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scafe.tool.cm.remove_strand_invader
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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 List::Util qw (sum shuffle min max);
use threads ('stack_size' => 64*4096);
use threads::shared;
use Cwd 'abs_path';
#<\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.cm.remove_strand_invader <---
<--- tool, common mode, remove strand invader artefact --->
Description:
This tool identify and remove strand invader artefact from a ctss bed file,
by aligning the sequence immediate upstream of a ctss to TS oligo sequence
Usage:
scafe.tool.cm.remove_strand_invader [options] --ctss_bed_path --genome --outputPrefix --outDir
--ctss_bed_path <required> [string] "collapse" ctss file from scafe.tool.sc.bam_to_ctss.pl,
4th column is number of cells and 5th column is number UMI
--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
--min_edit_distance (optional) [integer] edit distance threshold to define strand invader
the smaller value, the more stringent defintion of strand invader
(default=5)
--min_end_non_G_num (optional) [integer] immediate upstream non-G number threshold to define strand invader
the smaller value, the more stringent defintion of strand invader
(default=2)
--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)
--overwrite (optional) [yes/no] [yes/no] erase outDir/outputPrefix before running (default=no)
Dependencies:
bedtools
tabix
bgzip
For demo, cd to SCAFE dir and run,
scafe.tool.cm.remove_strand_invader \
--overwrite=yes \
--ctss_bed_path=./demo/output/sc.solo/bam_to_ctss/demo/bed/demo.collapse.ctss.bed.gz \
--genome=hg19.gencode_v32lift37 \
--outputPrefix=demo \
--outDir=./demo/output/sc.solo/remove_strand_invader/
=head1 VERSION
v0.9.0-beta [March 20, 2021]
-Initial pre-release
v1.0.0 [June 6, 2022]
-No update
=cut#
#<\doc>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<lastCmdCalled>
#
# notCalledBefore
#
# notCalledBefore
#
#<\lastCmdCalled>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<global>
my $scriptDirPath = dirname(rel2abs($0));
my $scriptAbsPath = abs_path($0);
my ($curntTimeStamp) = &timeStamp();#->1158
my $ARGVStr = join "\n", (¤tTime(), $scriptAbsPath, @ARGV);#->462
my $globalReadmeHsh_ref = {};
our $tmplog_fh;
#<\global>
#====================================================================================================================================================#
#====================================================================================================================================================#
{ #Main sections lexical scope starts
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 0_startingTasks
#
#<section ID="startingTasks" num="0">
my ($ctss_bed_path, $TS_oligo_seq, $min_edit_distance, $min_end_non_G_num, $max_thread, $genome, $outputPrefix, $outDir, $overwrite) = &readParameters();#->1083
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 1_defineHardCodedParam
#
#<section ID="defineHardCodedParam" num="1">
my $chunk_size = 100000000;
my $bgzip_sort_thread = 5;
$max_thread = 10 if $max_thread > 10;
my $output_cutoff_hsh_ref = {
'min_edit_distance' => $min_edit_distance, #---[2019/12/19 20:21] is strand invader if <= X edit distance in the whole 13 nucleotides (i.e. 13 to turn out)
'min_end_non_G_num' => $min_end_non_G_num, #---[2019/12/19 20:21] is strand invader if <= X nonGs in the last 3 nucleotides (i.e. 3 to turn out)
};
my $paramTag = "$outputPrefix";
#<\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 $result_fasta_dir = "$result_dir/fasta/"; push @mkDirAry, $result_fasta_dir;
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 $result_tmp_dir = "$result_dir/tmp/"; push @mkDirAry, $result_tmp_dir;
