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scafe.tool.sc.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.sc.bam_to_ctss <---
<--- tool, single-cell mode, convert bam to ctss --->
Description:
This tool converts a 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.sc.bam_to_ctss [options] --bamPath --genome --outputPrefix --outDir
--bamPath <required> [string] bam file from cellranger, 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)
--TS_oligo_seq (optional) [string] Template switching oligo sequence for identification of
5'end (default=TTTCTTATATGGG)
--detect_TS_oligo (optional) [match/trim/skip/auto] in bam_to_ctss step, the modes of detecting TS oligo. 1. match: search for
TS oligo sequence on the read, identify the TSO/cDNA junction as 5'end of
the read. This works only when the error rate of the TS oligo region on
the read is low, otherwise a considerable number of read will be invalid.
2. trim: assuming the 1st N bases of the reads are TS oligo, without
checking the actual sequence. N is determined by the length of TS oligo.
3. skip: assuming the TS oligo was not sequenced, the 1st base of the read
will be treated as the 1st base after the TS oligo. 4. auto: automatically
determines the best mode, best of the observed error rate of the TS oligo
and the frequency of 5'end softclipped bases by the aligner. If softcliped
bases is close to the length of TS oligo, mode 1 or 2 will be chosen,
depending on the observed error rate of the TS oligo (error rate <= 0.1,
mode 1 will be chosen or mode 2 otherwise). If softcliped base os close to
zero, mode 3 will be chosen. (default=auto).
--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.sc.bam_to_ctss \
--overwrite=yes \
--bamPath=./demo/input/sc.solo/demo.cellranger.bam \
--genome=hg19.gencode_v32lift37 \
--outputPrefix=demo \
--outDir=./demo/output/sc.solo/bam_to_ctss/
=head1 VERSION
v0.9.0-beta [March 20, 2021]
-Initial pre-release
v1.0.0 [June 6, 2022]
-updated the TSO detection and trimming method
-updated regular expression to get UMI and CB to be more permissive
-use tabix/bgzip for splitting be into chunks
=cut
#<\doc>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<lastCmdCalled>
#
# notCalledBefore
#
# notCalledBefore
#
#<\lastCmdCalled>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<global>
my $scriptDirPath = dirname(rel2abs($0));
my $scriptAbsPath = abs_path($0);
my ($curntTimeStamp) = &timeStamp();#->2013
my $ARGVStr = join "\n", (¤tTime(), $scriptAbsPath, @ARGV);#->790
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, $TS_oligo_seq, $detect_TS_oligo, $genome, $outputPrefix, $outDir, $overwrite) = &readParameters();#->1911
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 1_defineHardCodedParam
#
#<section ID="defineHardCodedParam" num="1">
my $paramTag = "$outputPrefix";
my $rm_tmp_file = 'yes';
my $sanity_check_toggle = 'yes';
my $num_chunk = 100;
my $TS_oligo_check_tail_length = 5;
my $max_mismatch = 1;
my $regex_match_margin = 5;
my $softclip_margin = 7;
my $min_match_length = 5;
my $max_non_G_mismatch = 10; #---[2021/08/15 15:44] maximum number of non-G mismatch allowed
my $num_subsample = 100000; #---[2021/08/15 15:39] number of read to be sampled for TSO presence detection
my $min_frac_subsample = 0.01; #---[2021/08/15 15:39] subsample fraction (-s option in samtools) for TSO presence detection
$max_thread = 20 if $max_thread > 20;
#<\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, $scriptAbsPath);#->1392
&printStartOrFinishMessage("startMessage");#->1783
my ($tabix_bin, $bgzip_bin, $bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin) = &checkAllExecutable();#->347
