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scafe.tool.cm.annotate
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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 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.annotate <---
<--- tool, common mode, define and annotate tCRE --->
Description:
This tool defines tCRE from TSS clusters and annotates them based their overlap with gene models.
Usage:
scafe.tool.cm.annotate [options] --tssCluster_bed_path --tssCluster_info_path --genome --outputPrefix --outDir
--tssCluster_bed_path <required> [string] bed file contains the ranges of filtered TSS clusters,
*.tssCluster.*.filtered.bed.gz from scafe.tool.cm.filter.pl
--tssCluster_info_path <required> [string] tsv file contains the information of all TSS clusters,
*.tssCluster.log.tsv from scafe.tool.cm.filter.pl
--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
--up_end5Rng (optional) [integer] TSS clusters will be classified as gene TSS, exonic, intron
and intergenic. $up_end5Rng determines the range upstream of
annotated gene TSS to be used for gene TSS assignment
(default = 500)
--dn_end5Rng (optional) [integer] TSS clusters will be classified as gene TSS, exonic, intron
and intergenic. $dn_end5Rng determines the range downstream of
annotated gene TSS to be used for gene TSS assignment
(default = 500)
--exon_slop_rng (optional) [integer] TSS clusters will be classified as gene TSS, exonic, intron
and intergenic. $exon_slop_rng determines the range to be extended
(i.e. slopped) from exon for assignment of exonic class.
Used -1 to NOT to extend (default = -1)
--merge_dist (optional) [integer] TSS clusters outside annotated gene promoters are grouped
as "dummy genes" (for operational uniformity) by merging closely
located TSS clusters. $merge_dist determines the maximum distances
between TSS clusters to be merged (default = 500)
--addon_length (optional) [integer] see $merge_dist. add-on "dummy transcrips" will assigned to TSS cluster of
"dummy genes" (for operational uniformity).$addon_length determines
the length of these add-on "dummy transcrips" (default = 500).
--proximity_slop_rng (optional) [integer] TSS clusters will be assigned to annotated gene TSS are "proximal"
TSS clusters. $proximity_slop_rng determines the range to be extended
(i.e. slopped) from gene TSS for assignment of proximal TSS clusters.
(default = 500)
--merge_strandness (optional) [string] see $merge_dist. $merge_strandness decides the merge to be
strand-aware ("stranded") or strand-agnostic "strandless".
(default = strandless)
--proximal_strandness (optional) [string] closely located proximal TSS clusters are merged
tCREs. $proximal_strandness decides the merge to be
strand-aware ("stranded") or strand-agnostic "strandless".
(default = stranded)
--CRE_extend_size (optional) [integer] tCREs were defined by merging the extended ranges of TSS clusters.
$CRE_extend_size determine the size of this range (both sides of
summit) (default = 500)
--CRE_extend_upstrm_ratio (optional) [float] see $CRE_extend_size. $CRE_extend_upstrm_ratio determines the ratio
(X:1) of flanking sizes on the upstream and downstream of summit.
e.g. $CRE_extend_upstrm_ratio=4, upstream and downstream size will be
taken as 4:1 ratio. $CRE_extend_size=500 and $CRE_extend_upstrm_ratio=4,
upstream and downstream will be 400 and 100 respectively
(default = 4)
--stitch_distance (optional) [integer] distance (nt) for stitching distal tCRE for defining hyperactive distal loci.
aka superenhancer candidates. If undefined, an optimized value will be
determined based on the tangent to rank-distance plot. As a note, the original
distance for stitching enhancer in
ROSE (http://younglab.wi.mit.edu/super_enhancer_code.html) is 12,500.
(default = undefined)
--min_total_exp_frac (optional) [fraction] minimum fraction of the expression amount (read/UMI) within a gene for an annotated
tCRE to be regarded as an unannotated promoter of the gene. The total expression amount
of a gene is defined as the total number of UMI/read of all its annotated promoters.
(default = 0.1)
--min_gene_strand_read_frac (optional) [fraction] minimum fraction of the expression amount (read/UMI) on the gene strand (in total number
of UMI/read both strand) of a tCRE to be regarded as an unannotated promoter of the gene.
(default = 0.8)
--min_spreadness (optional) [fraction] minimum spreadness of the distal CRE to be considered as hyperactive distal loci.
