")
if ( is_input_peak() && true_rep && get_num_rep() == 1 && is_TF_chipseq() ) \
error("Cannot perform IDR on with only one replicate when '-true_rep' is turned on!")
// if ( is_histone_chipseq() && sig_trk_for_pooled_rep_only ) \
// sig_trk_for_pooled_rep_only = false
if ( has_pe_input_tag() && parse_number( subsample_xcor ) > 0 ) \
print("Warning: PE tagaligns cannot be subsampled for cross-corr. analysis.\n")
if ( has_pe_input_tag() && parse_number( subsample_chip ) > 0 ) \
print("Warning: PE tagaligns cannot be subsampled!\n")
if ( has_pe_input_tag(1) && parse_number( subsample_ctl ) > 0 ) \
error("PE contol tagaligns cannot be subsampled!\n")
// if ( sig_trk_for_pooled_rep_only && macs2_for_idr ) \
// error("-sig_trk_for_pooled_rep_only and -macs2_for_idr are mutally exclusive parameters!\n")
}
void align() {
if ( is_input_peak() ) return
// distribute # of threads to each replicate/control according to their file sizes
// different weight for each data type 3 for fastq, 5 for tagaligns, 1 for others
int{} filesize
for ( int ctl=0; ctl <= 1; ctl++) { // iterate through inputs (ctl==0 : exp. replicate, ctl==1 : control)
if ( ctl==1 && !ctl_exists() ) continue
for ( int rep=1; rep <= get_num_rep( ctl ); rep++) {
if ( !input_file_exists( ctl, rep ) ) continue
// check file size to distribute_nonzero nth to each nth_app
// determine # threads for each app related to alignment
group := get_group_name( ctl, rep )
// get file size in bytes
if ( is_input_fastq( ctl, rep ) ) {
fastqs := get_fastqs( ctl, rep )
filesize{group} = (fastqs[0]).size()*3 // multiply 3 (weight) to allocate more cpus for align
if ( fastqs.size() > 1) filesize{group} += (fastqs[1]).size()
}
else if ( is_input_bam( ctl, rep ) ) filesize{group} = (get_bam( ctl, rep )).size()
else if ( is_input_filt_bam( ctl, rep ) ) filesize{group} = (get_filt_bam( ctl, rep )).size()
else if ( is_input_tag( ctl, rep ) ) filesize{group} = (get_tag( ctl, rep )).size()*5
}
}
nth_rep := distribute_nonzero( nth, filesize )
// align
for ( int ctl=0; ctl <= 1; ctl++) { // iterate through inputs (ctl==0 : exp. replicate, ctl==1 : control)
if ( ctl==1 && !ctl_exists() ) continue
for ( int rep=1; rep <= get_num_rep( ctl ); rep++) {
if ( !input_file_exists( ctl, rep ) ) continue
group := get_group_name( ctl, rep )
if ( no_par ) align( ctl, rep, nth_rep{group} ) // parallel jobs for align() for each replicate and each control
else par align( ctl, rep, nth_rep{group} ) // parallel jobs for align() for each replicate and each control
}
}
wait
print( "\n== Done align()\n" )
}
void jsd() { // plot fingerprint
if ( no_jsd ) return
if ( filt_bam.hasKey("ctl1") && filt_bam.hasKey("rep1") ) {
nth_sam_idx := min(2,nth)
string[] filt_bams, labels
for ( int rep=1; rep <= get_num_rep( 0 ); rep++) { // IP replicate
group := get_group_name( 0, rep )
filt_bam_ := filt_bam{group}
filt_bams.add(filt_bam_)
labels.add(group)
samtools_index(filt_bam_, group, nth_sam_idx)
}
group_ctl1 := get_group_name( 1, 1 ) // 1st control replicate
ctl_filt_bam1 := filt_bam{group_ctl1}
labels.add(group_ctl1)
samtools_index(ctl_filt_bam1, group_ctl1, nth_sam_idx)
wait
nth_jsd := min(4,nth)
string jsd_qc_all_reps
(jsd_png, jsd_qc_all_reps) = \