my ($chrom_name_path, $chrom_size_path, $genome_fasta_path) = &checkChromNameSizeGenomeFastaPath($genome);#->447
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 3_checkTSO
#
#<section ID="checkTSO" num="3">
my ($chosen_TSO_detection_mode, $CB_UMI_offset) = &chooseTSODetectionMode($bedtools_bin, $samtools_bin, $bamPath, $exclude_flag, $include_flag, $TS_oligo_seq, $TS_oligo_check_tail_length, $num_subsample, $min_frac_subsample, $detect_TS_oligo);#->616
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 4_convert
#
#<section ID="convert" num="4">
my ($chrom_name_size_hsh_ref, $out_chrom_size_hsh_ref) = &generateChromNameConversionHsh($chrom_name_path, $chrom_size_path, $samtools_bin, $bamPath);#->844
my ($chunk_size) = &generateChunkSize($out_chrom_size_hsh_ref, $num_chunk);#->968
my ($chunk_info_hsh_ref) = &generateChunkInfoHsh($chrom_name_size_hsh_ref, $chunk_size, $result_tmp_dir);#->914
&processPerChunk($bamPath, $bedtools_bin, $samtools_bin, $max_thread, $chunk_info_hsh_ref, $chrom_name_size_hsh_ref, $exclude_flag, $include_flag, $min_MAPQ, $TS_oligo_seq, $genome_fasta_path, $out_chrom_size_hsh_ref, $sanity_check_toggle, $TS_oligo_check_tail_length, $max_mismatch, $regex_match_margin, $softclip_margin, $min_match_length, $max_non_G_mismatch, $chosen_TSO_detection_mode, $CB_UMI_offset);#->1817
&poolChunkResults($tabix_bin, $bgzip_bin, $max_thread, $chunk_info_hsh_ref, $result_bed_dir, $result_log_dir, $paramTag, $result_tmp_dir, $rm_tmp_file);#->1532
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 5_finishingTasks
#
#<section ID="finishingTasks" num="5">
&printOutputFileListAndReadme($ARGVStr, $paramTag, $outDir);#->1668
&printStartOrFinishMessage("finishMessage");#->1783
#<\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=23]:
# calculateEditDistance, checkAllExecutable, checkChromNameSizeGenomeFastaPath
# checkChunkResults, checkTSOPresence, chooseTSODetectionMode
# defineTSOCheckParam, generateChromNameConversionHsh, generateChunkInfoHsh
# generateChunkSize, generateMisMatchRegex, getCTSS_match_TS_oligo
# getCTSS_skip_TS_oligo, getCTSS_trim_TS_oligo, getCheckSeq
# getChunkFasta, getEnd5StartUnencodedG, getSoftclipMatch
# getSubsampleFraction, getTSOMatchPosition, pileupCBDedupUMI
# poolChunkResults, processPerChunk
#
#====================================================================================================================================================#
sub calculateEditDistance {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: checkTSOPresence|494
# primaryAppearInSection: >none
# secondaryAppearInSection: >none
# input: $seq1, $seq2
# output: $edit
# toCall: my ($edit) = &calculateEditDistance($seq1, $seq2);
# calledInLine: 605
#....................................................................................................................................................#
my ($seq1, $seq2) = @_;
my $edit = '0';
my $match = '1';
my $mismatch = '-1';
my $gap = '-1';
my @matrix;
$matrix[0][0]{score} = 0;
$matrix[0][0]{pointer} = "none";
for(my $j = 1; $j <= length($seq1); $j++) {
$matrix[0][$j]{score} = $gap * $j;
$matrix[0][$j]{pointer} = "left";
}
for (my $i = 1; $i <= length($seq2); $i++) {
$matrix[$i][0]{score} = $gap * $i;
$matrix[$i][0]{pointer} = "up";
}
# fill
for(my $i = 1; $i <= length($seq2); $i++) {
for(my $j = 1; $j <= length($seq1); $j++) {
my ($diagonal_score, $left_score, $up_score);
# calculate match score
my $letter1 = substr($seq1, $j-1, 1);
my $letter2 = substr($seq2, $i-1, 1);
if ($letter1 eq $letter2) {
$diagonal_score = $matrix[$i-1][$j-1]{score} + $match;
} else {
$diagonal_score = $matrix[$i-1][$j-1]{score} + $mismatch;
}
# calculate gap scores
$up_score = $matrix[$i-1][$j]{score} + $gap;
