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scafe.tool.bk.bam_to_ctss
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#!/usr/bin/env perl
# This chunk of stuff was generated by App::FatPacker. To find the original
# file's code, look for the end of this BEGIN block or the string 'FATPACK'
BEGIN {
my %fatpacked;
s/^ //mg for values %fatpacked;
my $class = 'FatPacked::'.(0+\%fatpacked);
no strict 'refs';
*{"${class}::files"} = sub { keys %{$_[0]} };
if ($] < 5.008) {
*{"${class}::INC"} = sub {
if (my $fat = $_[0]{$_[1]}) {
my $pos = 0;
my $last = length $fat;
return (sub {
return 0 if $pos == $last;
my $next = (1 + index $fat, "\n", $pos) || $last;
$_ .= substr $fat, $pos, $next - $pos;
$pos = $next;
return 1;
});
}
};
}
else {
*{"${class}::INC"} = sub {
if (my $fat = $_[0]{$_[1]}) {
open my $fh, '<', \$fat
or die "FatPacker error loading $_[1] (could be a perl installation issue?)";
return $fh;
}
return;
};
}
unshift @INC, bless \%fatpacked, $class;
} # END OF FATPACK CODE
#====================================================================================================================================================#
#<use>
$|++; #---turn on the auto flush for the progress bar
no warnings 'utf8';
use warnings;
use strict;
use File::Path;
use File::Copy;
use File::Basename;
use File::Spec::Functions qw(rel2abs abs2rel);
use Time::HiRes qw( time );
use Getopt::Long 'HelpMessage';
use threads ('stack_size' => 64*4096);
use threads::shared;
use List::Util qw (sum shuffle min max);
use Cwd 'abs_path';
use AutoLoader qw/AUTOLOAD/;
#<\use>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<doc>
=head1 SYNOPSIS
5'-O~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~AAA-3'
O~~~AA O~~ O~ O~~~~~~~AO~~~~~~~~A
O~~ O~~ O~~ O~~ O~O~~ O~~ O~~
O~~ O~~ O~ O~~ O~~ O~~
O~~ O~~ O~~ O~~ O~~~~~AA O~~~~~~A
O~~ O~~ O~~~~~A O~~ O~~ O~~
O~~ O~~ O~~ O~~ O~~ O~~ O~~ O~~
O~~~~A O~~~ O~~ O~~O~~ O~~~~~~~AA
┌─ᐅ 5'-O~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-3'
...===┴========================================================================================...
Single Cell Analysis of Five-prime End (SCAFE) Tool Suite
---> scafe.tool.bk.bam_to_ctss <---
<--- tool, bulk mode, convert bam to ctss bed --->
Description:
This tool converts a bulk CAGE bam file to a ctss bed file, identifies read 5'end
(capped TSS, i.e. ctss), extracts the unencoded G information, pileup ctss,
and deduplicate the UMI
Usage:
scafe.tool.bk.bam_to_ctss [options] --bamPath --genome --outputPrefix --outDir
--bamPath <required> [string] bam file (of CAGE reads), can be read 1 only or pair-end
--genome <required> [string] name of genome reference, e.g. hg19.gencode_v32lift37
--outputPrefix <required> [string] prefix for the output files
--outDir <required> [string] directory for the output files
--include_flag (optional) [string] samflag to be included, comma delimited
e.g. '64' to include read1, (default=null)
--exclude_flag (optional) [string] samflag to be excluded, comma delimited,
e.g. '128,256,4' to exclude read2, secondary alignment
and unaligned reads (default=128,256,4)
--min_MAPQ (optional) [integer] minimum MAPQ to include (default=0)
--max_thread (optional) [integer] maximum number of parallel threads, capped at 10 to
avoid memory overflow (default=5)
--overwrite (optional) [yes/no] erase outDir/outputPrefix before running (default=no)
Dependencies:
bedtools
samtools
tabix
bgzip
For demo, cd to SCAFE dir and run,
scafe.tool.bk.bam_to_ctss \
--overwrite=yes \
--bamPath=./demo/input/bk.solo/demo.CAGE.bam \
--genome=hg19.gencode_v32lift37 \
--outputPrefix=demo \
--outDir=./demo/output/bk.solo/bam_to_ctss/
=head1 VERSION
v0.9.0-beta [March 20, 2021]
-Initial pre-release
v1.0.0 [June 6, 2022]
-implemented minimum bam alignment CIGAR match as 30 bases
=cut
#<\doc>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<lastCmdCalled>
#
# notCalledBefore
#
# notCalledBefore
#
#<\lastCmdCalled>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<global>
my $scriptDirPath = dirname(rel2abs($0));
my $scriptAbsPath = abs_path($0);
my ($curntTimeStamp) = &timeStamp();#->1303
my $ARGVStr = join "\n", (¤tTime(), $scriptAbsPath, @ARGV);#->455
my $globalReadmeHsh_ref = {};
our $tmplog_fh;
#<\global>
#====================================================================================================================================================#
#====================================================================================================================================================#
{ #Main sections lexical scope starts
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 0_startingTasks
#
#<section ID="startingTasks" num="0">
my ($bamPath, $exclude_flag, $include_flag, $min_MAPQ, $max_thread, $genome, $outputPrefix, $outDir, $overwrite) = &readParameters();#->1230
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 1_defineHardCodedParam