Spreadness is defined as "num-of-CRE/top-fraction". Top-fraction is defined as the expression
amount (read/UMI) on the highest expressed distal CRE in the expression amount of all distal CRE
within the locus. Num-of-CRE refers to the number of CRE within the distal CRE locus
(default = 4, equivalent to Num-of-CRE=3 and Top-fraction = 0.75, i.e. 3/0.75 = 4)
--Rscript_bin (optional) [string] path to the Rscript bin, aim to allow users to supply an R version other the
system wide R version. Package Caret must be installed. (default = Rscript)
--overwrite (optional) [yes/no] erase outDir/outputPrefix before running (default=no)
Dependencies:
bedtools,
R packages: 'ggplot2'
For demo, cd to SCAFE dir and run,
scafe.tool.cm.annotate \
--overwrite=yes \
--tssCluster_bed_path=./demo/output/sc.solo/filter/demo/bed/demo.tssCluster.default.filtered.bed.gz \
--tssCluster_info_path=./demo/output/sc.solo/filter/demo/log/demo.tssCluster.log.tsv \
--genome=hg19.gencode_v32lift37 \
--outputPrefix=demo \
--outDir=./demo/output/sc.solo/annotate/
=head1 VERSION
v0.9.0-beta [March 20, 2021]
-Initial pre-release
v1.0.0 [June 6, 2022]
-Added hyperactive distal tCRE locus definition with uniformity restritions (using --min_spreadness)
-Annotate potential alternative promoters
=cut
#<\doc>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<lastCmdCalled>
#
# notCalledBefore
#
# notCalledBefore
#
#<\lastCmdCalled>
#====================================================================================================================================================#
#====================================================================================================================================================#
#<global>
my $scriptDirPath = dirname(rel2abs($0));
my $scriptAbsPath = abs_path($0);
my ($curntTimeStamp) = &timeStamp();#->3161
my $ARGVStr = join "\n", (¤tTime(), $scriptAbsPath, @ARGV);#->695
my $globalReadmeHsh_ref = {};
our $tmplog_fh;
#<\global>
#====================================================================================================================================================#
#====================================================================================================================================================#
{ #Main sections lexical scope starts
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 0_startingTasks
#
#<section ID="startingTasks" num="0">
my ($exon_slop_rng, $addon_length, $merge_dist, $up_end5Rng, $dn_end5Rng, $tssCluster_bed_path, $tssCluster_info_path, $merge_strandness, $proximity_slop_rng, $CRE_extend_size, $CRE_extend_upstrm_ratio, $genome, $proximal_strandness, $stitch_distance, $min_total_exp_frac, $min_gene_strand_read_frac, $min_spreadness, $Rscript_bin, $outputPrefix, $outDir, $overwrite) = &readParameters();#->2676
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 1_defineHardCodedParam
#
#<section ID="defineHardCodedParam" num="1">
my $mask_class_hsh_ref = {
'Mt_rRNA' => 1,
'Mt_tRNA' => 1,
'rRNA' => 1,
'snRNA' => 1,
'snoRNA' => 1,
'structural_RNA' => 1,
'small_RNA' => 1,
'miRNA' => 1,
};
my $max_dist_rank = 100000; #---[2021/08/04 22:13] maximum distance to be used for optimize tangent for optimize distal tCRE stitching
my $paramTag = "$outputPrefix";
#$chrom_size_path, $gene_info_path, $transcript_bed_path, $transcript_info_path,
#<\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 $result_gtf_dir = "$result_dir/gtf/"; push @mkDirAry, $result_gtf_dir;
my $tmp_bed_dir = "$result_dir/bed/tmp/"; push @mkDirAry, $tmp_bed_dir;
my $result_log_dir = "$result_dir/log/"; push @mkDirAry, $result_log_dir;
my $result_plot_dir = "$result_dir/plot/"; push @mkDirAry, $result_plot_dir;
my $result_script_dir = "$result_dir/script/"; push @mkDirAry, $result_script_dir;
foreach my $dir (@mkDirAry) {system ("mkdir -pm 755 $dir");}
open $tmplog_fh, ">", "$result_dir/00_screen_log.$curntTimeStamp.log.txt";
&logCalledCMDAndScript($ARGVStr, $result_script_dir, $scriptAbsPath);#->1407
&printStartOrFinishMessage("startMessage");#->1760
my ($bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin) = &checkAllExecutable();#->553
my ($chrom_size_path, $gene_info_path, $transcript_info_path, $transcript_bed_path) = &checkTranscriptInfoBedChromSizeGeneInfoPath($genome);#->666
my ($optimize_tangent_R) = &checkRScriptVersion($Rscript_bin);#->633
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 3_readInfo
#
#<section ID="readInfo" num="3">
my ($chrom_size_hsh_ref) = &readChromSize($chrom_size_path);#->2454
my ($trnscptInfoHsh_ref, $transcript_in_scope_bed_path) = &readTranscriptBed($transcript_bed_path, $chrom_size_hsh_ref, $tmp_bed_dir, $outDir);#->2751
my ($geneInfoHsh_ref) = &readTranscriptInfo($transcript_info_path, $trnscptInfoHsh_ref);#->2830
&readGeneInfo($gene_info_path, $geneInfoHsh_ref);#->2604
my ($coding_class_hsh_ref) = &defineCodingClasses();#->787
my ($end5_rng_bed_path, $end5_point_bed_path) = &printEnd5Bed($trnscptInfoHsh_ref, $up_end5Rng, $dn_end5Rng, $tmp_bed_dir);#->1514
my ($clusterInfoHsh_ref) = &readClusterBed($tssCluster_bed_path);#->2479
&readClusterInfo($tssCluster_info_path, $clusterInfoHsh_ref);#->2543