$left_score = $matrix[$i][$j-1]{score} + $gap;
# choose best score
if ($diagonal_score >= $up_score) {
if ($diagonal_score >= $left_score) {
$matrix[$i][$j]{score} = $diagonal_score;
$matrix[$i][$j]{pointer} = "diagonal";
} else {
$matrix[$i][$j]{score} = $left_score;
$matrix[$i][$j]{pointer} = "left";
}
} else {
if ($up_score >= $left_score) {
$matrix[$i][$j]{score} = $up_score;
$matrix[$i][$j]{pointer} = "up";
} else {
$matrix[$i][$j]{score} = $left_score;
$matrix[$i][$j]{pointer} = "left";
}
}
}
}
# trace-back
my $align1 = "";
my $align2 = "";
# start at last cell of matrix
my $j = length($seq1);
my $i = length($seq2);
while (1) {
last if $matrix[$i][$j]{pointer} eq "none"; # ends at first cell of matrix
if ($matrix[$i][$j]{pointer} eq "diagonal") {
$align1 .= substr($seq1, $j-1, 1);
$align2 .= substr($seq2, $i-1, 1);
$i--;
$j--;
} elsif ($matrix[$i][$j]{pointer} eq "left") {
$align1 .= substr($seq1, $j-1, 1);
$align2 .= "-";
$j--;
} elsif ($matrix[$i][$j]{pointer} eq "up") {
$align1 .= "-";
$align2 .= substr($seq2, $i-1, 1);
$i--;
}
}
$align1 = reverse $align1;
$align2 = reverse $align2;
for (my $i=0; $i<length($align1); ++$i){
if (substr($align1,$i,1) ne substr($align2,$i,1)){
++$edit;
}
}
return($edit);
}
sub checkAllExecutable {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1991
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|155
# 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: 170
#....................................................................................................................................................#
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");#->1991
{
my $stdOut = `$tabix_bin --version 2>&1`;
if ($stdOut =~ m/tabix \(htslib\) (\S+)/) {
&reportAndLogStatus("Checking: tabix version: $1", 0, "\n");#->1991
} 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");#->1991
} 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");#->1991
} 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");#->1991
} 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");#->1991
} 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");#->1991
} 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");#->1991
} else {
die "bedGraphToBigWig not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$bigWigAverageOverBed_bin 2>&1`;
if ($stdOut =~ m/bigWigAverageOverBed v(\S+)/) {
&reportAndLogStatus("Checking: bigWigAverageOverBed version: $1", 0, "\n");#->1991
} else {
die "bigWigAverageOverBed is not installed properly. Quitting.\n";
}
}
return ($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|155
# 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: 171
#....................................................................................................................................................#
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|1991
# appearInSub: poolChunkResults|1532, processPerChunk|1817
# primaryAppearInSection: >none
# secondaryAppearInSection: 4_convert|184
# input: $chunk_info_hsh_ref, $chunk_num, $file_check_ary_ref
# output: $valid
# toCall: my ($valid) = &checkChunkResults($chunk_info_hsh_ref, $chunk_num, $file_check_ary_ref);
# calledInLine: 1565, 1854
#....................................................................................................................................................#
my ($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");#->1991
$valid = 'no' if not -f $file_path;
}
return ($valid);
}
sub checkTSOPresence {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: calculateEditDistance|240, reportAndLogStatus|1991
# appearInSub: chooseTSODetectionMode|616
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_checkTSO|176