jsd(filt_bams, ctl_filt_bam1, labels, "$out_dir/qc", nth_jsd )
wait
// get each line in jsd_qc_all_reps and save to individual qc file for each rep
lines := jsd_qc_all_reps.readLines()
for ( int rep=1; rep <= get_num_rep( 0 ); rep++) { // IP replicate
arr := lines[rep].trim().split("\t")
string[] arr_without_col_1 // remove sample name from log file
for ( int i=1; i < arr.size(); i++) {
arr_without_col_1.add(arr[i])
}
group := get_group_name( 0, rep )
long := get_long_group_name( 0, rep )
filt_bam_ := filt_bam{group}
prefix := replace_dir( rm_ext( filt_bam_, "bam" ), "$out_dir/qc/rep$rep" )
jsd_qc_ := "$prefix.jsd.qc"
if ( jsd_qc_ <- jsd_qc_all_reps ) {
jsd_qc_.write( arr_without_col_1.join("\t") )
}
jsd_qc{group} = jsd_qc_
add_file_to_table( jsd_qc_, "QC and logs/$long/JS Distance" )
}
// jsd_qc_all_reps.rm()
add_file_to_table( jsd_png, "QC and logs/Fingerprint JSD Plot")
print( "\n== Done jsd()\n" )
}
}
void align( int ctl, int rep, int nth_rep ) {
if ( is_se( ctl, rep ) ) {
align_SE( ctl, rep, nth_rep )
}
else {
align_PE( ctl, rep, nth_rep )
}
}
void align_SE( int ctl, int rep, int nth_rep ) {
// ctl==0: exp. replicate, ctl==1: control
group := get_group_name( ctl, rep )
long := get_long_group_name( ctl, rep )
aln_o_dir := mkdir( "$out_dir/align/$group" ) // create align output directory
qc_o_dir := mkdir( "$out_dir/qc/$group" ) // create qc output dir.
string bam_, flagstat_qc_
string mapped_reads // read count
if ( is_input_fastq( ctl, rep ) ) {
fastqs := get_fastqs( ctl, rep )
// pool if multiple fastqs
string pooled_fastq
if ( fastqs.size()==1 ) pooled_fastq = fastqs[0]
else {
pooled_fastq = pool_fastq( fastqs, aln_o_dir, group )
wait
}
if ( aligner == "bwa" ) {
( bam_, flagstat_qc_ ) = bwa( pooled_fastq, aln_o_dir, qc_o_dir, group, nth_rep )
}
else {
error("Unsupported aligner ($aligner)!")
}
bam{group} = bam_
flagstat_qc{group} = flagstat_qc_
add_file_to_table( flagstat_qc_, "QC and logs/$long/BWA map. flagstat log")
wait
// parse read counts
tmp_log := parse_flagstat( flagstat_qc_ )
raw_reads := metric_prefix( parse_int( tmp_log{"total"} ) )
mapped_reads = metric_prefix( parse_int( tmp_log{"mapped"} ) )
// apply read counts to report
if ( fastqs.size()==1 ) {
add_file_to_report( fastqs[0], "fastq\\n$raw_reads", group, "Raw reads/$long/Fastq ($raw_reads)" )
}
else {
for ( int i=0; i<fastqs.size(); i++) {
id := i+1
suffix := ":$id"
add_file_to_report( fastqs[i], "fastq$suffix", group, "Raw reads/$long/Fastq$suffix" )
}
add_file_to_report( pooled_fastq, "pooled\\nfastq\\n$raw_reads", group, \
"Raw reads/$long/Pooled fastq ($raw_reads)" )
}
}
wait
string filt_bam_, dup_qc_, pbc_qc_, flagstat_nodup_qc_
string deduped_reads
if ( is_input_bam( ctl, rep ) || is_input_fastq( ctl, rep ) ) {
if ( is_input_bam( ctl, rep ) ) bam_ = get_bam( ctl, rep )
add_file_to_report( bam_, "bam" + (mapped_reads ? "\\n$mapped_reads" : ""), group, \
"Alignment/$long/Bam" + (mapped_reads ? " ($mapped_reads)" : "") )
if ( is_final_stage_bam() ) return
if ( no_dup_removal ) {
string tmp
( filt_bam_, tmp ) \
= dedup_bam( bam_, aln_o_dir, qc_o_dir, group, nth_rep )
wait
}
else {
( filt_bam_, dup_qc_, flagstat_nodup_qc_, pbc_qc_ ) \
= dedup_bam( bam_, aln_o_dir, qc_o_dir, group, nth_rep )
dup_qc{group} = dup_qc_