my ($extended_tssCluster_bed_path) = &generateExtendedCluster($clusterInfoHsh_ref, $tmp_bed_dir, $CRE_extend_size, $CRE_extend_upstrm_ratio);#->1198
&intersectCluster($transcript_in_scope_bed_path, $tssCluster_bed_path, $end5_rng_bed_path, $chrom_size_path, $exon_slop_rng, $clusterInfoHsh_ref, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $bedtools_bin);#->1346
&filterUnannotatedCluster($clusterInfoHsh_ref);#->1119
my ($superClusterHsh_ref) = &getSuperCluster($tmp_bed_dir, $clusterInfoHsh_ref, $merge_dist, $merge_strandness, $bedtools_bin);#->1291
&addonTranscript($superClusterHsh_ref, $clusterInfoHsh_ref, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $addon_length, $chrom_size_hsh_ref);#->348
&summerizeGeneInfo($geneInfoHsh_ref, $trnscptInfoHsh_ref);#->3103
&addGeneMask($geneInfoHsh_ref, $trnscptInfoHsh_ref, $clusterInfoHsh_ref, $mask_class_hsh_ref);#->313
&assignClusterProximity($transcript_in_scope_bed_path, $tssCluster_bed_path, $end5_point_bed_path, $chrom_size_path, $proximity_slop_rng, $clusterInfoHsh_ref, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $bedtools_bin);#->445
&rankClusterWithinGene($clusterInfoHsh_ref, $geneInfoHsh_ref);#->2396
my ($CREInfoHsh_ref) = &defineCRE($CRE_extend_size, $CRE_extend_upstrm_ratio, $clusterInfoHsh_ref, $tmp_bed_dir, $chrom_size_path, $bedtools_bin, $proximal_strandness, $extended_tssCluster_bed_path);#->713
&summerizeCREInfo($clusterInfoHsh_ref, $CREInfoHsh_ref, $geneInfoHsh_ref);#->3025
&rankCREWithinGene($CREInfoHsh_ref, $geneInfoHsh_ref);#->2338
&removeUnsupportedGene($geneInfoHsh_ref, $trnscptInfoHsh_ref);#->2911
my ($gene_promoter_CRE_pair_hsh_ref) = &defineUnannoPromoter($CREInfoHsh_ref, $geneInfoHsh_ref, $min_total_exp_frac, $min_gene_strand_read_frac, $clusterInfoHsh_ref);#->1002
my ($distal_CRE_locus_info_hsh_ref) = &defineHyperactiveDistalLoci($CREInfoHsh_ref, $tmp_bed_dir, $bedtools_bin, $optimize_tangent_R, $max_dist_rank, $Rscript_bin, $stitch_distance, $min_spreadness, $result_plot_dir);#->861
my ($gene_promoter_info_hsh_ref) = &generateGenePromoterInfoHsh($CREInfoHsh_ref);#->1264
&printAllBed($result_bed_dir, $geneInfoHsh_ref, $trnscptInfoHsh_ref, $clusterInfoHsh_ref, $CREInfoHsh_ref, $distal_CRE_locus_info_hsh_ref, $gene_promoter_info_hsh_ref, $outputPrefix);#->1432
&printTableClusterInfo($result_log_dir, $geneInfoHsh_ref, $trnscptInfoHsh_ref, $clusterInfoHsh_ref, $coding_class_hsh_ref, $outputPrefix);#->1930
&printTableGeneInfo($result_log_dir, $geneInfoHsh_ref, $coding_class_hsh_ref, $outputPrefix);#->2099
&printTableIDMappingInfo($result_log_dir, $geneInfoHsh_ref, $trnscptInfoHsh_ref, $clusterInfoHsh_ref, $coding_class_hsh_ref, $outputPrefix);#->2161
&printTableTranscriptInfo($result_log_dir, $geneInfoHsh_ref, $trnscptInfoHsh_ref, $coding_class_hsh_ref, $outputPrefix);#->2221
&printTableCREInfo($result_log_dir, $geneInfoHsh_ref, $trnscptInfoHsh_ref, $clusterInfoHsh_ref, $CREInfoHsh_ref, $coding_class_hsh_ref, $outputPrefix);#->1794
&printTrnscptGtf($result_gtf_dir, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $outputPrefix);#->2278
&printTableDistalCRELocusInfo($result_log_dir, $distal_CRE_locus_info_hsh_ref, $outputPrefix);#->2045
&printGenePromoterInfo($result_log_dir, $gene_promoter_CRE_pair_hsh_ref, $geneInfoHsh_ref, $CREInfoHsh_ref, $outputPrefix);#->1595
&removeTmpBedDir($tmp_bed_dir);#->2892
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
# section 4_finishingTasks
#
#<section ID="finishingTasks" num="4">
&printOutputFileListAndReadme($ARGVStr, $paramTag, $outDir);#->1640
&printStartOrFinishMessage("finishMessage");#->1760
#<\section>
#====================================================================================================================================================#
#====================================================================================================================================================#
} #Main sections lexical scope ends
#====================================================================================================================================================#
#====================================================================================================================================================#
#List of subroutines by category
#
# checkTools [n=1]:
# checkRScriptVersion
#
# general [n=6]:
# checkRScriptVersion, currentTime, logCalledCMDAndScript
# printStartOrFinishMessage, readParameters, timeStamp
#
# getTextInfo [n=1]:
# readGeneInfo
#
# log [n=1]:
# reportAndLogStatus
#
# output [n=1]:
# printOutputFileListAndReadme
#
# specific [n=1]:
# printTrnscptGtf
#
# time [n=1]:
# timeStamp
#
# unassigned [n=37]:
# addGeneMask, addonTranscript, assignClusterProximity
# checkAllExecutable, checkTranscriptInfoBedChromSizeGeneInfoPath, defineCRE
# defineCodingClasses, defineHyperactiveDistalLoci, defineUnannoPromoter
# exonRngToGtf, filterUnannotatedCluster, generateAddonGene
# generateExtendedCluster, generateGenePromoterInfoHsh, getSuperCluster
# intersectCluster, printAllBed, printEnd5Bed
# printGenePromoterInfo, printTableCREInfo, printTableClusterInfo
# printTableDistalCRELocusInfo, printTableGeneInfo, printTableIDMappingInfo
# printTableTranscriptInfo, rankCREWithinGene, rankClusterWithinGene
# readChromSize, readClusterBed, readClusterInfo
# readTranscriptBed, readTranscriptInfo, removeTmpBedDir
# removeUnsupportedGene, storeCREInfoFromBed, summerizeCREInfo
# summerizeGeneInfo
#
#====================================================================================================================================================#