# input: $TS_oligo_check_tail_length, $TS_oligo_seq, $bamPath, $bedtools_bin, $exclude_flag, $frac_subsample, $include_flag, $num_subsample, $samtools_bin
# output: $CB_UMI_offset, $avg_softclip_length, $error_rate
# toCall: my ($error_rate, $avg_softclip_length, $CB_UMI_offset) = &checkTSOPresence($bedtools_bin, $samtools_bin, $bamPath, $exclude_flag, $include_flag, $TS_oligo_seq, $TS_oligo_check_tail_length, $num_subsample, $frac_subsample);
# calledInLine: 635
#....................................................................................................................................................#
my ($bedtools_bin, $samtools_bin, $bamPath, $exclude_flag, $include_flag, $TS_oligo_seq, $TS_oligo_check_tail_length, $num_subsample, $frac_subsample) = @_;
my $ex_F_param = '';
my $in_f_param = '';
my $subsample_s_param = '';
my $TSO_length = length($TS_oligo_seq);
my $check_start_pos = $TSO_length-$TS_oligo_check_tail_length;
my $check_TS_oligo_seq = substr $TS_oligo_seq, $check_start_pos, $TS_oligo_check_tail_length;
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";
}
$in_f_param = join " ", @flagAry;
}
if ($frac_subsample >= 0.75) {
$subsample_s_param = '';
} else {
$subsample_s_param = "-s $frac_subsample";
}
my $perl_covert_readname = 'while (<STDIN>) {chomp; @samAry = split /\t/;if ($_ =~ m/\tCB:Z:([^\t]+)\t.*UB:Z:([^\t]+)/) {$samAry[0] = $1."_".$2."_".$samAry[9];print join "", (join "\t", @samAry[0..10]), "\n";} elsif ($_ =~ m/\tUB:Z:([^\t]+)\t.*CB:Z:([^\t]+)/) {$samAry[0] = $2."_".$1."_".$samAry[9];print join "", (join "\t", @samAry[0..10]), "\n";} elsif ($_ =~ m/^\@/) {print "$_\n";}};';
my $read_hsh_ref = {};
open (BAMIN, "$samtools_bin view -h $subsample_s_param $in_f_param $ex_F_param $bamPath 2>/dev/null | perl -e \'$perl_covert_readname\' | $samtools_bin view -b - | $bedtools_bin bamtobed -cigar -i stdin | head -n $num_subsample |");
my $total_num = 0;
my $data_hsh_ref = {};
while (my $line = <BAMIN>) {
chomp $line;
my ($chrom, $bedStart, $bedEnd, $CB_UMI_Seq, $MAPQ, $strand, $cigar) = split /\t+/, $line;
my ($CB, $UMI, $rd_seq) = split /\_/, $CB_UMI_Seq;
$rd_seq =~ s/\/\d$//;
my $softclip_length = 0;
if ($strand eq '+') {
if ($cigar =~ m/^(\d+)S(\d+)M/) {
$softclip_length = $1;
}
} else {
$rd_seq = reverse $rd_seq;
$rd_seq =~ tr/ATGCatgc/TACGtacg/;
if ($cigar =~ m/(\d+)M(\d+)S$/) {
$softclip_length = $2;
}
}
$CB =~ s/\-.+$//;
my $UMI_length = length($UMI);
my $CB_length = length($CB);
push @{$data_hsh_ref->{'rd_seq'}}, $rd_seq;
push @{$data_hsh_ref->{'softclip_length'}}, $softclip_length;
push @{$data_hsh_ref->{'UMI_length'}}, $UMI_length;
push @{$data_hsh_ref->{'CB_length'}}, $CB_length;
$data_hsh_ref->{'count_softclip_length'}{$softclip_length}++;
$total_num++;
&reportAndLogStatus("$total_num read sampled", 10, "\n") if $total_num%10000==0;#->1991
}
close BAMIN;
my $avg_softclip_length = sprintf "%.5f", sum(@{$data_hsh_ref->{'softclip_length'}})/$total_num;
my $avg_CB_length = sprintf "%.5f", sum(@{$data_hsh_ref->{'CB_length'}})/$total_num;
my $avg_UMI_length = sprintf "%.5f", sum(@{$data_hsh_ref->{'UMI_length'}})/$total_num;
my $rank = 0;
my $rank_hsh_ref = {};
foreach my $softclip_length (sort {$data_hsh_ref->{'count_softclip_length'}{$b} <=> $data_hsh_ref->{'count_softclip_length'}{$a}} keys %{$data_hsh_ref->{'count_softclip_length'}}) {
$rank++;
my $pct = sprintf "%.5f", 100*($data_hsh_ref->{'count_softclip_length'}{$softclip_length}/$total_num);
$rank_hsh_ref->{$rank} = [$softclip_length, $pct];
}
my ($first_softclip_length, $first_softclip_length_pct) = @{$rank_hsh_ref->{1}};
my ($second_softclip_length, $second_softclip_length_pct) = @{$rank_hsh_ref->{2}};