my $result_script_dir = "$result_dir/script/"; push @mkDirAry, $result_script_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, $scriptAbsPath);#->711
&printStartOrFinishMessage("startMessage");#->940
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 3_count
#
#<section ID="count" num="3">
my ($tabix_bin, $bgzip_bin, $bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin) = &checkAllExecutable();#->311
my ($fasta_path, $chrom_size_path) = &checkFastaChromSizePath($genome);#->440
my ($chrom_size_hsh_ref) = &readChromSize($chrom_size_path);#->1058
my ($chunk_info_hsh_ref) = &generateChunkInfoHsh($chrom_size_hsh_ref, $chunk_size, $result_tmp_dir);#->587
&processPerChunk($ctss_bed_path, $fasta_path, $result_fasta_dir, $TS_oligo_seq, $bedtools_bin, $tabix_bin, $bgzip_bin, $bgzip_sort_thread, $max_thread, $output_cutoff_hsh_ref, $chunk_info_hsh_ref, $chrom_size_path, $paramTag);#->974
&poolChunkResults($tabix_bin, $chunk_info_hsh_ref, $result_bed_dir, $result_log_dir, $paramTag);#->736
system("rm -rf $result_tmp_dir");
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 4_finishingTasks
#
#<section ID="finishingTasks" num="4">
&printOutputFileListAndReadme($ARGVStr, $paramTag, $outDir);#->820
&printStartOrFinishMessage("finishMessage");#->940
#<\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
#
# time [n=1]:
# timeStamp
#
# unassigned [n=10]:
# calculateEditDistance, checkAllExecutable, checkChunkResults
# checkFastaChromSizePath, filterCTSS, generateChunkInfoHsh
# generateUpstreamSeq, poolChunkResults, processPerChunk
# readChromSize
#
#====================================================================================================================================================#
sub calculateEditDistance {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: filterCTSS|480
# primaryAppearInSection: >none
# secondaryAppearInSection: >none
# input: $seq1, $seq2
# output: $edit
# toCall: my ($edit) = &calculateEditDistance($seq1, $seq2);
# calledInLine: 535
#....................................................................................................................................................#
my ($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|1136
# appearInSub: >none
# primaryAppearInSection: 3_count|153
# 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: 156
#....................................................................................................................................................#
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");#->1136
{
my $stdOut = `$tabix_bin --version 2>&1`;
if ($stdOut =~ m/tabix \(htslib\) (\S+)/) {
&reportAndLogStatus("Checking: tabix version: $1", 0, "\n");#->1136
} 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");#->1136
} 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");#->1136
} 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");#->1136
} 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");#->1136
} 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");#->1136
} 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");#->1136
} else {
die "bedGraphToBigWig not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$bigWigAverageOverBed_bin 2>&1`;
if ($stdOut =~ m/bigWigAverageOverBed v(\S+)/) {
&reportAndLogStatus("Checking: bigWigAverageOverBed version: $1", 0, "\n");#->1136
} else {
die "bigWigAverageOverBed is not installed properly. Quitting.\n";
}
}
return ($tabix_bin, $bgzip_bin, $bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin);
}
sub checkChunkResults {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1136
# appearInSub: poolChunkResults|736, processPerChunk|974
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_count|153