#
#<section ID="defineHardCodedParam" num="1">
my $paramTag = "$outputPrefix";
my $rm_tmp_file = 'yes';
my $sanity_check_toggle = 'yes';
#my $sanity_check_toggle = 'no';
my $num_chunk = 100;
my $min_match_length = 5;
my $max_non_G_mismatch = 2;
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 2_defineOutDirPath
#
#<section ID="defineOutDirPath" num="2">
my @mkDirAry;
my $result_dir = "$outDir/$paramTag"; push @mkDirAry, $result_dir;
system "rm -rf $result_dir" if ($overwrite eq 'yes');
my $result_bed_dir = "$result_dir/bed/"; push @mkDirAry, $result_bed_dir;
my $result_log_dir = "$result_dir/log/"; push @mkDirAry, $result_log_dir;
my $result_script_dir = "$result_dir/script/"; push @mkDirAry, $result_script_dir;
my $result_tmp_dir = "$result_dir/tmp/"; push @mkDirAry, $result_tmp_dir;
foreach my $dir (@mkDirAry) {system ("mkdir -pm 777 $dir");}
open $tmplog_fh, ">", "$result_dir/00_screen_log.$curntTimeStamp.log.txt";
&logCalledCMDAndScript($ARGVStr, $result_script_dir, $scriptAbsPath);#->775
&printStartOrFinishMessage("startMessage");#->1102
my ($tabix_bin, $bgzip_bin, $bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin) = &checkAllExecutable();#->206
my ($chrom_name_path, $chrom_size_path, $genome_fasta_path) = &checkChromNameSizeGenomeFastaPath($genome);#->306
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 3_convert
#
#<section ID="convert" num="3">
my ($chrom_name_size_hsh_ref, $out_chrom_size_hsh_ref) = &generateChromNameConversionHsh($chrom_name_path, $chrom_size_path, $samtools_bin, $bamPath);#->473
my ($chunk_size) = &generateChunkSize($out_chrom_size_hsh_ref, $num_chunk);#->594
my ($chunk_info_hsh_ref) = &generateChunkInfoHsh($chrom_name_size_hsh_ref, $chunk_size, $result_tmp_dir);#->543
&processPerChunk($bamPath, $bedtools_bin, $samtools_bin, $max_thread, $chunk_info_hsh_ref, $chrom_name_size_hsh_ref, $exclude_flag, $include_flag, $min_MAPQ, $genome_fasta_path, $out_chrom_size_hsh_ref, $sanity_check_toggle, $min_match_length, $max_non_G_mismatch);#->1136
&poolChunkResults($chunk_info_hsh_ref, $result_bed_dir, $result_log_dir, $paramTag, $max_thread, $tabix_bin, $bgzip_bin, $result_tmp_dir, $rm_tmp_file);#->880
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 4_finishingTasks
#
#<section ID="finishingTasks" num="4">
&printOutputFileListAndReadme($ARGVStr, $paramTag, $outDir);#->987
&printStartOrFinishMessage("finishMessage");#->1102
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
} #Main sections lexical scope ends
#====================================================================================================================================================#
#====================================================================================================================================================#
#List of subroutines by category
#
# general [n=5]:
# currentTime, logCalledCMDAndScript, printStartOrFinishMessage
# readParameters, timeStamp
#
# log [n=1]:
# reportAndLogStatus
#
# output [n=1]:
# printOutputFileListAndReadme
#
# specific [n=1]:
# convertBamToCTSS
#
# time [n=1]:
# timeStamp
#
# unassigned [n=11]:
# checkAllExecutable, checkChromNameSizeGenomeFastaPath, checkChunkResults
# generateChromNameConversionHsh, generateChunkInfoHsh, generateChunkSize
# getCTSS, getChunkFasta, pileupCtss
# poolChunkResults, processPerChunk
#
#====================================================================================================================================================#
sub checkAllExecutable {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1281
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|133
# secondaryAppearInSection: >none
# input: none
# output: $bedGraphToBigWig_bin, $bedtools_bin, $bgzip_bin, $bigWigAverageOverBed_bin, $cut_sh_path, $paraclu_bin, $samtools_bin, $tabix_bin
# toCall: my ($tabix_bin, $bgzip_bin, $bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin) = &checkAllExecutable();
# calledInLine: 148
#....................................................................................................................................................#
my $dirPath = dirname(rel2abs($0));
my $tabix_bin = "$dirPath/../resources/bin/tabix/tabix";
my $bgzip_bin = "$dirPath/../resources/bin/bgzip/bgzip";
my $bedtools_bin = "$dirPath/../resources/bin/bedtools/bedtools";
my $samtools_bin = "$dirPath/../resources/bin/samtools/samtools";
my $paraclu_bin = "$dirPath/../resources/bin/paraclu/paraclu";
my $cut_sh_path = "$dirPath/../resources/bin/paraclu/paraclu-cut.sh";
my $bedGraphToBigWig_bin = "$dirPath/../resources/bin/bedGraphToBigWig/bedGraphToBigWig";
my $bigWigAverageOverBed_bin = "$dirPath/../resources/bin/bigWigAverageOverBed/bigWigAverageOverBed";
&reportAndLogStatus("Checking all SCAFE executables", 10, "\n");#->1281
{
my $stdOut = `$tabix_bin --version 2>&1`;