add_file_to_table( dup_qc_, "QC and logs/$long/Dedup. log")
pbc_qc{group} = pbc_qc_
add_file_to_table( pbc_qc_, "QC and logs/$long/PBC log")
flagstat_nodup_qc{group} = flagstat_nodup_qc_
add_file_to_table( flagstat_nodup_qc_, "QC and logs/$long/Filtered flagstat log")
wait
// add to report
tmp_log := parse_flagstat( flagstat_nodup_qc_ )
deduped_reads = metric_prefix( parse_int( tmp_log{"total"} ) )
}
}
string tag_
if ( is_input_filt_bam( ctl, rep ) || is_input_bam( ctl, rep ) || is_input_fastq( ctl, rep ) ) {
if ( is_input_filt_bam( ctl, rep ) ) filt_bam_ = get_filt_bam( ctl, rep )
add_file_to_report( filt_bam_, "filt. bam" + (deduped_reads ? "\\n$deduped_reads" : ""), group, \
"Alignment/$long/Filtered & deduped bam" + (deduped_reads ? " ($deduped_reads)" : "") )
filt_bam{group} = filt_bam_
if ( anon_filt_bam ) anonymize_filt_bam( filt_bam_, aln_o_dir, group )
if ( is_final_stage_filt_bam() ) return
tag_ = bam_to_tag( filt_bam_, aln_o_dir, group )
wait
}
if ( is_input_tag( ctl, rep ) || is_input_filt_bam( ctl, rep ) || is_input_bam( ctl, rep ) || is_input_fastq( ctl, rep ) ) {
if ( is_input_tag( ctl, rep ) ) tag_ = get_tag( ctl, rep )
add_file_to_report( tag_, "tag-align", group, "Alignment/$long/Tag-align" )
if ( ctl == 0 && parse_number( subsample_chip ) > 0 ) {
tag_ = subsample_tag( tag_, parse_number( subsample_chip ), false, aln_o_dir, group )
wait
}
else if ( ctl > 0 && parse_number( subsample_ctl ) > 0 ) {
tag_ = subsample_tag( tag_, parse_number( subsample_ctl ), false, aln_o_dir, group )
wait
}
tag{group} = tag_
if ( is_final_stage_tag() ) return
string xcor_qc_
if ( ctl == 0 ) { // if replicate
if ( !true_rep ) { // pseudo replicates
aln_pr1_o_dir := mkdir( "$out_dir/align/pseudo_reps/$group/pr1" )
aln_pr2_o_dir := mkdir( "$out_dir/align/pseudo_reps/$group/pr2" )
string tag_pr1_, tag_pr2_
(tag_pr1_, tag_pr2_ ) = spr( tag_, aln_pr1_o_dir, aln_pr2_o_dir, group ) // make self pseudo replicate
tag_pr1{group} = tag_pr1_
tag_pr2{group} = tag_pr2_
add_file_to_report( tag_pr1_, "tag-align", "$group-pr1", \
"Alignment/Pseudo-replicates/$long/Pseudo-replicate 1/Tag-align" )
add_file_to_report( tag_pr2_, "tag-align", "$group-pr2", \
"Alignment/Pseudo-replicates/$long/Pseudo-replicate 2/Tag-align" )
}
// wait
if ( !no_xcor ) {
string tag_xcor
if ( bam_ ) {
tag_xcor = bam_to_tag( bam_, aln_o_dir, group )
}
else {
tag_xcor = tag_
}
subsampled_tag_xcor := subsample_tag( tag_xcor, parse_number( subsample_xcor ), true, aln_o_dir, group )
wait
// xcor for true rep
string xcor_qc_, xcor_plot_
( xcor_qc_, xcor_plot_ ) = xcor( subsampled_tag_xcor, qc_o_dir, group, nth_rep )
xcor_qc{group} = xcor_qc_
xcor_plot{group} = xcor_plot_
add_file_to_report( xcor_qc_, "xcor log", group, "QC and logs/$long/Cross-corr. log" )
add_file_to_table( xcor_plot_, "QC and logs/$long/Cross-corr. plot" )
}
}
if ( is_final_stage_xcor() ) return
}
}
void align_PE( int ctl, int rep, int nth_rep ) {
if ( pe_xcor_only && ctl > 0 ) return
// ctl==0: exp. replicate, ctl==1: control
group := get_group_name( ctl, rep )
long := get_long_group_name( ctl, rep )
aln_o_dir := mkdir( "$out_dir/align/$group" ) // create align output directory
qc_o_dir := mkdir( "$out_dir/qc/$group" ) // create qc output dir.