# input: $chunk_info_hsh_ref, $chunk_num
# output: $valid
# toCall: my ($valid) = &checkChunkResults($chunk_info_hsh_ref, $chunk_num);
# calledInLine: 761, 1016
#....................................................................................................................................................#
my ($chunk_info_hsh_ref, $chunk_num) = @_;
my $valid = 'yes';
foreach my $file_to_check (qw/chunk_log_path/) {
my $file_path = $chunk_info_hsh_ref->{$chunk_num}{$file_to_check};
#&reportAndLogStatus("$file_path", 10, "\n");#->1136
$valid = 'no' if not -s $file_path;
}
foreach my $ctss_type (qw/chunk_pass_ctss chunk_invader_ctss/) {
my $file_path = $chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'sorted'}{$ctss_type};
#&reportAndLogStatus("$file_path", 10, "\n");#->1136
$valid = 'no' if not -s $file_path;
}
return ($valid);
}
sub checkFastaChromSizePath {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 3_count|153
# secondaryAppearInSection: >none
# input: $genome
# output: $chrom_size_path, $fasta_path
# toCall: my ($fasta_path, $chrom_size_path) = &checkFastaChromSizePath($genome);
# calledInLine: 157
#....................................................................................................................................................#
my ($genome) = @_;
my $dirPath = dirname(rel2abs($0));
my $fasta_path = "$dirPath/../resources/genome/$genome/fasta/genome.fa";
my $chrom_size_path = "$dirPath/../resources/genome/$genome/tsv/chrom.sizes.tsv";
die "genome $genome does not have fasta_path. Please rerun prep_genome step\n" if not -s $fasta_path;
die "genome $genome does not have chrom_sizes_path. Please rerun prep_genome step\n" if not -s $chrom_size_path;
return ($fasta_path, $chrom_size_path);
}
sub currentTime {
#....................................................................................................................................................#
# subroutineCategory: general
# dependOnSub: >none
# appearInSub: printStartOrFinishMessage|940, reportAndLogStatus|1136
# primaryAppearInSection: >none
# secondaryAppearInSection: 2_defineOutDirPath|133, 4_finishingTasks|167
# input: none
# output: $runTime
# toCall: my ($runTime) = ¤tTime();
# calledInLine: 99, 956, 960, 965, 969, 1152, 1153
#....................................................................................................................................................#
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 filterCTSS {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: calculateEditDistance|204, reportAndLogStatus|1136
# appearInSub: processPerChunk|974
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_count|153
# input: $TS_oligo_seq, $bgzip_bin, $bgzip_sort_thread, $chunk_info_hsh_ref, $chunk_num, $count_cutoff_hsh_ref, $output_cutoff_hsh_ref
# output:
# toCall: &filterCTSS($chunk_info_hsh_ref, $chunk_num, $TS_oligo_seq, $bgzip_bin, $bgzip_sort_thread, $output_cutoff_hsh_ref, $count_cutoff_hsh_ref);
# calledInLine: 1024
#....................................................................................................................................................#
my ($chunk_info_hsh_ref, $chunk_num, $TS_oligo_seq, $bgzip_bin, $bgzip_sort_thread, $output_cutoff_hsh_ref, $count_cutoff_hsh_ref) = @_;
my $chunk_pass_ctss_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'sorted'}{'chunk_pass_ctss'};
my $chunk_unsort_pass_ctss_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'unsort'}{'chunk_pass_ctss'};
my $chunk_invader_ctss_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'sorted'}{'chunk_invader_ctss'};
my $chunk_unsort_invader_ctss_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'unsort'}{'chunk_invader_ctss'};