if ($stdOut =~ m/tabix \(htslib\) (\S+)/) {
&reportAndLogStatus("Checking: tabix version: $1", 0, "\n");#->1281
} else {
die "tabix is not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$bgzip_bin --version 2>&1`;
if ($stdOut =~ m/bgzip \(htslib\) (\S+)/) {
&reportAndLogStatus("Checking: bgzip version: $1", 0, "\n");#->1281
} else {
die "bgzip is not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$bedtools_bin --version 2>&1`;
if ($stdOut =~ m/bedtools v(\S+)/) {
&reportAndLogStatus("Checking: bedtools version: $1", 0, "\n");#->1281
} else {
die "bedtools is not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$samtools_bin 2>&1`;
if ($stdOut =~ m/\s+(Version: \S+)\s+/) {
&reportAndLogStatus("Checking: samtools version: $1", 0, "\n");#->1281
} else {
die "samtools is not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$paraclu_bin 2>&1`;
if ($stdOut =~ m/paraclu: I need a minValue and a fileName/) {
&reportAndLogStatus("Checking: paraclu found.", 0, "\n");#->1281
} else {
die "paraclu is not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$cut_sh_path 55C7128A 2>&1`;
if ($stdOut =~ m/awk:/) {
&reportAndLogStatus("Checking: paraclu-cut found.", 0, "\n");#->1281
} else {
die "paraclu-cut is not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$bedGraphToBigWig_bin 2>&1`;
if ($stdOut =~ m/bedGraphToBigWig v (\S+)/) {
&reportAndLogStatus("Checking: bedGraphToBigWig version: $1", 0, "\n");#->1281
} else {
die "bedGraphToBigWig not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$bigWigAverageOverBed_bin 2>&1`;
if ($stdOut =~ m/bigWigAverageOverBed v(\S+)/) {
&reportAndLogStatus("Checking: bigWigAverageOverBed version: $1", 0, "\n");#->1281
} else {
die "bigWigAverageOverBed is not installed properly. Quitting.\n";
}
}
return ($tabix_bin, $bgzip_bin, $bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin);
}
sub checkChromNameSizeGenomeFastaPath {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|133
# secondaryAppearInSection: >none
# input: $genome
# output: $chrom_name_path, $chrom_size_path, $genome_fasta_path
# toCall: my ($chrom_name_path, $chrom_size_path, $genome_fasta_path) = &checkChromNameSizeGenomeFastaPath($genome);
# calledInLine: 149
#....................................................................................................................................................#
my ($genome) = @_;
my $dirPath = dirname(rel2abs($0));
my $chrom_name_path = "$dirPath/../resources/genome/$genome/tsv/chrom.bam_name.tsv";
my $chrom_size_path = "$dirPath/../resources/genome/$genome/tsv/chrom.sizes.tsv";
my $genome_fasta_path = "$dirPath/../resources/genome/$genome/fasta/genome.fa";
die "genome $genome does not have chrom_name_path. Please rerun prep_genome step\n" if not -s $chrom_name_path;
die "genome $genome does not have chrom_size_path. Please rerun prep_genome step\n" if not -s $chrom_size_path;
die "genome $genome does not have genome_fasta_path. Please rerun prep_genome step\n" if not -s $genome_fasta_path;
return ($chrom_name_path, $chrom_size_path, $genome_fasta_path);
}
sub checkChunkResults {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1281
# appearInSub: poolChunkResults|880, processPerChunk|1136
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_convert|154
# input: $chunk_info_hsh_ref, $chunk_num, $file_check_ary_ref
# output: $valid
# toCall: my ($valid) = &checkChunkResults($chunk_info_hsh_ref, $chunk_num, $file_check_ary_ref);
# calledInLine: 912, 1174
#....................................................................................................................................................#
my ($chunk_info_hsh_ref, $chunk_num, $file_check_ary_ref) = @_;
my $valid = 'yes';
foreach my $file_to_check (@{$file_check_ary_ref}) {
my $file_path = $chunk_info_hsh_ref->{$chunk_num}{$file_to_check};
#&reportAndLogStatus("$file_path", 10, "\n");#->1281
$valid = 'no' if not -f $file_path;
}
return ($valid);
}
sub convertBamToCTSS {
#....................................................................................................................................................#
# subroutineCategory: specific
# dependOnSub: getCTSS|619, reportAndLogStatus|1281
# appearInSub: processPerChunk|1136
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_convert|154
# input: $bamPath, $bedtools_bin, $chrom_name_size_hsh_ref, $chunk_endPos, $chunk_fasta_offset_pos, $chunk_fasta_path, $chunk_log_path, $chunk_long_ctss_bed_path, $chunk_sanity_check_path, $chunk_seq_str, $chunk_startPos, $chunk_unsort_long_ctss_bed_path, $exclude_flag, $include_flag, $max_non_G_mismatch, $min_MAPQ, $min_match_length, $region_str, $samtools_bin, $sanity_check_toggle
# output:
# toCall: &convertBamToCTSS($bamPath, $chrom_name_size_hsh_ref, $exclude_flag, $include_flag, $bedtools_bin, $samtools_bin, $min_MAPQ, $chunk_long_ctss_bed_path, $chunk_unsort_long_ctss_bed_path, $region_str, $chunk_log_path, $chunk_startPos, $chunk_endPos, $chunk_fasta_path, $chunk_seq_str, $chunk_fasta_offset_pos, $chunk_sanity_check_path, $sanity_check_toggle, $min_match_length, $max_non_G_mismatch);
# calledInLine: 1194
#....................................................................................................................................................#