my ($third_softclip_length, $third_softclip_length_pct) = @{$rank_hsh_ref->{3}};
&reportAndLogStatus("average CB length=$avg_CB_length", 10, "\n");#->1991
&reportAndLogStatus("average UMI length=$avg_UMI_length", 10, "\n");#->1991
&reportAndLogStatus("average softclip length=$avg_softclip_length", 10, "\n");#->1991
&reportAndLogStatus("1st ranked softclip length=$first_softclip_length [$first_softclip_length_pct%]", 10, "\n");#->1991
&reportAndLogStatus("2nd ranked softclip length=$second_softclip_length [$second_softclip_length_pct%]", 10, "\n");#->1991
&reportAndLogStatus("3rd ranked softclip length=$third_softclip_length [$third_softclip_length_pct%]", 10, "\n");#->1991
my $CB_UMI_offset = 0;
my $CB_UMI_TSO_length = $TSO_length+$avg_CB_length+$avg_UMI_length;
if ($avg_softclip_length >= $CB_UMI_TSO_length - 2 and $avg_softclip_length <= $CB_UMI_TSO_length + 2) {
$CB_UMI_offset = $avg_CB_length+$avg_UMI_length;
}
my $offset_check_start_pos = $check_start_pos+$CB_UMI_offset;
&reportAndLogStatus("calculating error rate with offset_check_start_pos = $offset_check_start_pos", 10, "\n");#->1991
foreach my $rd_seq (@{$data_hsh_ref->{'rd_seq'}}) {
my $check_rd_seq = substr $rd_seq, $offset_check_start_pos, $TS_oligo_check_tail_length;
my ($edit_dist) = &calculateEditDistance($check_rd_seq, $check_TS_oligo_seq);#->240
my $dist_per_nt = sprintf "%.5f", $edit_dist/$TS_oligo_check_tail_length;
push @{$data_hsh_ref->{'dist_per_nt'}}, $dist_per_nt;
}
my $error_rate = sprintf "%.5f", sum(@{$data_hsh_ref->{'dist_per_nt'}})/$total_num;
&reportAndLogStatus("TSO error_rate=$error_rate", 10, "\n");#->1991
return ($error_rate, $avg_softclip_length, $CB_UMI_offset);
}
sub chooseTSODetectionMode {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: checkTSOPresence|494, getSubsampleFraction|1336, reportAndLogStatus|1991
# appearInSub: >none
# primaryAppearInSection: 3_checkTSO|176
# secondaryAppearInSection: >none
# input: $TS_oligo_check_tail_length, $TS_oligo_seq, $bamPath, $bedtools_bin, $detect_TS_oligo, $exclude_flag, $include_flag, $min_frac_subsample, $num_subsample, $samtools_bin
# output: $CB_UMI_offset, $chosen_TSO_detection_mode
# toCall: my ($chosen_TSO_detection_mode, $CB_UMI_offset) = &chooseTSODetectionMode($bedtools_bin, $samtools_bin, $bamPath, $exclude_flag, $include_flag, $TS_oligo_seq, $TS_oligo_check_tail_length, $num_subsample, $min_frac_subsample, $detect_TS_oligo);
# calledInLine: 179
#....................................................................................................................................................#
my ($bedtools_bin, $samtools_bin, $bamPath, $exclude_flag, $include_flag, $TS_oligo_seq, $TS_oligo_check_tail_length, $num_subsample, $min_frac_subsample, $detect_TS_oligo) = @_;
my $chosen_TSO_detection_mode;
my $max_error_rate = 0.10; #---[2021/08/15 16:19] max error_rate
my $softclip_margin = 3;
&reportAndLogStatus("Checking TSO presence in bam", 10, "\n");#->1991
my ($frac_subsample) = &getSubsampleFraction($min_frac_subsample, $num_subsample, $samtools_bin, $bamPath);#->1336
my ($error_rate, $avg_softclip_length, $CB_UMI_offset) = &checkTSOPresence($bedtools_bin, $samtools_bin, $bamPath, $exclude_flag, $include_flag, $TS_oligo_seq, $TS_oligo_check_tail_length, $num_subsample, $frac_subsample);#->494
if ($detect_TS_oligo eq 'auto') {
if ($avg_softclip_length <= $softclip_margin) {
$chosen_TSO_detection_mode = 'skip';
&reportAndLogStatus("chosen_TSO_detection_mode is auto defined as $chosen_TSO_detection_mode", 10, "\n");#->1991
} elsif ($avg_softclip_length >= ($CB_UMI_offset+length($TS_oligo_seq)-$softclip_margin) and $avg_softclip_length <= ($CB_UMI_offset+length($TS_oligo_seq)+$softclip_margin)) {
if ($error_rate <= $max_error_rate) {