my $chunk_log_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_log_path'};
my $chunk_upstream_seq_fasta_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_upstream_seq_fasta_path'};
open PASSCTSS, ">", $chunk_unsort_pass_ctss_bed_path;
open STRNDINVADCTSS, ">", $chunk_unsort_invader_ctss_bed_path;
my $total_pos = 0;
my $total_read = 0;
my $filter_count_hsh_ref = {};
foreach my $min_edit_distance (@{$count_cutoff_hsh_ref->{'min_edit_distance'}}) {
foreach my $min_end_non_G_num (@{$count_cutoff_hsh_ref->{'min_end_non_G_num'}}) {
$filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'read'} = 0;
$filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'pos'} = 0;
}
}
open FASTA, "< $chunk_upstream_seq_fasta_path";
while (<FASTA>) {
chomp;
if ($_ =~ m/^>(.+)$/) {
my $header = $1;
#>chr15.20020713.+.1.1(+)
$header =~ s/\(.\)$//;
my ($chrom, $chromEnd, $strand, $original_ctssID, $score) = split /\.|:/, $header;
my $chromStart = $chromEnd - 1;
die "$score is not integer. Problem with getfasta.\n" if $score !~ m/^\d+$/;
$total_pos++;
$total_read += $score;
&reportAndLogStatus("getting edit distance for chunk $chunk_num. $total_pos ctss position processed", 10, "\n") if $total_pos%100000==0;#->1136
chomp (my $upstream_seq = <FASTA>);
$upstream_seq = uc($upstream_seq);
my ($edit_distance) = &calculateEditDistance($TS_oligo_seq, $upstream_seq);#->204
my $last_3_nt = substr($upstream_seq, -3);
my $end_non_G_num = ($last_3_nt ^ 'GGG') =~ tr/\0//c;
if (($edit_distance <= $output_cutoff_hsh_ref->{'min_edit_distance'} and $end_non_G_num <= $output_cutoff_hsh_ref->{'min_end_non_G_num'})) {
print STRNDINVADCTSS join "", (join "\t", ($chrom, $chromStart, $chromEnd, $original_ctssID, $score, $strand)), "\n";
} else {
print PASSCTSS join "", (join "\t", ($chrom, $chromStart, $chromEnd, $original_ctssID, $score, $strand)), "\n";
}
foreach my $min_edit_distance (@{$count_cutoff_hsh_ref->{'min_edit_distance'}}) {
foreach my $min_end_non_G_num (@{$count_cutoff_hsh_ref->{'min_end_non_G_num'}}) {
if (($edit_distance <= $min_edit_distance and $end_non_G_num <= $min_end_non_G_num)) {
$filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'read'} += $score;
$filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'pos'}++;
}
}
}
}
};
close FASTA;
close PASSCTSS;
close STRNDINVADCTSS;
system "sort --parallel $bgzip_sort_thread -k2,2n -k6,6 $chunk_unsort_pass_ctss_bed_path | $bgzip_bin -@ $bgzip_sort_thread >$chunk_pass_ctss_bed_path";
system "rm $chunk_unsort_pass_ctss_bed_path";
system "sort --parallel $bgzip_sort_thread -k2,2n -k6,6 $chunk_unsort_invader_ctss_bed_path | $bgzip_bin -@ $bgzip_sort_thread >$chunk_invader_ctss_bed_path";
system "rm $chunk_unsort_invader_ctss_bed_path";
&reportAndLogStatus("done getting edit distance for chunk $chunk_num. $total_pos ctss position processed", 10, "\n") if $total_pos%100000==0;#->1136
open LOG, ">", $chunk_log_path;
print LOG join "", (join "\t", ('min_edit_distance', 'min_end_non_G_num', 'pos', 'read', 'pos_pct', 'read_pct', 'total_read', 'total_pos')), "\n";
foreach my $min_edit_distance (@{$count_cutoff_hsh_ref->{'min_edit_distance'}}) {
foreach my $min_end_non_G_num (@{$count_cutoff_hsh_ref->{'min_end_non_G_num'}}) {
my $pos = $filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'pos'};
my $read = $filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'read'};
my $pos_pct = 0;
my $read_pct = 0;
if ($total_pos > 0) {
$pos_pct = sprintf "%.5f", 100*$pos/$total_pos;
$read_pct = sprintf "%.5f", 100*$read/$total_read;
}
print LOG join "", (join "\t", ($min_edit_distance, $min_end_non_G_num, $pos, $read, $pos_pct, $read_pct, $total_read, $total_pos)), "\n";
}
}
close LOG;
return ();
}
sub generateChunkInfoHsh {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1136