sub addGeneMask {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 3_readInfo|217
# secondaryAppearInSection: >none
# input: $clusterInfoHsh_ref, $geneInfoHsh_ref, $mask_class_hsh_ref, $trnscptInfoHsh_ref
# output:
# toCall: &addGeneMask($geneInfoHsh_ref, $trnscptInfoHsh_ref, $clusterInfoHsh_ref, $mask_class_hsh_ref);
# calledInLine: 234
#....................................................................................................................................................#
my ($geneInfoHsh_ref, $trnscptInfoHsh_ref, $clusterInfoHsh_ref, $mask_class_hsh_ref) = @_;
foreach my $geneID (keys %{$geneInfoHsh_ref}) {
my $geneClass = $geneInfoHsh_ref->{$geneID}{'geneClass'};
my $mask = 'no';
$mask = 'yes' if exists $mask_class_hsh_ref->{$geneClass};
$geneInfoHsh_ref->{$geneID}{'mask'} = $mask;
foreach my $trnscptID (keys %{$geneInfoHsh_ref->{$geneID}{'trnscptID'}}) {
#die "$trnscptID\n" if not exists $trnscptInfoHsh_ref->{$trnscptID}{'bedLine'};
$trnscptInfoHsh_ref->{$trnscptID}{'mask'} = $mask;
}
}
foreach my $clusterID (keys %{$clusterInfoHsh_ref}) {
my $mask = 'yes';
foreach my $geneID (keys %{$clusterInfoHsh_ref->{$clusterID}{'geneID'}}) {
$mask = 'no' if $geneInfoHsh_ref->{$geneID}{'mask'} eq 'no';
}
$clusterInfoHsh_ref->{$clusterID}{'mask'} = $mask;
}
return ();
}
sub addonTranscript {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: generateAddonGene|1150, reportAndLogStatus|2953
# appearInSub: >none
# primaryAppearInSection: 3_readInfo|217
# secondaryAppearInSection: >none
# input: $addon_length, $chrom_size_hsh_ref, $clusterInfoHsh_ref, $geneInfoHsh_ref, $superClusterHsh_ref, $trnscptInfoHsh_ref
# output: none
# toCall: &addonTranscript($superClusterHsh_ref, $clusterInfoHsh_ref, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $addon_length, $chrom_size_hsh_ref);
# calledInLine: 232
#....................................................................................................................................................#
my ($superClusterHsh_ref, $clusterInfoHsh_ref, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $addon_length, $chrom_size_hsh_ref) = @_;
my $trnscptIDHsh_ref = {};
&reportAndLogStatus("creating addon transcripts", 10, "\n");#->2953
foreach my $superclusterID (keys %{$superClusterHsh_ref}) {
my ($geneID, $addon_chrom) = &generateAddonGene($superClusterHsh_ref, $clusterInfoHsh_ref, $superclusterID);#->1150
$geneInfoHsh_ref->{$geneID}{'geneName'} = $geneID;
$geneInfoHsh_ref->{$geneID}{'geneClass'} = 'unanno_tss';
foreach my $clusterID (@{$superClusterHsh_ref->{$superclusterID}}) {
my $strand = $clusterInfoHsh_ref->{$clusterID}{'strand'};
my $chrom = $clusterInfoHsh_ref->{$clusterID}{'chrom'};
my $start = $clusterInfoHsh_ref->{$clusterID}{'start'};
my $end = $clusterInfoHsh_ref->{$clusterID}{'end'};
my $peakPos = $clusterInfoHsh_ref->{$clusterID}{'peakPos'};
my @t_strandAry = ();
if ($strand eq '.') {
push @t_strandAry, '+';
push @t_strandAry, '-';
} else {
push @t_strandAry, $strand;
}
foreach my $t_strand (@t_strandAry) {
my $t_start;
my $t_end;
my $addon_t_strand;
if ($t_strand eq '+') {
$t_start = $peakPos;
$t_end = $t_start + $addon_length;
$addon_t_strand = 'F';
} else {
$t_start = $peakPos - $addon_length;
$t_end = $peakPos;
$addon_t_strand = 'R';
}
$t_start = 1 if $t_start < 1;
$t_end = $chrom_size_hsh_ref->{$chrom}-1 if $t_end >= $chrom_size_hsh_ref->{$chrom};
my $addon_t_start = sprintf "%.9d", $t_start;
my $trnscptID = "ADDT".$addon_chrom.$addon_t_start.'.'.$addon_t_strand;
if (exists $trnscptInfoHsh_ref->{$trnscptID}) {
$trnscptIDHsh_ref->{$trnscptID}++;
$trnscptID = "ADDT".$addon_chrom.$addon_t_start.'.'.$addon_t_strand.$trnscptIDHsh_ref->{$trnscptID};
}
$trnscptIDHsh_ref->{$trnscptID}++;
$trnscptInfoHsh_ref->{$trnscptID}{'chrom'} = $chrom;
$trnscptInfoHsh_ref->{$trnscptID}{'strand'} = $t_strand;
@{$trnscptInfoHsh_ref->{$trnscptID}{'exonRng'}} = ($t_start, $t_end);
$clusterInfoHsh_ref->{$clusterID}{'trnscptID'}{$trnscptID} = 'addon';
$clusterInfoHsh_ref->{$clusterID}{'geneID'}{$geneID}++;
$trnscptInfoHsh_ref->{$trnscptID}{'clusterID'}{$clusterID} = 'addon';
$trnscptInfoHsh_ref->{$trnscptID}{'geneID'} = $geneID;
$geneInfoHsh_ref->{$geneID}{'clusterID'}{$clusterID} = 'addon';
$geneInfoHsh_ref->{$geneID}{'trnscptID'}{$trnscptID}++;
my $chromStart = $t_start - 1;
my $chromEnd = $t_end;
my $thickStart = $chromStart;
my $thickEnd = $chromEnd;
my $blockSizes = $chromEnd - $chromStart;
my $blockCount = 1;
my $blockStarts = 0;
$trnscptInfoHsh_ref->{$trnscptID}{'bedLine'} = [$chrom, $chromStart, $chromEnd, $trnscptID, '0', $t_strand, $thickStart, $thickEnd, ".", $blockCount, $blockSizes, $blockStarts];
}
}
}
}
sub assignClusterProximity {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|2953