my ($bamPath, $chrom_name_size_hsh_ref, $exclude_flag, $include_flag, $bedtools_bin, $samtools_bin, $min_MAPQ, $chunk_long_ctss_bed_path, $chunk_unsort_long_ctss_bed_path, $region_str, $chunk_log_path, $chunk_startPos, $chunk_endPos, $chunk_fasta_path, $chunk_seq_str, $chunk_fasta_offset_pos, $chunk_sanity_check_path, $sanity_check_toggle, $min_match_length, $max_non_G_mismatch) = @_;
my $force_rerun = 'yes';
if (not -s $chunk_long_ctss_bed_path or $force_rerun eq 'yes') {
#&reportAndLogStatus("converting $paramTag bam to ctss bed", 10, "\n");#->1281
my $perl_covert_readname = 'while (<STDIN>) {chomp; if ($_ =~ m/^\@/) {print "$_\n";} else {@samAry = split /\t/; $samAry[0] = $samAry[9]; print join "", (join "\t", @samAry[0..10]), "\n";}}';
my $min_softclip_length = 0;
my $max_softclip_length = 3;
my $min_total_match = 30;
my $margin = 20;
my $num_proc = 0;
my $ex_F_param = '';
my $in_f_param = '';
if (defined $exclude_flag) {
my @flagAry = ();
foreach my $flag (split /,/, $exclude_flag) {
push @flagAry, "-F $flag";
}
$ex_F_param = join " ", @flagAry;
}
if (defined $include_flag) {
my @flagAry = ();
foreach my $flag (split /,/, $include_flag) {
push @flagAry, "-f $flag";
}
$in_f_param = join " ", @flagAry;
}
#---[6/18/15 14:23] get all
#my $cmd = "$samtools_bin view -h $in_f_param $ex_F_param $bamPath $region_str 2>/dev/null | perl -e \'$perl_covert_readname\' | $samtools_bin view -b - | $bedtools_bin bamtobed -cigar -i stdin |";
#print $cmd."\n";
open (BAMIN, "$samtools_bin view -h $in_f_param $ex_F_param $bamPath $region_str 2>/dev/null | perl -e \'$perl_covert_readname\' | $samtools_bin view -b - | $bedtools_bin bamtobed -cigar -i stdin |");
#open (CTSSOUT, "| sort -k1,1 -k2,2n -k6,6 >$chunk_long_ctss_bed_path");
open (CTSSOUT, ">", $chunk_unsort_long_ctss_bed_path);
open SANITYCHECK, ">", $chunk_sanity_check_path;
print SANITYCHECK join "", (join "\t", ('out_chrom', 'bedStart', 'bedEnd', 'strand', 'rd_seq', 'check_read_seq', 'check_genome_seq', 'cigar', 'softclip_length', 'match_length', 'check_read_pos', 'trim_length', 'num_unencoded_G', 'non_G_mismatch')), "\n";
my $chunk_total_read_num = 0;
my $chunk_passed_read_num = 0;
while (my $line = <BAMIN>) {
chomp $line;
my ($chrom, $bedStart, $bedEnd, $bam_seq, $MAPQ, $strand, $cigar) = split /\t+/, $line;
$chunk_total_read_num++;
my $total_match = 0;
$total_match = sum($cigar =~ m/(\d+)M/g) if $cigar =~ /\d+M/;
next if $MAPQ < $min_MAPQ;
next if $total_match < $min_total_match;
$bam_seq =~ s/\/\d$//;
my $out_chrom = $chrom_name_size_hsh_ref->{$chrom}{'out_chrom'};
my $size = $chrom_name_size_hsh_ref->{$chrom}{'size'};
#&reportAndLogStatus("passed_read_num = $passed_read_num", 10, "\n") if ($passed_read_num%10000 == 0);#->1281
my ($end5Start, $unencoded_G, $sanity_check_ary_ref) = &getCTSS($bedStart, $bedEnd, $strand, $bam_seq, $chunk_startPos, $chunk_endPos, $chunk_seq_str, $chunk_fasta_offset_pos, $samtools_bin, $out_chrom, $cigar, $min_softclip_length, $max_softclip_length, $min_match_length);#->619
if (defined $end5Start) {
my $end5End = $end5Start+1;
if ($end5Start >= $chunk_startPos and $end5End <= $chunk_endPos) {
if ($end5Start >= $margin and $end5End <= ($size - $margin)) {
$chunk_passed_read_num++;
print CTSSOUT join "", (join "\t", ($out_chrom, $end5Start, $end5End, $MAPQ, $unencoded_G, $strand)), "\n";
if ($sanity_check_toggle eq 'yes') {
print SANITYCHECK join "", (join "\t", (@{$sanity_check_ary_ref})), "\n";
}
}
}
}
}
open (LOG, ">$chunk_log_path");
print LOG "$chunk_passed_read_num\t$chunk_total_read_num\n";
close LOG;
close BAMIN;
close CTSSOUT;
close SANITYCHECK;
system "sort -k1,1 -k2,2n -k6,6 $chunk_unsort_long_ctss_bed_path >$chunk_long_ctss_bed_path";
system "rm $chunk_unsort_long_ctss_bed_path";
} else {
&reportAndLogStatus("chunk_long_ctss_bed_path found. Skipping conversion.", 10, "\n");#->1281
}
return ();
}
sub currentTime {
#....................................................................................................................................................#
# subroutineCategory: general
# dependOnSub: >none
# appearInSub: printStartOrFinishMessage|1102, reportAndLogStatus|1281
# primaryAppearInSection: >none
# secondaryAppearInSection: 2_defineOutDirPath|133, 4_finishingTasks|166
# input: none
# output: $runTime
# toCall: my ($runTime) = ¤tTime();
# calledInLine: 100, 1118, 1122, 1127, 1131, 1297, 1298
#....................................................................................................................................................#