# appearInSub: >none
# primaryAppearInSection: 3_count|153
# secondaryAppearInSection: >none
# input: $chrom_size_hsh_ref, $chunk_size, $result_tmp_dir
# output: $chunk_info_hsh_ref
# toCall: my ($chunk_info_hsh_ref) = &generateChunkInfoHsh($chrom_size_hsh_ref, $chunk_size, $result_tmp_dir);
# calledInLine: 159
#....................................................................................................................................................#
my ($chrom_size_hsh_ref, $chunk_size, $result_tmp_dir) = @_;
my $chunk_num = 1;
my $chunk_info_hsh_ref = {};
foreach my $chrom (sort keys %{$chrom_size_hsh_ref}) {
my $size = $chrom_size_hsh_ref->{$chrom};
foreach (my $startPos = 0; $startPos <= $size; $startPos += $chunk_size) {
my $endPos = $startPos + $chunk_size;
$endPos = $size if ($endPos > $size);
my $file_tag = "$chrom.$startPos.$endPos";
my $size_in_chunk = $endPos - $startPos;
&reportAndLogStatus("chunk $chunk_num contains $size_in_chunk nt covering $chrom $startPos to $endPos", 10, "\n");#->1136
my $chunk_upstream_seq_fasta_path = "$result_tmp_dir/$file_tag.upstream_seq.fasta";
my $chunk_ctss_in_scope_bed_path = "$result_tmp_dir/$file_tag.ctss_in_scope.bed";
my $chunk_scope_bed_path = "$result_tmp_dir/$file_tag.scope.bed";
my $chunk_pass_ctss_bed_path = "$result_tmp_dir/$file_tag.pass.ctss.bed.gz";
my $chunk_invader_ctss_bed_path = "$result_tmp_dir/$file_tag.invader.ctss.bed.gz";
my $chunk_unsort_pass_ctss_bed_path = "$result_tmp_dir/$file_tag.unsort.pass.ctss.bed";
my $chunk_unsort_invader_ctss_bed_path = "$result_tmp_dir/$file_tag.unsort.invader.ctss.bed";
my $chunk_log_path = "$result_tmp_dir/$file_tag.log.txt";
my ($chunk_chrom, $chunk_startPos, $chunk_endPos) = ($chrom, $startPos, $endPos);
my $tabix_startPos = $chunk_startPos + 1; #---[2021/09/01 22:58] everything is zero based except tabix
$chunk_info_hsh_ref->{$chunk_num}{'region_str'} = "$chunk_chrom:$chunk_startPos-$chunk_endPos";
$chunk_info_hsh_ref->{$chunk_num}{'loc'} = [$chunk_chrom, $chunk_startPos, $chunk_endPos];
$chunk_info_hsh_ref->{$chunk_num}{'chunk_upstream_seq_fasta_path'} = $chunk_upstream_seq_fasta_path;
$chunk_info_hsh_ref->{$chunk_num}{'chunk_ctss_in_scope_bed_path'} = $chunk_ctss_in_scope_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'tabix_region_str'} = "$chunk_chrom:$tabix_startPos-$chunk_endPos";
$chunk_info_hsh_ref->{$chunk_num}{'chunk_log_path'} = $chunk_log_path;
$chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'sorted'}{'chunk_pass_ctss'} = $chunk_pass_ctss_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'unsort'}{'chunk_pass_ctss'} = $chunk_unsort_pass_ctss_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'sorted'}{'chunk_invader_ctss'} = $chunk_invader_ctss_bed_path;
$chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'unsort'}{'chunk_invader_ctss'} = $chunk_unsort_invader_ctss_bed_path;
$chunk_num++;
}
}
return ($chunk_info_hsh_ref);
}
sub generateUpstreamSeq {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|1136
# appearInSub: processPerChunk|974
# primaryAppearInSection: >none
# secondaryAppearInSection: 3_count|153
# input: $TS_oligo_seq, $bedtools_bin, $chrom_size_path, $chunk_info_hsh_ref, $chunk_num, $ctss_bed_path, $fasta_path, $tabix_bin
# output: $chunk_upstream_seq_fasta_path
# toCall: my ($chunk_upstream_seq_fasta_path) = &generateUpstreamSeq($ctss_bed_path, $fasta_path, $TS_oligo_seq, $bedtools_bin, $tabix_bin, $chunk_num, $chunk_info_hsh_ref, $chrom_size_path);
# calledInLine: 1023
#....................................................................................................................................................#
my ($ctss_bed_path, $fasta_path, $TS_oligo_seq, $bedtools_bin, $tabix_bin, $chunk_num, $chunk_info_hsh_ref, $chrom_size_path) = @_;