$chosen_TSO_detection_mode = 'match';
&reportAndLogStatus("chosen_TSO_detection_mode is auto defined as $chosen_TSO_detection_mode", 10, "\n");#->1991
} else {
$chosen_TSO_detection_mode = 'trim';
&reportAndLogStatus("chosen_TSO_detection_mode is auto defined as $chosen_TSO_detection_mode", 10, "\n");#->1991
}
} else {
die "Quitting: Auto detection of TS oligo failed. Average softclip length is ambiguous.\n";
}
} else {
$chosen_TSO_detection_mode = $detect_TS_oligo;
&reportAndLogStatus("chosen_TSO_detection_mode is user defined as $chosen_TSO_detection_mode", 10, "\n");#->1991
}
return ($chosen_TSO_detection_mode, $CB_UMI_offset);
}
sub convertBamToCTSS {
#....................................................................................................................................................#
# subroutineCategory: specific
# dependOnSub: generateMisMatchRegex|993, getCTSS_match_TS_oligo|1062, getCTSS_skip_TS_oligo|1096, getCTSS_trim_TS_oligo|1128, reportAndLogStatus|1991
# appearInSub: processPerChunk|1817
# primaryAppearInSection: >none
# secondaryAppearInSection: 4_convert|184
# input: $CB_UMI_offset, $TS_oligo_check_tail_length, $TS_oligo_seq, $bamPath, $bedtools_bin, $chosen_TSO_detection_mode, $chrom_name_size_hsh_ref, $chunk_UMI_CB_ctss_bed_path, $chunk_endPos, $chunk_fasta_offset_pos, $chunk_fasta_path, $chunk_log_path, $chunk_sanity_check_path, $chunk_seq_str, $chunk_startPos, $chunk_unsort_UMI_CB_ctss_bed_path, $exclude_flag, $include_flag, $max_mismatch, $max_non_G_mismatch, $min_MAPQ, $min_match_length, $regex_match_margin, $region_str, $samtools_bin, $sanity_check_toggle, $softclip_margin
# output:
# toCall: &convertBamToCTSS($bamPath, $chrom_name_size_hsh_ref, $exclude_flag, $include_flag, $bedtools_bin, $samtools_bin, $min_MAPQ, $chunk_UMI_CB_ctss_bed_path, $chunk_unsort_UMI_CB_ctss_bed_path, $region_str, $chunk_log_path, $chunk_startPos, $chunk_endPos, $TS_oligo_seq, $chunk_fasta_path, $chunk_seq_str, $chunk_fasta_offset_pos, $chunk_sanity_check_path, $sanity_check_toggle, $TS_oligo_check_tail_length, $max_mismatch, $regex_match_margin, $softclip_margin, $min_match_length, $max_non_G_mismatch, $chosen_TSO_detection_mode, $CB_UMI_offset);
# calledInLine: 1875
#....................................................................................................................................................#
my ($bamPath, $chrom_name_size_hsh_ref, $exclude_flag, $include_flag, $bedtools_bin, $samtools_bin, $min_MAPQ, $chunk_UMI_CB_ctss_bed_path, $chunk_unsort_UMI_CB_ctss_bed_path, $region_str, $chunk_log_path, $chunk_startPos, $chunk_endPos, $TS_oligo_seq, $chunk_fasta_path, $chunk_seq_str, $chunk_fasta_offset_pos, $chunk_sanity_check_path, $sanity_check_toggle, $TS_oligo_check_tail_length, $max_mismatch, $regex_match_margin, $softclip_margin, $min_match_length, $max_non_G_mismatch, $chosen_TSO_detection_mode, $CB_UMI_offset) = @_;
if (not -s $chunk_UMI_CB_ctss_bed_path) {
#&reportAndLogStatus("converting $paramTag bam to ctss bed", 10, "\n");#->1991
my $perl_covert_readname = 'while (<STDIN>) {chomp; @samAry = split /\t/;if ($_ =~ m/\tCB:Z:([^\t]+)\t.*UB:Z:([^\t]+)/) {$samAry[0] = $1."_".$2."_".$samAry[9];print join "", (join "\t", @samAry[0..10]), "\n";} elsif ($_ =~ m/\tUB:Z:([^\t]+)\t.*CB:Z:([^\t]+)/) {$samAry[0] = $2."_".$1."_".$samAry[9];print join "", (join "\t", @samAry[0..10]), "\n";} elsif ($_ =~ m/^\@/) {print "$_\n";}};';
my $margin = 20;
my $min_total_match = 30;
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";
}
$in_f_param = join " ", @flagAry;
}
#---[6/18/15 14:23] get all
open (BAMIN, "$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_UMI_CB_ctss_bed_path");