my ($sec,$min,$hour,$mday,$mon,$year,$wday,$yday,$isdst)=localtime(time);
my $runTime = sprintf "%04d-%02d-%02d %02d:%02d", $year+1900, $mon+1,$mday,$hour,$min;
return $runTime;
}
sub generateChromNameConversionHsh {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1281
# appearInSub: >none
# primaryAppearInSection: 3_convert|154
# secondaryAppearInSection: >none
# input: $bamPath, $chrom_name_path, $chrom_size_path, $samtools_bin
# output: $chrom_name_size_hsh_ref, $out_chrom_size_hsh_ref
# toCall: my ($chrom_name_size_hsh_ref, $out_chrom_size_hsh_ref) = &generateChromNameConversionHsh($chrom_name_path, $chrom_size_path, $samtools_bin, $bamPath);
# calledInLine: 157
#....................................................................................................................................................#
my ($chrom_name_path, $chrom_size_path, $samtools_bin, $bamPath) = @_;
my $min_hit_read_pct = 10;
&reportAndLogStatus("getting bam header chromosome", 10, "\n");#->1281
my $actual_bam_chrom_name_hsh_ref = {};
my $total_num_read = 0;
open SAMTOOLS, "$samtools_bin idxstats $bamPath|";
while (<SAMTOOLS>) {
chomp;
my ($actual_bam_chrom_name, $size, $num_read) = split /\t/;
if ($actual_bam_chrom_name ne "*") {
$actual_bam_chrom_name_hsh_ref->{$actual_bam_chrom_name} = $num_read;
$total_num_read+=$num_read;
&reportAndLogStatus("chrom $actual_bam_chrom_name found in bam with size $size and num_read $num_read", 10, "\n");#->1281
}
}
close SAMTOOLS;
my $chrom_name_size_hsh_ref = {};
my $tmp_chrom_hsh_ref = {};
my $out_chrom_size_hsh_ref = {};
my $hit_num_read = 0;
open (CHROMNAME, "<", $chrom_name_path);
while (<CHROMNAME>) {
chomp;
my ($out_chrom, $bam_chrom_name) = split /\s+/;
if (exists $actual_bam_chrom_name_hsh_ref->{$out_chrom}) {
$bam_chrom_name = $out_chrom;
$hit_num_read += $actual_bam_chrom_name_hsh_ref->{$out_chrom};
} elsif (exists $actual_bam_chrom_name_hsh_ref->{$bam_chrom_name}) {
$hit_num_read += $actual_bam_chrom_name_hsh_ref->{$bam_chrom_name};
}
$chrom_name_size_hsh_ref->{$bam_chrom_name}{'out_chrom'} = $out_chrom;
$tmp_chrom_hsh_ref->{$out_chrom} = $bam_chrom_name;
}
close CHROMNAME;
my $hit_read_pct = 100*($hit_num_read/$total_num_read);
&reportAndLogStatus("$hit_read_pct % of read in bam is within chrom list scope", 10, "\n");#->1281
if ($hit_read_pct < $min_hit_read_pct) {
die "Quitting due to low % of read in bam is within chrom list scope.\n";
}
open (CHROMSIZE, "<", $chrom_size_path);
while (<CHROMSIZE>) {
chomp;
my ($out_chrom, $size) = split /\s+/;
my $bam_chrom_name = $tmp_chrom_hsh_ref->{$out_chrom};
$chrom_name_size_hsh_ref->{$bam_chrom_name}{'size'} = $size;
$out_chrom_size_hsh_ref->{$out_chrom} = $size;
}
close CHROMSIZE;
return ($chrom_name_size_hsh_ref, $out_chrom_size_hsh_ref);
}
sub generateChunkInfoHsh {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1281
# appearInSub: >none
# primaryAppearInSection: 3_convert|154
# secondaryAppearInSection: >none
# input: $chrom_name_size_hsh_ref, $chunk_size, $result_tmp_dir
# output: $chunk_info_hsh_ref
# toCall: my ($chunk_info_hsh_ref) = &generateChunkInfoHsh($chrom_name_size_hsh_ref, $chunk_size, $result_tmp_dir);
# calledInLine: 159
#....................................................................................................................................................#
my ($chrom_name_size_hsh_ref, $chunk_size, $result_tmp_dir) = @_;
my $chunk_num = 1;
my $chunk_info_hsh_ref = {};
foreach my $chrom (sort {$chrom_name_size_hsh_ref->{$a}{'out_chrom'} cmp $chrom_name_size_hsh_ref->{$b}{'out_chrom'}} keys %{$chrom_name_size_hsh_ref}) {
my $size = $chrom_name_size_hsh_ref->{$chrom}{'size'};
my $out_chrom = $chrom_name_size_hsh_ref->{$chrom}{'out_chrom'};
foreach (my $startPos = 0; $startPos <= $size; $startPos += $chunk_size) {
my $endPos = $startPos + $chunk_size;
$endPos = $size if ($endPos > $size);
my $file_tag = "$out_chrom.$startPos.$endPos";
my $size_in_chunk = $endPos - $startPos;
&reportAndLogStatus("chunk $chunk_num contains $size_in_chunk nt covering $out_chrom $startPos to $endPos", 10, "\n");#->1281
my $chunk_unsort_long_ctss_bed_path = "$result_tmp_dir/$file_tag.unsorted.long.ctss.bed";
my $chunk_long_ctss_bed_path = "$result_tmp_dir/$file_tag.long.ctss.bed";
my $chunk_collapse_ctss_bed_path = "$result_tmp_dir/$file_tag.collapse.ctss.bed";
my $chunk_ung_collapse_ctss_bed_path = "$result_tmp_dir/$file_tag.unencoded_G.collapse.ctss.bed";
my $chunk_log_path = "$result_tmp_dir/$file_tag.log.txt";
my $chunk_fasta_path = "$result_tmp_dir/$file_tag.seq.fasta";
my $chunk_sanity_check_path = "$result_tmp_dir/$file_tag.unencoded_G.sanity_check.txt";
my ($chunk_chrom, $chunk_startPos, $chunk_endPos) = ($chrom, $startPos, $endPos);
$chunk_info_hsh_ref->{$chunk_num}{'loc'} = [$chunk_chrom, $chunk_startPos, $chunk_endPos];