my $length = length($TS_oligo_seq);
my ($chunk_chrom, $chunk_startPos, $chunk_endPos) = @{$chunk_info_hsh_ref->{$chunk_num}{'loc'}};
my $chunk_upstream_seq_fasta_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_upstream_seq_fasta_path'};
my $chunk_ctss_in_scope_bed_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_ctss_in_scope_bed_path'};
my $tabix_region_str = $chunk_info_hsh_ref->{$chunk_num}{'tabix_region_str'};
&reportAndLogStatus("extracting ctss upstream seq for chunk $chunk_num", 10, "\n");#->1136
open (TABIX, "$tabix_bin $ctss_bed_path $tabix_region_str |");
open CTSSINSCOPE, ">", "$chunk_ctss_in_scope_bed_path";
my $num_ctss_proc = 0;
while (<TABIX>) {
chomp;
my ($chrom, $chromStart, $chromEnd, $original_ctssID, $score, $strand) = split /\t/;
$num_ctss_proc++;
$original_ctssID =~ s/\./_/g;
$original_ctssID =~ s/\:/_/g;
my $ctss_ID = "$chrom.$chromEnd.$strand.$original_ctssID.$score";
&reportAndLogStatus("getting upstream seq for $chrom. $num_ctss_proc ctss position processed for chunk $chunk_num", 10, "\n") if $num_ctss_proc%1000000==0;#->1136
my $upstream_start;
my $upstream_end;
if ($strand eq '+') {
$upstream_start = $chromStart - $length;
$upstream_end = $chromStart;
if ($upstream_start < 0) {
my $shift = abs($upstream_start);
$upstream_start = $upstream_start + $shift;
$upstream_end = $upstream_end + $shift;
}
} elsif ($strand eq '-') {
$upstream_start = $chromEnd;
$upstream_end = $chromEnd + $length;
} else {
die;
}
print CTSSINSCOPE join "", (join "\t", ($chrom, $upstream_start, $upstream_end, $ctss_ID, $score, $strand)), "\n";
}
close TABIX;
close CTSSINSCOPE;
&reportAndLogStatus("getting upstream seq for chunk $chunk_num", 10, "\n");#->1136
system "$bedtools_bin getfasta -s -name -fi $fasta_path -bed $chunk_ctss_in_scope_bed_path -fo $chunk_upstream_seq_fasta_path";
&reportAndLogStatus("done getting upstream seq for chunk $chunk_num. $num_ctss_proc ctss position processed", 10, "\n");#->1136
return ($chunk_upstream_seq_fasta_path);
}
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, $scriptAbsPath);
# calledInLine: 147
#....................................................................................................................................................#
my ($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 poolChunkResults {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: checkChunkResults|411, reportAndLogStatus|1136
# appearInSub: >none
# primaryAppearInSection: 3_count|153
# secondaryAppearInSection: >none
# input: $chunk_info_hsh_ref, $paramTag, $result_bed_dir, $result_log_dir, $tabix_bin
# output:
# toCall: &poolChunkResults($tabix_bin, $chunk_info_hsh_ref, $result_bed_dir, $result_log_dir, $paramTag);
# calledInLine: 161
#....................................................................................................................................................#
my ($tabix_bin, $chunk_info_hsh_ref, $result_bed_dir, $result_log_dir, $paramTag) = @_;
my $all_log_path = "$result_log_dir/$paramTag.invader.log.tsv";
my $out_bed_hsh_ref = {
'chunk_pass_ctss' => "$result_bed_dir/$paramTag.pass.ctss.bed.gz",
'chunk_invader_ctss' => "$result_bed_dir/$paramTag.invader.ctss.bed.gz",
};
#---[2021/09/01 23:25] pool chunk bed
foreach my $ctss_type (keys %{$out_bed_hsh_ref}) {
my $out_ctss_type = $out_bed_hsh_ref->{$ctss_type};
my @file_path_ary = ();
foreach my $chunk_num (sort {$a <=> $b} keys %{$chunk_info_hsh_ref}) {
my ($valid) = &checkChunkResults($chunk_info_hsh_ref, $chunk_num);#->411
if ($valid eq 'yes') {
my $chunk_sorted_ctss = $chunk_info_hsh_ref->{$chunk_num}{'ctss'}{'sorted'}{$ctss_type};
push @file_path_ary, $chunk_sorted_ctss;
&reportAndLogStatus("$ctss_type of chunk $chunk_num is found", 10, "\n");#->1136
} else {