# appearInSub: >none
# primaryAppearInSection: 3_readInfo|217
# secondaryAppearInSection: >none
# input: $bedtools_bin, $chrom_size_path, $clusterInfoHsh_ref, $end5_point_bed_path, $geneInfoHsh_ref, $proximity_slop_rng, $transcript_in_scope_bed_path, $trnscptInfoHsh_ref, $tssCluster_bed_path
# output: none
# toCall: &assignClusterProximity($transcript_in_scope_bed_path, $tssCluster_bed_path, $end5_point_bed_path, $chrom_size_path, $proximity_slop_rng, $clusterInfoHsh_ref, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $bedtools_bin);
# calledInLine: 235
#....................................................................................................................................................#
my ($transcript_in_scope_bed_path, $tssCluster_bed_path, $end5_point_bed_path, $chrom_size_path, $proximity_slop_rng, $clusterInfoHsh_ref, $trnscptInfoHsh_ref, $geneInfoHsh_ref, $bedtools_bin) = @_;
&reportAndLogStatus("assigning region to cluster", 10, "\n");#->2953
my $region_cmd_hsh_ref = {
'end5' => {
'in_bed6_cmd' => "cut -f 1-6 $end5_point_bed_path",
'slop_rng' => $proximity_slop_rng,
},
'exon' => {
'in_bed6_cmd' => "$bedtools_bin bed12tobed6 -i $transcript_in_scope_bed_path",
'slop_rng' => 0,
},
'intron' => {
'in_bed6_cmd' => "cut -f 1-6 $transcript_in_scope_bed_path",
'slop_rng' => 0,
},
};
foreach my $regionType (qw/end5 exon intron/) {
foreach my $oritentation (qw/ss as/) {
&reportAndLogStatus("Getting cluster at $regionType $oritentation", 10, "\n");#->2953
my $in_bed6_cmd = $region_cmd_hsh_ref->{$regionType}{'in_bed6_cmd'};
my $slop_rng = $region_cmd_hsh_ref->{$regionType}{'slop_rng'};
open BEDTOOLS, "$in_bed6_cmd | $bedtools_bin slop -b $slop_rng -i stdin -g $chrom_size_path | $bedtools_bin intersect -wo -a stdin -b $tssCluster_bed_path |" ;
while (<BEDTOOLS>) {
#chr10 13141663 13142302 ENST00000482140.5_1 1 + chr10 13142075 13142193 p1@OPTN 247174 + 13142162 13142163 255,0,0 118
my (@splt) = split /\t/;
my $clusterID = $splt[9];
next if exists $clusterInfoHsh_ref->{$clusterID}{'regionType'};
my $cluster_strand = $splt[11];
my $trnscpt_strand = $splt[5];
my $trnscptID = $splt[3];
my $oritentation_matched = 'no';
my $assigned_orientation;
if ($cluster_strand eq '.') {
$oritentation_matched = 'yes';
$assigned_orientation = 'ns';
} elsif ($cluster_strand eq $trnscpt_strand and $oritentation eq 'ss') {
$oritentation_matched = 'yes';
$assigned_orientation = 'ss';
} elsif ($cluster_strand ne $trnscpt_strand and $oritentation eq 'as') {
$oritentation_matched = 'yes';
$assigned_orientation = 'as';
}
if ($oritentation_matched eq 'yes') {
die "$trnscptID does not exists in trnscptInfoHsh_ref\n" if not exists $trnscptInfoHsh_ref->{$trnscptID};
die "$clusterID does not exists in clusterInfoHsh_ref\n" if not exists $clusterInfoHsh_ref->{$clusterID};
my $geneID = $trnscptInfoHsh_ref->{$trnscptID}{'geneID'};
$clusterInfoHsh_ref->{$clusterID}{'regionGeneID'} = $geneID;
$clusterInfoHsh_ref->{$clusterID}{'regionType'} = $regionType;
$clusterInfoHsh_ref->{$clusterID}{'regionOritentation'} = $oritentation;
}
}
close BEDTOOLS;
}
}
&reportAndLogStatus("Adding intergenic region type", 10, "\n");#->2953
foreach my $clusterID (keys %{$clusterInfoHsh_ref}) {
if (not exists $clusterInfoHsh_ref->{$clusterID}{'regionType'}) {
$clusterInfoHsh_ref->{$clusterID}{'regionGeneID'} = '__na';
$clusterInfoHsh_ref->{$clusterID}{'regionType'} = 'intergenic';
$clusterInfoHsh_ref->{$clusterID}{'regionOritentation'} = 'ns';
}
}
&reportAndLogStatus("Adding proximity", 10, "\n");#->2953
foreach my $clusterID (keys %{$clusterInfoHsh_ref}) {
if ($clusterInfoHsh_ref->{$clusterID}{'regionType'} eq 'end5') {
$clusterInfoHsh_ref->{$clusterID}{'proximity'} = 'proximal';
#} elsif ($clusterInfoHsh_ref->{$clusterID}{'regionType'} eq 'exon') {
#$clusterInfoHsh_ref->{$clusterID}{'proximity'} = 'exonic';
} else {
$clusterInfoHsh_ref->{$clusterID}{'proximity'} = 'distal';
}
}
}
sub checkAllExecutable {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|2953
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|193
# secondaryAppearInSection: >none
# input: none
# output: $bedGraphToBigWig_bin, $bedtools_bin, $bigWigAverageOverBed_bin, $cut_sh_path, $paraclu_bin, $samtools_bin
# toCall: my ($bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin) = &checkAllExecutable();
# calledInLine: 210
#....................................................................................................................................................#
my $dirPath = dirname(rel2abs($0));
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");#->2953
{
my $stdOut = `$bedtools_bin --version 2>&1`;
if ($stdOut =~ m/bedtools v(\S+)/) {
&reportAndLogStatus("Checking: bedtools version: $1", 0, "\n");#->2953
} 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");#->2953
} 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");#->2953
} 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");#->2953
} 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");#->2953
} else {
die "bedGraphToBigWig not installed properly. Quitting.\n";
}
}
{
my $stdOut = `$bigWigAverageOverBed_bin 2>&1`;
if ($stdOut =~ m/bigWigAverageOverBed v(\S+)/) {
&reportAndLogStatus("Checking: bigWigAverageOverBed version: $1", 0, "\n");#->2953
} else {
die "bigWigAverageOverBed is not installed properly. Quitting.\n";
}
}
return ($bedtools_bin, $samtools_bin, $paraclu_bin, $cut_sh_path, $bedGraphToBigWig_bin, $bigWigAverageOverBed_bin);