$chunk_info_hsh_ref->{$chunk_num}{'region_str'} = "$chunk_chrom:$chunk_startPos-$chunk_endPos";
$chunk_info_hsh_ref->{$chunk_num}{'chunk_unsort_long_ctss_bed_path'} = $chunk_unsort_long_ctss_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'chunk_long_ctss_bed_path'} = $chunk_long_ctss_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'chunk_collapse_ctss_bed_path'} = $chunk_collapse_ctss_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'chunk_ung_collapse_ctss_bed_path'} = $chunk_ung_collapse_ctss_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'chunk_log_path'} = $chunk_log_path;
$chunk_info_hsh_ref->{$chunk_num}{'chunk_sanity_check_path'} = $chunk_sanity_check_path;
$chunk_info_hsh_ref->{$chunk_num}{'chunk_fasta_path'} = $chunk_fasta_path;
$chunk_num++;
}
}
return ($chunk_info_hsh_ref);
}
sub generateChunkSize {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 3_convert|154
# secondaryAppearInSection: >none
# input: $num_chunk, $out_chrom_size_hsh_ref
# output: $chunk_size
# toCall: my ($chunk_size) = &generateChunkSize($out_chrom_size_hsh_ref, $num_chunk);
# calledInLine: 158
#....................................................................................................................................................#
my ($out_chrom_size_hsh_ref, $num_chunk) = @_;
my $total_size = 0;
foreach my $out_chrom (keys %{$out_chrom_size_hsh_ref}) {
$total_size += $out_chrom_size_hsh_ref->{$out_chrom};
}
my $chunk_size = int ($total_size/$num_chunk);
return ($chunk_size);
}
sub getCTSS {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1281
# appearInSub: convertBamToCTSS|353
# primaryAppearInSection: >none
# secondaryAppearInSection: >none
# input: $bam_seq, $bedEnd, $bedStart, $chunk_endPos, $chunk_fasta_offset_pos, $chunk_seq_str, $chunk_startPos, $cigar, $max_softclip_length, $min_match_length, $min_softclip_length, $out_chrom, $samtools_bin, $strand
# output: $end5Start, $num_unencoded_G, $sanity_check_ary_ref
# toCall: my ($end5Start, $num_unencoded_G, $sanity_check_ary_ref) = &getCTSS($bedStart, $bedEnd, $strand, $bam_seq, $chunk_startPos, $chunk_endPos, $chunk_seq_str, $chunk_fasta_offset_pos, $samtools_bin, $out_chrom, $cigar, $min_softclip_length, $max_softclip_length, $min_match_length);
# calledInLine: 421
#....................................................................................................................................................#
my ($bedStart, $bedEnd, $strand, $bam_seq, $chunk_startPos, $chunk_endPos, $chunk_seq_str, $chunk_fasta_offset_pos, $samtools_bin, $out_chrom, $cigar, $min_softclip_length, $max_softclip_length, $min_match_length) = @_;
my ($end5Start, $num_unencoded_G);
my $sanity_check_ary_ref = [];
my $num_nt_extract_check = 10;
my $max_non_G_mismatch = 3;
my ($readStart, $readEnd) = ($bedStart, $bedEnd);
my $rd_seq = $bam_seq;
my $softclip_valid = 'no';
my $match_valid = 'no';
my $TSS_within_chunk = 'no';
my $softclip_length = 0;
my $match_length = 0;
if ($strand eq '+') {
$TSS_within_chunk = 'yes' if $readStart >= $chunk_startPos;
if ($cigar =~ m/^(\d+)S(\d+)M/) {
$softclip_length = $1;
$match_length = $2;
$readStart -= $softclip_length;
} elsif ($cigar =~ m/(\d+)M$/) {
$match_length = $1;
}
} else {
$rd_seq = reverse $rd_seq;
$rd_seq =~ tr/ATGCatgc/TACGtacg/;
$TSS_within_chunk = 'yes' if $readEnd <= $chunk_endPos;
if ($cigar =~ m/(\d+)M(\d+)S$/) {
$softclip_length = $2;
$match_length = $1;
$readEnd += $softclip_length;
} elsif ($cigar =~ m/(\d+)M$/) {
$match_length = $1;
}
}
$softclip_valid = 'yes' if ($softclip_length >= $min_softclip_length and $softclip_length <= $max_softclip_length);
$match_valid = 'yes' if ($match_length >= $min_match_length);
if ($softclip_valid eq 'yes' and $match_valid eq 'yes' and $TSS_within_chunk eq 'yes') {
#---take 10nt for checking
my $check_genome_seq;
my $check_genome_pos_start;
if ($strand eq '+') {
$check_genome_pos_start = $readStart - $chunk_fasta_offset_pos + 1;
$check_genome_seq = substr ($chunk_seq_str, $check_genome_pos_start, $num_nt_extract_check);
} else {
my $check_genome_pos_end = $readEnd - $chunk_fasta_offset_pos;
$check_genome_pos_start = $check_genome_pos_end - $num_nt_extract_check + 1;
$check_genome_seq = substr ($chunk_seq_str, $check_genome_pos_start, $num_nt_extract_check);
$check_genome_seq = reverse($check_genome_seq);
$check_genome_seq =~ tr/ATGC/TACG/;
}
my $check_read_pos = 0;
my $check_read_seq = substr $rd_seq, $check_read_pos, $num_nt_extract_check;
my $trim_length = 0;
$num_unencoded_G = 0;
my $matched = 'no';
my $non_G_mismatch = 0;
foreach my $shift_pos (0..$num_nt_extract_check) {
my $read_nt = substr($check_read_seq, $shift_pos, 1);
my $genome_nt = substr($check_genome_seq, $shift_pos, 1);
if ($read_nt ne $genome_nt) {
if ($read_nt eq 'G') {
$num_unencoded_G++;
} else {
$non_G_mismatch++;
};
$trim_length++;
last if $non_G_mismatch > $max_non_G_mismatch;