string bam_, flagstat_qc_, bam_R1
string mapped_reads // read count
string tmp
if ( is_input_fastq( ctl, rep ) ) {
fastqs_pair1 := get_fastq( ctl, rep, 1 )
string[] fastqs_pair2
if ( !pe_xcor_only ) {
fastqs_pair2 = get_fastq( ctl, rep, 2 )
if ( fastqs_pair1.size() != fastqs_pair2.size() ) {
error("Number of fastqs to be pooled for pair 1 and pair 2 do not match!\n")
}
}
string pooled_fastq_pair1, pooled_fastq_pair2
if ( fastqs_pair1.size()==1 ) {
pooled_fastq_pair1 = fastqs_pair1[0]
if ( !pe_xcor_only ) {
pooled_fastq_pair2 = fastqs_pair2[0]
}
}
else {
pooled_fastq_pair1 = pool_fastq( fastqs_pair1, aln_o_dir, group )
if ( !pe_xcor_only ) {
pooled_fastq_pair2 = pool_fastq( fastqs_pair2, aln_o_dir, group )
}
wait
}
// allocate cpu for bwa_SE and bwa_PE
int[] nth_map
if ( !pe_xcor_only ) {
nth_map = distribute_nonzero( nth_rep, [1,2] )
}
else {
nth_map = [nth_rep]
}
// align on R1 fastq
if ( ctl==0 && !pe_no_trim_fastq ) {
trimmed_fastq_R1 := trim_fastq( pooled_fastq_pair1, aln_o_dir, group )
add_file_to_report( trimmed_fastq_R1, "trim. fastq R1", group, "Raw reads/$long/Trimmed fastq R1" )
wait
( bam_R1, tmp ) \
= bwa( trimmed_fastq_R1, aln_o_dir, qc_o_dir, group, nth_map[0] )
add_file_to_report( bam_R1, "bam R1", group, "Alignment/$long/Bam R1" )
}
if ( !pe_xcor_only ) {
if ( aligner == "bwa" ) {
( bam_, flagstat_qc_ ) \
= bwa_PE( pooled_fastq_pair1, pooled_fastq_pair2, aln_o_dir, qc_o_dir, group, nth_map[1] )
}
else {
error("Unsupported aligner ($aligner)!")