}
sub checkRScriptVersion {
#....................................................................................................................................................#
# subroutineCategory: general, checkTools
# dependOnSub: reportAndLogStatus|2953
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|193
# secondaryAppearInSection: >none
# input: $Rscript_bin
# output: $optimize_tangent_R
# toCall: my ($optimize_tangent_R) = &checkRScriptVersion($Rscript_bin);
# calledInLine: 212
#....................................................................................................................................................#
my ($Rscript_bin) = @_;
my $dirPath = dirname(rel2abs($0));
my $optimize_tangent_R = "$dirPath/../resources/R/optimize_tangent.R";
my $stdOut = `$Rscript_bin --version 2>&1`;
if ($stdOut =~ m/R scripting front-end version (\S+)/) {
&reportAndLogStatus("Checking: Rscript version: $1", 0, "\n");#->2953
} else {
die "Rscript is not installed properly at $Rscript_bin. Quitting.\n";
}
if (-s $optimize_tangent_R) {
&reportAndLogStatus("optimize_tangent_R found.", 0, "\n");#->2953
} else {
die "Rscript optimize_tangent_R is not found. Quitting.\n";
}
return($optimize_tangent_R);
}
sub checkTranscriptInfoBedChromSizeGeneInfoPath {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 2_defineOutDirPath|193
# secondaryAppearInSection: >none
# input: $genome
# output: $chrom_size_path, $gene_info_path, $transcript_bed_path, $transcript_info_path
# toCall: my ($chrom_size_path, $gene_info_path, $transcript_info_path, $transcript_bed_path) = &checkTranscriptInfoBedChromSizeGeneInfoPath($genome);
# calledInLine: 211
#....................................................................................................................................................#
my ($genome) = @_;
#$chrom_size_path, $gene_info_path, $transcript_bed_path, $transcript_info_path,
my $dirPath = dirname(rel2abs($0));
my $chrom_size_path = "$dirPath/../resources/genome/$genome/tsv/chrom.sizes.tsv";
my $gene_info_path = "$dirPath/../resources/genome/$genome/tsv/gene.info.tsv";
my $transcript_info_path = "$dirPath/../resources/genome/$genome/tsv/transcript.info.tsv";
my $transcript_bed_path = "$dirPath/../resources/genome/$genome/bed/transcript.bed.gz";
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 gene_info_path. Please rerun prep_genome step\n" if not -s $gene_info_path;
die "genome $genome does not have transcript_info_path. Please rerun prep_genome step\n" if not -s $transcript_info_path;
die "genome $genome does not have transcript_bed_path. Please rerun prep_genome step\n" if not -s $transcript_bed_path;
return ($chrom_size_path, $gene_info_path, $transcript_info_path, $transcript_bed_path);
}
sub currentTime {
#....................................................................................................................................................#
# subroutineCategory: general
# dependOnSub: >none
# appearInSub: printStartOrFinishMessage|1760, reportAndLogStatus|2953
# primaryAppearInSection: >none
# secondaryAppearInSection: 2_defineOutDirPath|193, 4_finishingTasks|258
# input: none
# output: $runTime
# toCall: my ($runTime) = ¤tTime();
# calledInLine: 154, 1776, 1780, 1785, 1789, 2969, 2970
#....................................................................................................................................................#
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 defineCRE {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|2953, storeCREInfoFromBed|2975
# appearInSub: >none
# primaryAppearInSection: 3_readInfo|217
# secondaryAppearInSection: >none
# input: $CRE_extend_size, $CRE_extend_upstrm_ratio, $bedtools_bin, $chrom_size_path, $clusterInfoHsh_ref, $extended_tssCluster_bed_path, $proximal_strandness, $tmp_bed_dir
# output: $CREInfoHsh_ref
# toCall: my ($CREInfoHsh_ref) = &defineCRE($CRE_extend_size, $CRE_extend_upstrm_ratio, $clusterInfoHsh_ref, $tmp_bed_dir, $chrom_size_path, $bedtools_bin, $proximal_strandness, $extended_tssCluster_bed_path);
# calledInLine: 237
#....................................................................................................................................................#
my ($CRE_extend_size, $CRE_extend_upstrm_ratio, $clusterInfoHsh_ref, $tmp_bed_dir, $chrom_size_path, $bedtools_bin, $proximal_strandness, $extended_tssCluster_bed_path) = @_;
my $CREInfoHsh_ref = {};
{
&reportAndLogStatus("defining proximal CRE", 10, "\n");#->2953
my $proximity = 'proximal';
my $strand_opt = '-s';
$strand_opt = '' if $proximal_strandness eq 'strandless';
my $tmp_merged_CRE_bed = "$tmp_bed_dir/cluster_merged_CRE.$proximity.bed";
my $tmp_indiv_CRE_bed = "$tmp_bed_dir/cluster_indiv_CRE.$proximity.bed";
my $tmp_indiv_CRE_plus_bed = "$tmp_bed_dir/cluster_indiv_CRE.$proximity.plus_edge_distal.bed";
open INDIVCRE, "| sort -k1,1 -k2,2n >$tmp_indiv_CRE_bed";
foreach my $clusterID (keys %{$clusterInfoHsh_ref}) {
if ($clusterInfoHsh_ref->{$clusterID}{'proximity'} eq $proximity) {
my $extendedStart = $clusterInfoHsh_ref->{$clusterID}{'extendedStart'};
my $extendedEnd = $clusterInfoHsh_ref->{$clusterID}{'extendedEnd'};