die "results of chunk $chunk_num is not found. quitting\n";
}
}
&reportAndLogStatus("Concatenating $ctss_type files", 10, "\n");#->1136
my $file_path_str = join " ", @file_path_ary;
system "cat $file_path_str >$out_ctss_type";
&reportAndLogStatus("Tabix indexing combined $ctss_type", 10, "\n");#->1136
system "$tabix_bin -p bed $out_ctss_type";
}
#---[2021/09/01 23:25] pool chunk log
my $filter_count_hsh_ref = {};
my $all_total_pos = 0;
my $all_total_read = 0;
foreach my $chunk_num (sort {$a <=> $b} keys %{$chunk_info_hsh_ref}) {
my $chunk_log_path = $chunk_info_hsh_ref->{$chunk_num}{'chunk_log_path'};
my $chunk_total_pos;
my $chunk_total_read;
open (CHUNKLOG, "<", $chunk_log_path);
<CHUNKLOG>;
while (<CHUNKLOG>) {
chomp;
my ($min_edit_distance, $min_end_non_G_num, $pos, $read, $pos_pct, $read_pct, $total_read, $total_pos) = split /\t/;
$filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'read'} += $read;
$filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'pos'} += $pos;
$chunk_total_pos = $total_pos;
$chunk_total_read = $total_read;
}
close CHUNKLOG;
$all_total_pos += $chunk_total_pos;
$all_total_read += $chunk_total_read;
}
open ALLLOG, ">", $all_log_path;
print ALLLOG join "", (join "\t", ('min_edit_distance', 'min_end_non_G_num', 'pos', 'read', 'pos_pct', 'read_pct')), "\n";
foreach my $min_edit_distance (sort {$a <=> $b} keys %{$filter_count_hsh_ref}) {
foreach my $min_end_non_G_num (sort {$a <=> $b} keys %{$filter_count_hsh_ref->{$min_edit_distance}}) {
my $pos = $filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'pos'};
my $read = $filter_count_hsh_ref->{$min_edit_distance}{$min_end_non_G_num}{'read'};
my $pos_pct = sprintf "%.5f", 100*$pos/$all_total_pos;
my $read_pct = sprintf "%.5f", 100*$read/$all_total_read;
print ALLLOG join "", (join "\t", ($min_edit_distance, $min_end_non_G_num, $pos, $read, $pos_pct, $read_pct)), "\n";
}
}
close ALLLOG;
return ();
}
sub printOutputFileListAndReadme {
#....................................................................................................................................................#
# subroutineCategory: output
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 4_finishingTasks|167
# secondaryAppearInSection: >none
# input: $ARGVStr, $outDir, $paramTag
# output:
# toCall: &printOutputFileListAndReadme($ARGVStr, $paramTag, $outDir);
# calledInLine: 170
#....................................................................................................................................................#
my ($ARGVStr, $paramTag, $outDir) = @_;
my $outputFileListPath = "$outDir/$paramTag/output.file.list.txt";
open (OUTFILELIST, ">", $outputFileListPath);
my %dirHsh = ();
my %filelistLenCountHsh = ();
push @{$filelistLenCountHsh{'dir'}}, length 'Directory';
push @{$filelistLenCountHsh{'name'}}, length 'Name';
push @{$filelistLenCountHsh{'description'}}, length 'Description';
foreach my $outputFilePath (sort keys %{$globalReadmeHsh_ref}) {
my $fileDescription = $globalReadmeHsh_ref->{$outputFilePath}{'description'};
my $cleandOutputFilePath = $outputFilePath;
$cleandOutputFilePath =~ s/\/+/\//g;
my ($filePrefix, $fileDir, $fileSuffix) = fileparse($cleandOutputFilePath, qr/\.[^.]*/);
$fileDir =~ s/^$outDir//;
my $fileName = $filePrefix.$fileSuffix;
$dirHsh{$fileDir}{$fileName} = $fileDescription;
push @{$filelistLenCountHsh{'dir'}}, length $fileDir;
push @{$filelistLenCountHsh{'name'}}, length $fileName;
push @{$filelistLenCountHsh{'description'}}, length $fileDescription;
open README, ">", "$outputFilePath.readme.txt";
print README "=================\n";
print README "File descriptions\n";
print README "=================\n";
print README "$fileDescription\n";
if (exists $globalReadmeHsh_ref->{$outputFilePath}{'headerAry'}) {