}
bam{group} = bam_
flagstat_qc{group} = flagstat_qc_
add_file_to_table( flagstat_qc_, "QC and logs/$long/BWA map. flagstat log")
wait
// parse read counts
tmp_log := parse_flagstat( flagstat_qc_ )
raw_reads := metric_prefix( parse_int( tmp_log{"total"} ) )
half_raw_reads := metric_prefix( parse_int( tmp_log{"total"} )/2 )
mapped_reads = metric_prefix( parse_int( tmp_log{"mapped"} ) )
// apply read counts to report
if ( fastqs_pair1.size()==1 ) {
add_file_to_report( fastqs_pair1[0], "fastq1\\n$half_raw_reads", group, \
"Raw reads/$long/Fastq 1 ($half_raw_reads)" )
add_file_to_report( fastqs_pair2[0], "fastq2\\n$half_raw_reads", group, \
"Raw reads/$long/Fastq 2 ($half_raw_reads)" )
}
else {
for ( int i=0; i<fastqs_pair1.size(); i++) {
id := i+1
suffix := ":$id"
add_file_to_report( fastqs_pair1[i], "fastq$suffix", group, "Raw reads/$long/Fastq 1$suffix" )
add_file_to_report( fastqs_pair2[i], "fastq$suffix", group, "Raw reads/$long/Fastq 2$suffix" )
}
add_file_to_report( pooled_fastq_pair1, "pooled\\nfastq1\\n$raw_reads", group, \
"Raw reads/$long/Pooled fastq 1 ($raw_reads)" )
add_file_to_report( pooled_fastq_pair2, "pooled\\nfastq2\\n$raw_reads", group, \
"Raw reads/$long/Pooled fastq 2 ($raw_reads)" )
}
}
}
wait
string filt_bam_, dup_qc_, pbc_qc_, flagstat_nodup_qc_
string deduped_reads
if ( is_input_bam( ctl, rep ) || is_input_fastq( ctl, rep ) ) {
if ( !pe_xcor_only ) {
if ( is_input_bam( ctl, rep ) ) bam_ = get_bam( ctl, rep )
add_file_to_report( bam_, "bam" + (mapped_reads ? "\\n$mapped_reads" : ""), group, \
"Alignment/$long/Bam" + (mapped_reads ? " ($mapped_reads)" : "") )
if ( is_final_stage_bam() ) return
if ( no_dup_removal ) {
( filt_bam_, tmp ) \
= dedup_bam_PE( bam_, aln_o_dir, qc_o_dir, group, nth_rep )
wait
}
else {
( filt_bam_, dup_qc_, flagstat_nodup_qc_, pbc_qc_ ) \
= dedup_bam_PE( bam_, aln_o_dir, qc_o_dir, group, nth_rep )
dup_qc{group} = dup_qc_
add_file_to_table( dup_qc_, "QC and logs/$long/Dedup. log")
pbc_qc{group} = pbc_qc_
add_file_to_table( pbc_qc_, "QC and logs/$long/PBC log")
flagstat_nodup_qc{group} = flagstat_nodup_qc_
add_file_to_table( flagstat_nodup_qc_, "QC and logs/$long/Filtered flagstat log")
wait
// add to report
tmp_log := parse_flagstat( flagstat_nodup_qc_ )
deduped_reads = metric_prefix( parse_int( tmp_log{"total"} ) )
}
}
}
string tag_, bedpe, tag_R1
if ( is_input_filt_bam( ctl, rep ) || is_input_bam( ctl, rep ) || is_input_fastq( ctl, rep ) ) {
if ( ctl==0 && bam_R1 ) {
tag_R1 = bam_to_tag( bam_R1, aln_o_dir, group )
add_file_to_report( tag_R1, "tag-align-R1", group, "Alignment/$long/Tag-align R1" )
}
if ( !pe_xcor_only ) {
if ( is_input_filt_bam( ctl, rep ) ) filt_bam_ = get_filt_bam( ctl, rep )
add_file_to_report( filt_bam_, "filt. bam" + (deduped_reads ? "\\n$deduped_reads" : ""), group, \
"Alignment/$long/Filtered & deduped bam" + (deduped_reads ? " ($deduped_reads)" : "") )
filt_bam{group} = filt_bam_
if ( anon_filt_bam ) anonymize_filt_bam( filt_bam_, aln_o_dir, group )
if ( is_final_stage_filt_bam() ) return
bedpe = bam_to_bedpe( filt_bam_, aln_o_dir, group )
wait
add_file_to_graph( bedpe, "bedpe", group )
tag_ = bedpe_to_tag( bedpe, aln_o_dir, group )
wait
}
}
if ( is_input_tag( ctl, rep ) || is_input_filt_bam( ctl, rep ) || is_input_bam( ctl, rep ) || is_input_fastq( ctl, rep ) ) {