open (CTSSOUT, ">", $chunk_unsort_UMI_CB_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 $TS_oligo_length = length $TS_oligo_seq;
my $check_TS_oligo_substr = -1*$TS_oligo_check_tail_length;
my $check_TS_oligo_seq = substr $TS_oligo_seq, $check_TS_oligo_substr;
my $min_softclip_length = $CB_UMI_offset + $TS_oligo_length - $softclip_margin;
my $max_softclip_length = $CB_UMI_offset + $TS_oligo_length + $softclip_margin;
my $min_regex_match_pos = $CB_UMI_offset + $TS_oligo_length - $regex_match_margin;
my $max_regex_match_pos = $CB_UMI_offset + $TS_oligo_length + $regex_match_margin;
my $match_string = $check_TS_oligo_seq;
my ($regex_string) = &generateMisMatchRegex($match_string, $max_mismatch);#->993
my $check_TS_oligo_regex = $regex_string;
#&reportAndLogStatus("REGEX for TS_oligo check:$check_TS_oligo_regex", 10, "\n");#->1991
my $chunk_total_read_num = 0;
my $chunk_passed_read_num = 0;
while (my $line = <BAMIN>) {
chomp $line;
my ($chrom, $bedStart, $bedEnd, $CB_UMI_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 < $min_total_match;
my ($CB, $UMI, $bam_seq) = split /\_/, $CB_UMI_Seq;
my $readname = join "_", ($CB, $UMI);
$bam_seq =~ s/\/\d$//;
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);#->1991
my ($end5Start, $num_unencoded_G, $sanity_check_ary_ref);
if ($chosen_TSO_detection_mode eq 'match') {
($end5Start, $num_unencoded_G, $sanity_check_ary_ref) = &getCTSS_match_TS_oligo($bedStart, $bedEnd, $strand, $bam_seq, $check_TS_oligo_seq, $chunk_startPos, $chunk_endPos, $chunk_seq_str, $chunk_fasta_offset_pos, $samtools_bin, $out_chrom, $cigar, $min_softclip_length, $max_softclip_length, $min_regex_match_pos, $max_regex_match_pos, $check_TS_oligo_regex, $min_match_length, $max_non_G_mismatch, $CB_UMI_offset);#->1062
} elsif ($chosen_TSO_detection_mode eq 'trim') {
($end5Start, $num_unencoded_G, $sanity_check_ary_ref) = &getCTSS_trim_TS_oligo($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, $max_non_G_mismatch, $TS_oligo_length, $CB_UMI_offset);#->1128
} elsif ($chosen_TSO_detection_mode eq 'skip') {
($end5Start, $num_unencoded_G, $sanity_check_ary_ref) = &getCTSS_skip_TS_oligo($bedStart, $bedEnd, $strand, $bam_seq, $chunk_startPos, $chunk_endPos, $chunk_seq_str, $chunk_fasta_offset_pos, $samtools_bin, $out_chrom, $cigar, $min_match_length, $max_non_G_mismatch);#->1096
} else {
die "Quitting: chosen_TSO_detection_mode is not probalbly defined\n";
}
if ($sanity_check_toggle eq 'yes') {
print SANITYCHECK join "", (join "\t", (@{$sanity_check_ary_ref})), "\n";
}
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, $readname, $num_unencoded_G, $strand)), "\n";
}
}
}
}
open (LOG, ">$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 $chunk_unsort_UMI_CB_ctss_bed_path >$chunk_UMI_CB_ctss_bed_path";
system "rm $chunk_unsort_UMI_CB_ctss_bed_path";
} else {
&reportAndLogStatus("chunk_UMI_CB_ctss_bed_path found. Skipping conversion.", 10, "\n");#->1991
}
return ();
}
sub currentTime {
#....................................................................................................................................................#
# subroutineCategory: general
# dependOnSub: >none
# appearInSub: printStartOrFinishMessage|1783, reportAndLogStatus|1991
# primaryAppearInSection: >none
# secondaryAppearInSection: 2_defineOutDirPath|155, 5_finishingTasks|196
# input: none
# output: $runTime
# toCall: my ($runTime) = ¤tTime();
# calledInLine: 116, 1799, 1803, 1808, 1812, 2007, 2008
#....................................................................................................................................................#
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 defineTSOCheckParam {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: getCTSS_match_TS_oligo|1062, getCTSS_skip_TS_oligo|1096, getCTSS_trim_TS_oligo|1128