} else {
$matched = 'yes';
last;
}
}
if ($matched eq 'yes') {
$sanity_check_ary_ref = [$out_chrom, $bedStart, $bedEnd, $strand, $rd_seq, $check_read_seq, $check_genome_seq, $cigar, $softclip_length, $match_length, $check_read_pos, $trim_length, $num_unencoded_G, $non_G_mismatch];
if ($strand eq '+') {
$end5Start = $readStart + $trim_length;
} elsif ($strand eq '-') {
$end5Start = $readEnd - $trim_length - 1;
} else {
die "strand=$strand, must be + or -\n";
}
$end5Start = 0 if $end5Start < 0;
} else {
#&reportAndLogStatus("$check_genome_seq $check_read_seq $non_G_mismatch $num_unencoded_G", 10, "\n");#->1281
}
} else {
#&reportAndLogStatus("softclip_length=$softclip_length match_length=$match_length cigar=$cigar", 10, "\n");#->1281
}
return ($end5Start, $num_unencoded_G, $sanity_check_ary_ref);
}
sub getChunkFasta {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1281
# appearInSub: processPerChunk|1136
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_convert|154
# input: $chunk_endPos, $chunk_fasta_path, $chunk_startPos, $genome_fasta_path, $out_chrom, $samtools_bin, $size
# output: $chunk_fasta_offset_pos, $chunk_seq_str
# toCall: my ($chunk_seq_str, $chunk_fasta_offset_pos) = &getChunkFasta($chunk_fasta_path, $out_chrom, $chunk_startPos, $chunk_endPos, $genome_fasta_path, $size, $samtools_bin);
# calledInLine: 1193
#....................................................................................................................................................#
my ($chunk_fasta_path, $out_chrom, $chunk_startPos, $chunk_endPos, $genome_fasta_path, $size, $samtools_bin) = @_;
&reportAndLogStatus("Extracting chunk fasta", 10, "\n");#->1281
my $fasta_start_pos = $chunk_startPos - 500;
$fasta_start_pos = 1 if $fasta_start_pos < 1;
my $fasta_end_pos = $chunk_endPos + 500;
$fasta_end_pos = $size if $fasta_end_pos > $size;
my $chunk_fasta_name = "$out_chrom:$fasta_start_pos-$fasta_end_pos";
system ("$samtools_bin faidx $genome_fasta_path $chunk_fasta_name >$chunk_fasta_path");
my $chunk_seq_str = '';
open CHUNKFASTA, "<", $chunk_fasta_path;
<CHUNKFASTA>;
while (<CHUNKFASTA>) {
chomp;
$chunk_seq_str = $chunk_seq_str.$_;
}
close CHUNKFASTA;
$chunk_seq_str = uc $chunk_seq_str;
my $chunk_fasta_offset_pos = $fasta_start_pos;
return ($chunk_seq_str, $chunk_fasta_offset_pos);
}
sub logCalledCMDAndScript {
#....................................................................................................................................................#
# subroutineCategory: general
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|133
# secondaryAppearInSection: >none
# input: $ARGVStr, $result_script_dir, $scriptAbsPath
# output:
# toCall: &logCalledCMDAndScript($ARGVStr, $result_script_dir, $scriptAbsPath);
# calledInLine: 146
#....................................................................................................................................................#
my ($ARGVStr, $result_script_dir, $scriptAbsPath) = @_;
my $cpScriptPath = "$result_script_dir/script.ran.pl";
my $calledCMDPath = "$result_script_dir/called.cmd.txt";
system "cp -f $scriptAbsPath $cpScriptPath";
system "chmod 0444 $cpScriptPath"; #---[07/03/2014 18:02] make it read-only to make sure there'll be accodental change of parameters
open CALLEDCMD, ">", $calledCMDPath;
print CALLEDCMD join "", ($ARGVStr), "\n";
close CALLEDCMD;
return ();
}
sub pileupCtss {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1281
# appearInSub: processPerChunk|1136
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_convert|154
# input: $chunk_collapse_ctss_bed_path, $chunk_long_ctss_bed_path, $chunk_num, $chunk_ung_collapse_ctss_bed_path
# output:
# toCall: &pileupCtss($chunk_long_ctss_bed_path, $chunk_collapse_ctss_bed_path, $chunk_ung_collapse_ctss_bed_path, $chunk_num);
# calledInLine: 1195
#....................................................................................................................................................#
my ($chunk_long_ctss_bed_path, $chunk_collapse_ctss_bed_path, $chunk_ung_collapse_ctss_bed_path, $chunk_num) = @_;
my $num_proc = 0;
my $tmp_CTSS_hsh_ref = {};
my $last_chrom = 'initial';
my $last_start = 'initial';
my $last_strand = 'initial';
open INCTSS, "<", $chunk_long_ctss_bed_path;
open OUTCBCOLLAPSECTSS, ">", $chunk_collapse_ctss_bed_path;
open UNGOUTCBCOLLAPSECTSS, ">", $chunk_ung_collapse_ctss_bed_path;
while (<INCTSS>) {
chomp;
my ($chrom, $start, $end, $MAPQ, $num_unencoded_G, $strand) = split /\t/;
$tmp_CTSS_hsh_ref->{$chrom}{$start}{$strand}{'all'}++;
$tmp_CTSS_hsh_ref->{$chrom}{$start}{$strand}{'ung'}++ if $num_unencoded_G > 0;
if ($last_chrom eq 'initial') {
$last_chrom = $chrom;
$last_start = $start;
$last_strand = $strand;
}
if (($chrom ne $last_chrom or $start ne $last_start or $strand ne $last_strand)) {
my $last_end = $last_start + 1;