if ( !pe_xcor_only ) {
if ( is_input_tag( ctl, rep ) ) tag_ = get_tag( ctl, rep )
add_file_to_report( tag_, "tag-align", group, "Alignment/$long/Tag-align" )
if ( ctl == 0 && parse_number( subsample_chip ) > 0 ) {
tag_ = subsample_tag_PE( tag_, parse_number( subsample_chip ), false, aln_o_dir, group )
wait
}
else if ( ctl > 0 && parse_number( subsample_ctl ) > 0 ) {
tag_ = subsample_tag_PE( tag_, parse_number( subsample_ctl ), false, aln_o_dir, group )
wait
}
tag{group} = tag_
if ( is_final_stage_tag() ) return
if ( ctl == 0 ) { // if replicate
if ( !true_rep ) { // if replicate
aln_pr1_o_dir := mkdir( "$out_dir/align/pseudo_reps/$group/pr1" )
aln_pr2_o_dir := mkdir( "$out_dir/align/pseudo_reps/$group/pr2" )
string tag_pr1_, tag_pr2_
( tag_pr1_, tag_pr2_ ) = spr_tag_PE( tag_, aln_pr1_o_dir, aln_pr2_o_dir, group )
tag_pr1{group} = tag_pr1_
tag_pr2{group} = tag_pr2_
add_file_to_report( tag_pr1_, "tag-align", "$group-pr1", \
"Alignment/Pseudo-replicates/$long/Pseudo-replicate 1/Tag-align" )
add_file_to_report( tag_pr2_, "tag-align", "$group-pr2", \
"Alignment/Pseudo-replicates/$long/Pseudo-replicate 2/Tag-align" )
}
}
// wait
}
if ( !no_xcor && ctl==0 ) {
string subsampled_tag_xcor
if ( tag_R1 ) {
subsampled_tag_xcor = subsample_tag( tag_R1, parse_number( subsample_xcor ), true, aln_o_dir, group )
}
else if ( bam_ ) {
bedpe_ := bam_to_bedpe( bam_, aln_o_dir, group )
wait
tag_xcor := bedpe_to_tag( bedpe_, aln_o_dir, group )
wait
subsampled_tag_xcor = subsample_tag_PE_for_xcor( tag_xcor, parse_number( subsample_xcor ), true, aln_o_dir, group )
}
else {
subsampled_tag_xcor = subsample_tag_PE_for_xcor( tag_, parse_number( subsample_xcor ), true, aln_o_dir, group )
}
wait
string xcor_qc_, xcor_plot_
( xcor_qc_, xcor_plot_ ) = xcor( subsampled_tag_xcor, qc_o_dir, group, nth_rep )
xcor_qc{group} = xcor_qc_
xcor_plot{group} = xcor_plot_
add_file_to_report( xcor_qc_, "xcor log", group, "QC and logs/$long/Cross-corr. log" )
add_file_to_table( xcor_plot_, "QC and logs/$long/Cross-corr. plot" )
}
if ( is_final_stage_xcor() ) return
}
}
void pool_tags() {
if ( !(is_final_stage_peak() || is_final_stage_idr()) ) return
if ( is_input_peak() ) return
////////////// pool replicates and controls, then create ppr (pooled pseudoreplicates) before peak calling
// make a temporary array of tagaligns, control tagaligns
string[] tags, tags_pr1, tags_pr2, tags_ctl
for ( int rep=1; rep<=get_num_rep(); rep++ ) {
tags.add( tag{"rep$rep"} )
if ( !true_rep ) {
tags_pr1.add( tag_pr1{"rep$rep"} )
tags_pr2.add( tag_pr2{"rep$rep"} )
}
if ( input_file_exists( 1, rep ) ) { // if control exists
tags_ctl.add( tag{"ctl$rep"} )
}
}
// if multiple tagalign for replicates, pool them
if ( tags.size() > 1 ) {
aln_pooled_o_dir := mkdir( "$out_dir/align/pooled_rep" )
tag_pooled := pool_tag( tags, aln_pooled_o_dir, "pooled_rep" )
add_file_to_report( tag_pooled, "tag-align", "pooled_rep", "Alignment/Pooled replicate/Tag-align" )
tag{"pooled_rep"} = tag_pooled
if ( !true_rep ) {
aln_ppr1_o_dir := mkdir( "$out_dir/align/pooled_pseudo_reps/ppr1" )
aln_ppr2_o_dir := mkdir( "$out_dir/align/pooled_pseudo_reps/ppr2" )
qc_ppr1_o_dir := mkdir( "$out_dir/qc/pooled_pseudo_reps/ppr1" ) // create qc output dir.