# primaryAppearInSection: >none
# secondaryAppearInSection: >none
# input: $bam_seq, $bedEnd, $bedStart
# output: $TSO_match_valid, $TSS_within_chunk, $check_genome_seq, $check_read_pos, $check_read_seq, $end5Start, $match_length, $match_valid, $non_G_mismatch, $num_nt_extract_check, $num_unencoded_G, $overall_valid, $rd_seq, $readEnd, $readStart, $softclip_length, $softclip_valid, $trim_length
# toCall: my ($rd_seq, $readStart, $readEnd, $end5Start, $num_nt_extract_check, $check_read_seq, $check_genome_seq, $check_read_pos, $trim_length, $num_unencoded_G, $non_G_mismatch, $softclip_valid, $match_valid, $TSS_within_chunk, $TSO_match_valid, $overall_valid, $softclip_length, $match_length) = &defineTSOCheckParam($bedStart, $bedEnd, $bam_seq);
# calledInLine: 1075, 1112, 1141
#....................................................................................................................................................#
my ($bedStart, $bedEnd, $bam_seq) = @_;
my $end5Start = undef;
my $num_nt_extract_check = 10;
my $overall_valid = 'no';
my $check_read_seq = '__na';
my $check_genome_seq = '__na';
my $check_read_pos = '__na';
my $trim_length = '__na';
my $num_unencoded_G = '__na';
my $non_G_mismatch = '__na';
my $softclip_valid = '__na';
my $match_valid = '__na';
my $TSS_within_chunk = '__na';
my $TSO_match_valid = '__na';
my $softclip_length = '__na';
my $match_length = '__na';
my ($readStart, $readEnd) = ($bedStart, $bedEnd);
my $rd_seq = $bam_seq;
return ($rd_seq, $readStart, $readEnd, $end5Start, $num_nt_extract_check, $check_read_seq, $check_genome_seq, $check_read_pos, $trim_length, $num_unencoded_G, $non_G_mismatch, $softclip_valid, $match_valid, $TSS_within_chunk, $TSO_match_valid, $overall_valid, $softclip_length, $match_length);
}
sub generateChromNameConversionHsh {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1991
# appearInSub: >none
# primaryAppearInSection: 4_convert|184
# 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, $bamPath);
# calledInLine: 187
#....................................................................................................................................................#
my ($chrom_name_path, $chrom_size_path, $samtools_bin, $bamPath) = @_;
my $min_hit_read_pct = 10;
&reportAndLogStatus("getting bam header chromosome", 10, "\n");#->1991
my $actual_bam_chrom_name_hsh_ref = {};
my $total_num_read = 0;
open SAMTOOLS, "$samtools_bin idxstats $bamPath 2>/dev/null |";
while (<SAMTOOLS>) {
chomp;
my ($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");#->1991
}
}
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");#->1991
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|1991
# appearInSub: >none
# primaryAppearInSection: 4_convert|184
# 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, $result_tmp_dir);
# calledInLine: 189
#....................................................................................................................................................#
my ($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");#->1991
my $chunk_CB_ctss_bed_path = "$result_tmp_dir/$file_tag.CB.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 $chunk_UMI_CB_ctss_bed_path = "$result_tmp_dir/$file_tag.UMI_CB.ctss.bed";
my $chunk_unsort_UMI_CB_ctss_bed_path = "$result_tmp_dir/$file_tag.unsorted.UMI_CB.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}{'file_tag'} = $file_tag;
$chunk_info_hsh_ref->{$chunk_num}{'loc'} = [$chunk_chrom, $chunk_startPos, $chunk_endPos];
$chunk_info_hsh_ref->{$chunk_num}{'region_str'} = "$chunk_chrom:$chunk_startPos-$chunk_endPos";