my $num_total_ctss = $tmp_CTSS_hsh_ref->{$last_chrom}{$last_start}{$last_strand}{'all'};
print OUTCBCOLLAPSECTSS join "", (join "\t", ($last_chrom, $last_start, $last_end, $num_total_ctss, $num_total_ctss, $last_strand)), "\n";
if (exists $tmp_CTSS_hsh_ref->{$last_chrom}{$last_start}{$last_strand}{'ung'}) {
my $num_ung_ctss = $tmp_CTSS_hsh_ref->{$last_chrom}{$last_start}{$last_strand}{'ung'};
print UNGOUTCBCOLLAPSECTSS join "", (join "\t", ($last_chrom, $last_start, $last_end, $num_total_ctss, $num_ung_ctss, $last_strand)), "\n";
}
delete $tmp_CTSS_hsh_ref->{$last_chrom}{$last_start}{$last_strand};
$last_chrom = $chrom;
$last_start = $start;
$last_strand = $strand;
$num_proc++;
if ($num_proc%10000 == 0) {
&reportAndLogStatus("$num_proc CTSS piled up for chunk $chunk_num", 10, "\n");#->1281
}
}
if (eof(INCTSS)) {
foreach my $chrom (keys %{$tmp_CTSS_hsh_ref}) {
foreach my $start (keys %{$tmp_CTSS_hsh_ref->{$chrom}}) {
my $end = $start + 1;
foreach my $strand (keys %{$tmp_CTSS_hsh_ref->{$chrom}{$start}}) {
my $num_total_ctss = $tmp_CTSS_hsh_ref->{$chrom}{$start}{$strand}{'all'};
print OUTCBCOLLAPSECTSS join "", (join "\t", ($chrom, $start, $end, $num_total_ctss, $num_total_ctss, $strand)), "\n";
if (exists $tmp_CTSS_hsh_ref->{$chrom}{$start}{$strand}{'ung'}) {
my $num_ung_ctss = $tmp_CTSS_hsh_ref->{$chrom}{$start}{$strand}{'ung'};
print UNGOUTCBCOLLAPSECTSS join "", (join "\t", ($chrom, $start, $end, $num_total_ctss, $num_ung_ctss, $strand)), "\n";
}
}
}
}
}
}
close INCTSS;
close OUTCBCOLLAPSECTSS;
close UNGOUTCBCOLLAPSECTSS;
return ();
}
sub poolChunkResults {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: checkChunkResults|330, reportAndLogStatus|1281
# appearInSub: >none
# primaryAppearInSection: 3_convert|154
# secondaryAppearInSection: >none
# input: $chunk_info_hsh_ref, $paramTag, $result_bed_dir, $result_log_dir, $result_tmp_dir, $rm_tmp_file
# output:
# toCall: &poolChunkResults($chunk_info_hsh_ref, $result_bed_dir, $result_log_dir, $paramTag, $result_tmp_dir, $rm_tmp_file);
# calledInLine: 161
#....................................................................................................................................................#
my ($chunk_info_hsh_ref, $result_bed_dir, $result_log_dir, $paramTag, $max_thread, $tabix_bin, $bgzip_bin, $result_tmp_dir, $rm_tmp_file) = @_;
my $ung_collapse_ctss_bed_path = "$result_bed_dir/$paramTag.unencoded_G.collapse.ctss.bed";
my $collapse_ctss_bed_path = "$result_bed_dir/$paramTag.collapse.ctss.bed";
my $long_ctss_bed_path = "$result_bed_dir/$paramTag.long.ctss.bed";
my $log_path = "$result_log_dir/$paramTag.read_count.txt";
my $sanity_check_path = "$result_tmp_dir/$paramTag.unencoded_G.sanity_check.txt";
open SANITYCHECK, ">", $sanity_check_path;
print SANITYCHECK join "", (join "\t", ('out_chrom', 'bedStart', 'bedEnd', 'strand', 'rd_seq', 'check_read_seq', 'check_genome_seq', 'cigar', 'softclip_length', 'match_length', 'check_read_pos', 'trim_length', 'num_unencoded_G', 'non_G_mismatch')), "\n";
my $total_read_num = 0;
my $passed_read_num = 0;
my $num_sanity_print = 1000;
open LONGCTSS, ">$long_ctss_bed_path";
open COLLAPSECTSS, ">$collapse_ctss_bed_path";
open UNGCOLLAPSECTSS, ">$ung_collapse_ctss_bed_path";
foreach my $chunk_num (sort {$a <=> $b} keys %{$chunk_info_hsh_ref}) {
my $file_check_ary_ref = [qw/chunk_long_ctss_bed_path chunk_collapse_ctss_bed_path/];
my ($valid) = &checkChunkResults($chunk_info_hsh_ref, $chunk_num, $file_check_ary_ref);#->330
if ($valid eq 'yes') {
&reportAndLogStatus("results of chunk $chunk_num is found. start parsing.", 10, "\n");#->1281
my $chunk_long_ctss_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_long_ctss_bed_path'};
my $chunk_collapse_ctss_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_collapse_ctss_bed_path'};
my $chunk_ung_collapse_ctss_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_ung_collapse_ctss_bed_path'};
my $chunk_log_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_log_path'};
my $chunk_sanity_check_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_sanity_check_path'};
my $chunk_fasta_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_fasta_path'};
my $num_sanity_check = 0;
open (CHUNKSANITYCHECK, "<", $chunk_sanity_check_path);
<CHUNKSANITYCHECK>;
while (<CHUNKSANITYCHECK>) {
print SANITYCHECK $_;
$num_sanity_check++;
last if $num_sanity_check >= $num_sanity_print;
}
close CHUNKSANITYCHECK;
open (CHUNKLONG, "<", $chunk_long_ctss_bed_path);
while (<CHUNKLONG>) {
print LONGCTSS $_;
}
close CHUNKLONG;
open (UNGCHUNKCOLLAPSE, "<", $chunk_ung_collapse_ctss_bed_path);
while (<UNGCHUNKCOLLAPSE>) {
print UNGCOLLAPSECTSS $_;
}
close UNGCHUNKCOLLAPSE;
open (CHUNKCOLLAPSE, "<", $chunk_collapse_ctss_bed_path);
while (<CHUNKCOLLAPSE>) {
print COLLAPSECTSS $_;
}
close CHUNKCOLLAPSE;
open (CHUNKLOG, "<", $chunk_log_path);
while (<CHUNKLOG>) {
chomp;