qc_ppr2_o_dir := mkdir( "$out_dir/qc/pooled_pseudo_reps/ppr2" ) // create qc output dir.
tag_ppr1_ := pool_tag( tags_pr1, aln_ppr1_o_dir, "ppr1" )
tag_ppr2_ := pool_tag( tags_pr2, aln_ppr2_o_dir, "ppr2" )
tag_ppr1 = tag_ppr1_ // for thread safety
tag_ppr2 = tag_ppr2_
add_file_to_report( tag_ppr1_, "tag-align", "ppr1", \
"Alignment/Pooled pseudo-replicates/Pooled pseudo-replicate 1/Tag-align" )
add_file_to_report( tag_ppr2_, "tag-align", "ppr2", \
"Alignment/Pooled pseudo-replicates/Pooled pseudo-replicate 2/Tag-align" )
wait
}
}
// if multiple tagalign for controls, pool them
if ( tags_ctl.size() > 1 ) {
aln_pooled_ctl_o_dir := mkdir( "$out_dir/align/pooled_ctl" )
tag_pooled := pool_tag( tags_ctl, aln_pooled_ctl_o_dir, "pooled_ctl" )
tag{"pooled_ctl"} = tag_pooled
add_file_to_report( tag_pooled, "tag-align\\n(pooled ctl.)", "pooled_ctl", "Alignment/Pooled control/Tag-align" )
}
wait
print( "\n== Done pool_tags()\n" )
}
void call_peaks() {
if ( !(is_final_stage_peak() || is_final_stage_idr()) ) return
if ( is_input_peak() ) return
// make a temporary array of tagaligns, control tagaligns
string[] tags, tags_pr1, tags_pr2, tags_ctl
for ( int rep=1; rep<=get_num_rep(); rep++ ) {
tags.add( tag{"rep$rep"} )
if ( !true_rep ) {
tags_pr1.add( tag_pr1{"rep$rep"} )
tags_pr2.add( tag_pr2{"rep$rep"} )
}
if ( input_file_exists( 1, rep ) ) { // if control exists
tags_ctl.add( tag{"ctl$rep"} )
}
}
// choose control (between true and pooled controls) for each replicate
string{} tag_ctl, tag_ctl_label // key: replicate id, value: actual control used for each replicate (according to control depth ratio)
if ( tags_ctl.size() == 0 ) { // if no control at all
tag_ctl{"pooled"} = ""
for ( int rep=1; rep<=get_num_rep(); rep++ ) {
tag_ctl{rep} = ""
}
}
else if ( tags_ctl.size() == 1 ) { // if only one control, use it for all replicates
tag_ctl{"pooled"} = tags_ctl[0]
for ( int rep=1; rep<=get_num_rep(); rep++ ) {
tag_ctl{rep} = tags_ctl[0]
}
}
else { // if multiple controls, check # of lines in replicate/control tagaligns and apply ctl_depth_ratio
tag_ctl{"pooled"} = tag{"pooled_ctl"}
real{} nlines // # of lines in tagaligns, key: 0,rep for replicate, 1,rep for control
real[] nlines_ctl // # of lines in control tagaligns
for ( int rep=1; rep<=get_num_rep(); rep++ ) {
nlines{"rep$rep"} = get_num_lines( tag{"rep$rep"} )
//print("DEBUG: # lines rep $rep tag.: " + nlines{"rep$rep"} + ", " + tag{"rep$rep"} + "\n")
if ( input_file_exists( 1, rep ) ) { // if control exists
nlines{"ctl$rep"} = get_num_lines( tag{"ctl$rep"} )
nlines_ctl.add( nlines{"ctl$rep"} )
//print("DEBUG: # lines ctl $rep tag.: " + nlines{"ctl$rep"} + ", " + tag{"ctl$rep"} + "\n")
}
}
use_pooled_ctl_ := use_pooled_ctl
for ( int i=0; i<nlines_ctl.size(); i++ ) { // check every # lines in every pair of control tagaligns
for ( int j=i+1; j<nlines_ctl.size(); j++ ) {
if ( nlines_ctl[i] / nlines_ctl[j] > ctl_depth_ratio || \