my $chrom = $clusterInfoHsh_ref->{$clusterID}{'chrom'};
my $score = $clusterInfoHsh_ref->{$clusterID}{'score'};
my $strand = $clusterInfoHsh_ref->{$clusterID}{'strand'};
print INDIVCRE join "", (join "\t", ($chrom, $extendedStart, $extendedEnd, $clusterID, $score, $strand)), "\n";
}
}
close INDIVCRE;
#---[2021/01/13 1:13] get the distal clusters at the edge of the proximal clusters
system "$bedtools_bin intersect $strand_opt -wa -a $extended_tssCluster_bed_path -b $tmp_indiv_CRE_bed >$tmp_indiv_CRE_plus_bed";
system "$bedtools_bin merge $strand_opt -c 4,5,6 -o distinct,sum,distinct -delim \";\" -i $tmp_indiv_CRE_plus_bed >$tmp_merged_CRE_bed";
&storeCREInfoFromBed($tmp_merged_CRE_bed, $proximity, $CREInfoHsh_ref, $clusterInfoHsh_ref);#->2975
}
{
&reportAndLogStatus("defining distal CRE", 10, "\n");#->2953
my $proximity = 'distal';
my $strand_opt = '';
my $tmp_merged_CRE_bed = "$tmp_bed_dir/cluster_merged_CRE.$proximity.bed";
my $tmp_indiv_CRE_bed = "$tmp_bed_dir/cluster_indiv_CRE.$proximity.bed";
open INDIVCRE, "| sort -k1,1 -k2,2n >$tmp_indiv_CRE_bed";
foreach my $clusterID (keys %{$clusterInfoHsh_ref}) {
#---[2021/01/13 1:21] get the unassigned CREID
if (not exists $clusterInfoHsh_ref->{$clusterID}{'CREID'}) {
my $extendedStart = $clusterInfoHsh_ref->{$clusterID}{'extendedStart'};
my $extendedEnd = $clusterInfoHsh_ref->{$clusterID}{'extendedEnd'};
my $chrom = $clusterInfoHsh_ref->{$clusterID}{'chrom'};
my $score = $clusterInfoHsh_ref->{$clusterID}{'score'};
my $strand = $clusterInfoHsh_ref->{$clusterID}{'strand'};
print INDIVCRE join "", (join "\t", ($chrom, $extendedStart, $extendedEnd, $clusterID, $score, $strand)), "\n";
}
}
close INDIVCRE;
system "$bedtools_bin merge $strand_opt -c 4,5,6 -o distinct,sum,distinct -delim \";\" -i $tmp_indiv_CRE_bed >$tmp_merged_CRE_bed";
&storeCREInfoFromBed($tmp_merged_CRE_bed, $proximity, $CREInfoHsh_ref, $clusterInfoHsh_ref);#->2975
}
return ($CREInfoHsh_ref);
}
sub defineCodingClasses {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: >none
# appearInSub: >none
# primaryAppearInSection: 3_readInfo|217
# secondaryAppearInSection: >none
# input: none
# output: $coding_class_hsh_ref
# toCall: my ($coding_class_hsh_ref) = &defineCodingClasses();
# calledInLine: 224
#....................................................................................................................................................#
my $coding_class_hsh_ref = {
"antisense" => "noncoding",
"IG_C_gene" => "coding",
"IG_C_pseudogene" => "pseudogene",
"IG_D_gene" => "coding",
"IG_J_gene" => "coding",
"IG_J_pseudogene" => "pseudogene",
"IG_pseudogene" => "pseudogene",
"IG_V_gene" => "coding",
"IG_V_pseudogene" => "pseudogene",
"lincRNA" => "noncoding",
"lncRNA" => "noncoding",
"miRNA" => "others",
"misc_RNA" => "others",
"Mt_rRNA" => "others",
"Mt_tRNA" => "others",
"polymorphic_pseudogene" => "pseudogene",
"processed_pseudogene" => "pseudogene",
"processed_transcript" => "others",
"protein_coding" => "coding",
"pseudogene" => "pseudogene",
"rRNA" => "others",
"rRNA_pseudogene" => "others",
"scRNA" => "others",
"sense_intronic" => "noncoding",
"sense_overlapping" => "noncoding",
"snoRNA" => "others",
"snRNA" => "others",
"TEC" => "noncoding",
"transcribed_processed_pseudogene" => "pseudogene",
"transcribed_unitary_pseudogene" => "pseudogene",
"transcribed_unprocessed_pseudogene" => "pseudogene",
"translated_processed_pseudogene" => "pseudogene",
"translated_unprocessed_pseudogene" => "pseudogene",
"TR_C_gene" => "coding",
"TR_D_gene" => "coding",
"TR_J_gene" => "coding",
"TR_J_pseudogene" => "pseudogene",
"TR_V_gene" => "coding",
"TR_V_pseudogene" => "pseudogene",
"unitary_pseudogene" => "pseudogene",
"unprocessed_pseudogene" => "pseudogene",
"vaultRNA" => "others",
"coding_mRNA" => "coding",
"lncRNA_antisense" => "noncoding",
"lncRNA_divergent" => "noncoding",
"lncRNA_intergenic" => "noncoding",
"lncRNA_sense_intronic" => "noncoding",
"pseudogene" => "pseudogene",
"sense_overlap_RNA" => "noncoding",
"short_ncRNA" => "noncoding",
"small_RNA" => "others",
"structural_RNA" => "others",
"uncertain_coding" => "others",
#"unanno_tss" => "others",
};
return ($coding_class_hsh_ref);
}
sub defineHyperactiveDistalLoci {
#....................................................................................................................................................#
# subroutineCategory: unassigned
# dependOnSub: reportAndLogStatus|2953
# appearInSub: >none
# primaryAppearInSection: 3_readInfo|217
# secondaryAppearInSection: >none
# input: $CREInfoHsh_ref, $Rscript_bin, $bedtools_bin, $max_dist_rank, $min_spreadness, $optimize_tangent_R, $result_plot_dir, $stitch_distance, $tmp_bed_dir
# output: $distal_CRE_locus_info_hsh_ref
# toCall: my ($distal_CRE_locus_info_hsh_ref) = &defineHyperactiveDistalLoci($CREInfoHsh_ref, $tmp_bed_dir, $bedtools_bin, $optimize_tangent_R, $max_dist_rank, $Rscript_bin, $stitch_distance, $min_spreadness, $result_plot_dir);
# calledInLine: 242
#....................................................................................................................................................#
my ($CREInfoHsh_ref, $tmp_bed_dir, $bedtools_bin, $optimize_tangent_R, $max_dist_rank, $Rscript_bin, $stitch_distance, $min_spreadness, $result_plot_dir) = @_;