my @colLenCountHsh = (length 'column');
push @colLenCountHsh, length $_ foreach (@{$globalReadmeHsh_ref->{$outputFilePath}{'headerAry'}});
@colLenCountHsh = sort {$b <=> $a} @colLenCountHsh;
my $headerColLen = $colLenCountHsh[0]+2;
print README "\n";
print README "\n";
print README "===================\n";
print README "Column descriptions\n";
print README "===================\n";
print README "\n";
printf README "%-".$headerColLen."s", 'column';
print README "description\n";
printf README "%-".$headerColLen."s", '------';
print README "-----------\n";
foreach my $header (@{$globalReadmeHsh_ref->{$outputFilePath}{'headerAry'}}) {
my $columnDescription = 'self-explanatory';
$columnDescription = $globalReadmeHsh_ref->{$outputFilePath}{'header'}{$header} if exists $globalReadmeHsh_ref->{$outputFilePath}{'header'}{$header};
printf README "%-".$headerColLen."s", $header;
print README $columnDescription."\n";
}
}
if (exists $globalReadmeHsh_ref->{$outputFilePath}{'extra_info'}) {
print README "\n";
print README "\n";
print README "=================\n";
print README "Extra information\n";
print README "=================\n";
print README "\n";
foreach my $title (sort keys %{$globalReadmeHsh_ref->{$outputFilePath}{'extra_info'}}) {
print README "$title\n";
print README "-" foreach (1..length $title);
print README "\n";
print README "$_\n" foreach @{$globalReadmeHsh_ref->{$outputFilePath}{'extra_info'}{$title}};
}
}
print README "\n";
print README "\n";
print README "~" foreach (1..length "$fileName was created from running,");
print README "\n";
print README "$fileName was created from running,\n";
print README "\n";
print README "$ARGVStr\n";
print README "\n";
close README;
}
@{$filelistLenCountHsh{'dir'}} = sort {$b <=> $a} @{$filelistLenCountHsh{'dir'}};
@{$filelistLenCountHsh{'name'}} = sort {$b <=> $a} @{$filelistLenCountHsh{'name'}};
@{$filelistLenCountHsh{'description'}} = sort {$b <=> $a} @{$filelistLenCountHsh{'description'}};
my $fileDir_colLen = $filelistLenCountHsh{'dir'}[0]+2;
my $fileName_colLen = $filelistLenCountHsh{'name'}[0]+2;
my $fileDescription_colLen = $filelistLenCountHsh{'description'}[0]+2;
printf OUTFILELIST ("%-".$fileDir_colLen."s %-".$fileName_colLen."s %-".$fileDescription_colLen."s\n", 'directory', 'name', 'description');
printf OUTFILELIST ("%-".$fileDir_colLen."s %-".$fileName_colLen."s %-".$fileDescription_colLen."s\n", '=========', '====', '===========');
foreach my $fileDir (sort {$a cmp $b} keys %dirHsh) {
foreach my $fileName (sort {$a cmp $b} keys %{$dirHsh{$fileDir}}) {
my $fileDescription = $dirHsh{$fileDir}{$fileName};
printf OUTFILELIST ("%-".$fileDir_colLen."s %-".$fileName_colLen."s %-".$fileDescription_colLen."s\n", $fileDir, $fileName, $fileDescription);
}
}
print OUTFILELIST "\n";
print OUTFILELIST "\n";
print OUTFILELIST "~" foreach (1..length "The above files were generated by running,");
print OUTFILELIST "\n";
print OUTFILELIST "The above files were generated by running,\n";
print OUTFILELIST "\n";
print OUTFILELIST "$ARGVStr\n";
print OUTFILELIST "\n";
close OUTFILELIST;
return ();
}
sub printStartOrFinishMessage {
#....................................................................................................................................................#
# subroutineCategory: general
# dependOnSub: currentTime|462
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|133, 4_finishingTasks|167
# secondaryAppearInSection: >none
# input: $StartOrFinishMessage
# output: none
# toCall: &printStartOrFinishMessage($StartOrFinishMessage);
# calledInLine: 148, 171
#....................................................................................................................................................#
my ($StartOrFinishMessage) = @_;