#time perl /osc-fs_home/hon-chun/analysis/tenX_single_cell/scafe/dev/scripts/1.2/scafe.tool.cm.annotate.pl --overwrite=yes --tssCluster_bed_path=/osc-fs_home/hon-chun/analysis/tenX_single_cell/paper_run/cellranger_unified_proc/pooled_celltype/scafe_run/PBMC/filter/PBMC/bed/PBMC.tssCluster.default.filtered.bed.gz --tssCluster_info_path=/osc-fs_home/hon-chun/analysis/tenX_single_cell/paper_run/cellranger_unified_proc/pooled_celltype/scafe_run/PBMC/filter/PBMC/log/PBMC.tssCluster.log.tsv --genome=hg19.gencode_v32lift37 --outputPrefix=demo --outDir=/osc-fs_home/hon-chun/analysis/tenX_single_cell/scafe/dev/test_run/annotate_super_enhancer/
&reportAndLogStatus("defining hyperactive distal loci", 10, "\n");#->2953
my $distal_CRE_bed = "$tmp_bed_dir/distal_CRE.bed";
open OUTBED, "| sort -k1,1 -k2,2n >$distal_CRE_bed";
foreach my $CREID (keys %{$CREInfoHsh_ref}) {
my $proximity = $CREInfoHsh_ref->{$CREID}{'proximity'};
my $gene_promoter = $CREInfoHsh_ref->{$CREID}{'gene_promoter'};
if ($proximity eq 'distal' and $gene_promoter eq 'none') {
my $sumClusterRead = $CREInfoHsh_ref->{$CREID}{'sumClusterRead'};
my ($chrom, $chromStart, $chromEnd) = @{$CREInfoHsh_ref->{$CREID}{'bedLine'}};
print OUTBED join "", (join "\t", ($chrom, $chromStart, $chromEnd, $CREID, $sumClusterRead)), "\n";
}
}
close OUTBED;
if ($stitch_distance eq 'undefined') {
my $distal_CRE_dist_rank = "$tmp_bed_dir/distal_CRE_dist_rank.txt";
open DIST, ">", $distal_CRE_dist_rank;
open BEDTOOLSCLOSEST, "$bedtools_bin closest -io -d -a $distal_CRE_bed -b $distal_CRE_bed |";
while (<BEDTOOLSCLOSEST>) {
chomp;
my @bedtools_ary = split /\t/;
my $dist = $bedtools_ary[-1];
print DIST "$dist\n" if $dist < $max_dist_rank;
}
close DIST;
close BEDTOOLSCLOSEST;
&reportAndLogStatus("getting optimized stitch distance", 10, "\n");#->2953
my $stitch_dist_plot_path = "$result_plot_dir/optimize.stitch_distance.pdf";
$globalReadmeHsh_ref->{$stitch_dist_plot_path}{'description'} = "Plot of the closest distance of a distal CRE to another, ranked in ascending order, a tangent line is used to define the cutoff for stitching within a distal CRE locus";
chomp ($stitch_distance = `$Rscript_bin $optimize_tangent_R $distal_CRE_dist_rank $stitch_dist_plot_path optimize_stitch_distance CRE_rank_by_distance distance`);
&reportAndLogStatus("optimized stitch_distance = $stitch_distance", 10, "\n");#->2953
} else {
&reportAndLogStatus("stitch_distance is user-defined at $stitch_distance", 10, "\n");#->2953
}
&reportAndLogStatus("stitching distal CREs", 10, "\n");#->2953
my $tmp_distal_CRE_locus_hsh_ref = {};
open BEDTOOLSCLOSEST, "$bedtools_bin cluster -d $stitch_distance -i $distal_CRE_bed |";
while (<BEDTOOLSCLOSEST>) {
chomp;
my ($chrom, $start, $end, $CREID, $count, $distal_CRE_locus_num) = split /\t/;
push @{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'count'}}, $count;
push @{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'CREID'}}, $CREID;
push @{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'start'}}, $start;
push @{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'end'}}, $end;
$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'chrom'} = $chrom;
}
close BEDTOOLSCLOSEST;
my @count_ary = ();
my $distal_CRE_locus_info_hsh_ref = {};
foreach my $distal_CRE_locus_num (keys %{$tmp_distal_CRE_locus_hsh_ref}) {
my $start = min(@{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'start'}});
my $end = max(@{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'end'}});
my $size = $end - $start;
my $chrom = $tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'chrom'};
my $total_count = sum(@{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'count'}});
my $top_count = max(@{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'count'}});
my $top_frac = $top_count/$total_count;
my $num_CRE = @{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'CREID'}};
my $spreadness = $num_CRE/$top_frac;
my $distal_CRE_locus_ID = join "_", ($chrom, $start, $end);
@{$distal_CRE_locus_info_hsh_ref->{$distal_CRE_locus_ID}{'CREID'}} = @{$tmp_distal_CRE_locus_hsh_ref->{$distal_CRE_locus_num}{'CREID'}};
$distal_CRE_locus_info_hsh_ref->{$distal_CRE_locus_ID}{'loc'} = [$chrom, $start, $end];
$distal_CRE_locus_info_hsh_ref->{$distal_CRE_locus_ID}{'total_count'} = $total_count;
$distal_CRE_locus_info_hsh_ref->{$distal_CRE_locus_ID}{'top_count'} = $top_count;
$distal_CRE_locus_info_hsh_ref->{$distal_CRE_locus_ID}{'top_frac'} = $top_frac;
$distal_CRE_locus_info_hsh_ref->{$distal_CRE_locus_ID}{'spreadness'} = $spreadness;
$distal_CRE_locus_info_hsh_ref->{$distal_CRE_locus_ID}{'size'} = $size;
push @count_ary, $total_count;
}
my $num_loci = @count_ary;
@count_ary = sort {$a <=> $b} @count_ary;
my $max_index = $#count_ary - int($num_loci*0.001);
my $distal_CRE_locus_count_rank = "$tmp_bed_dir/distal_CRE_locus_count_rank.txt";
open COUNT, ">", $distal_CRE_locus_count_rank;
foreach my $i (0..$max_index) {
print COUNT join "", (join "\t", ($count_ary[$i])), "\n";
}
close COUNT;
&reportAndLogStatus("getting hyperactive locus cutoff", 10, "\n");#->2953
my $hyperactive_count_plot_path = "$result_plot_dir/optimize.hyperactive_count.pdf";