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rf-peakcall
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#!/usr/bin/env perl
##
# RF PeakCall
# RNA Framework [http://www.rnaframework.com]
#
# Author: Danny Incarnato (dincarnato[at]rnaframework.com)
# Summary: Performs peak calling of RNA immunoprecipitation (IP) experiments
#
# This program is free software, and can be redistribute and/or modified
# under the terms of the GNU General Public License as published by
# the Free Software Foundation, either version 3 of the License, or
# any later version.
#
# Please see <http://www.gnu.org/licenses/> for more informations.
##
use strict;
use Config;
use File::Basename;
use File::Path qw(mkpath);
use FindBin qw($Bin);
use Getopt::Long qw(:config no_ignore_case);
use threads;
use threads::shared;
use lib $Bin . "/lib";
use Core::Mathematics qw(:all);
use Core::Statistics;
use Core::Utils;
use Data::IO;
use Data::Sequence::Utils;
use Graphics::Chart::Linedot;
use Graphics::Image;
use RF::Data::IO::RC;
use RF::Utils;
use Term::Constants qw(:screen);
use Term::Progress;
use constant SIZE5UTR => 75;
use constant SIZECDS => 650;
use constant SIZE3UTR => 275;
use constant SIZERNA => 1000;
$|++;
die "\n [!] Error: This program requires ithreads." .
"\n Please recompile Perl with ithreads support and try again\n\n" unless(defined $Config{useithreads});
my ($help, $output, $overwrite, $win, $tmpDir,
$pvalue, $pseudocount, $meancov, $mediancov,
$control, $ip, $index, $enrich, $ctrlIndex,
$ipIndex, $threads, $iprc, $ctrlrc, $decimals,
$mergedist, $img, $error, $metaplot,
$refine, $summit, $relaxed, $offset,
$metaonlyenriched, $whitelist, $metacoding,
$minorflen, $altstart, $anystart, $gencode,
$codsize, $orfFile, $R, @pool, @imeans, %orfs);
my $ctrlnreads : shared;
my $ipnreads : shared;
my $progressBar : shared;
my @qcaller : shared;
my @qresults : shared;
my @metaip : shared;
my @metactrl : shared;
my @metapeaks : shared;
my @metaipcod : shared;
my @metactrlcod : shared;
my @metapeakscod : shared;
my %results : shared;
%results = ( cov => 0,
incov => 0,
diffcseq => 0,
nocid => 0,
pass => 0,
peaks => 0,
codpeaks => 0 );
do {
local $SIG{__WARN__} = sub { };
GetOptions( "h|help" => \$help,
"o|output=s" => \$output,
"ow|overwrite" => \$overwrite,
"g|img" => \$img,
"mp|meta-plot" => \$metaplot,
"eo|plot-enriched-only" => \$metaonlyenriched,
"w|window=i" => \$win,
"l|whitelist=s" => \$whitelist,
"mc|mean-coverage=s" => \$meancov,
"ec|median-coverage=s" => \$mediancov,
"c|control=s" => \$control,
"I|IP=s" => \$ip,
"i|index=s" => \$index,
"D|decimals=i" => \$decimals,
"p|processors=i" => \$threads,
"f|offset=i" => \$offset,
"e|enrichment=s" => \$enrich,
"v|p-value=s" => \$pvalue,
"r|refine" => \$refine,
"x|relaxed" => \$relaxed,
"s|summit" => \$summit,
"of|orf-file=s" => \$orfFile,
"pc|pseudocount=s" => \$pseudocount,
"md|merge-distance=i" => \$mergedist,
"mcp|meta-coding-plot" => \$metacoding,
"mo|min-orf-len=i" => \$minorflen,
"als|alt-start" => \$altstart,
"ans|any-start" => \$anystart,
"gc|genetic-code" => \$gencode ) or help(1);
};
help() if ($help);
# Default
$codsize = SIZE5UTR + SIZECDS + SIZE3UTR;
$threads ||= 1;
$win ||= 150;
$offset ||= round($win / 2);
$enrich ||= 3;
$mergedist //= 50;
$pvalue //= 0.05;
$pseudocount ||= 1;
$decimals ||= 3;
$meancov //= 0;
$mediancov //= 0;
$gencode ||= 1;
$minorflen ||= 50;
$R = checkRinstall($R) if ($img || $metacoding || $metaplot);
die "\n [!] Error: No IP sample RC file provided\n\n" if (!defined $ip);
die "\n [!] Error: Provided IP sample RC file does not exist\n\n" if (!-e $ip);
die "\n [!] Error: Provided RCI index does not exist\n\n" if (defined $index && !-e $index);
die "\n [!] Error: Provided ORF file does not exist\n\n" if (defined $orfFile && !-e $orfFile);
die "\n [!] Error: Provided control sample RC file does not exist\n\n" if (defined $control && !-e $control);
die "\n [!] Error: Number of processors must be an integer greater than 0\n\n" if ($threads < 1);
die "\n [!] Error: Decimals value must be an integer comprised between 1 and 10\n\n" if ($decimals < 1 || $decimals > 10);
die "\n [!] Error: Window's size must be an integer >= 10\n\n" if ($win < 10);
die "\n [!] Error: Offset cannot exceed window's size\n\n" if ($offset > $win);
die "\n [!] Error: P-value must be numeric\n\n" if (!isnumeric($pvalue));
die "\n [!] Error: P-value must be comprised between 0 and 1\n\n" if (!ispositive($pvalue) || $pvalue > 1);
die "\n [!] Error: Pseudocount value must be numeric\n\n" if (!isnumeric($pseudocount));
die "\n [!] Error: Pseudocount value must be > 0\n\n" if (!$pseudocount);
die "\n [!] Error: Enrichment value must be numeric\n\n" if (!isnumeric($enrich));
die "\n [!] Error: Enrichment value must be > 0\n\n" if (!$enrich);
die "\n [!] Error: Mean coverage value must be numeric\n\n" if (!isnumeric($meancov));
die "\n [!] Error: Mean coverage value must be >= 0\n\n" if (!ispositive($meancov));
die "\n [!] Error: Median coverage value must be numeric\n\n" if (!isnumeric($mediancov));
die "\n [!] Error: Median coverage value must be >= 0\n\n" if (!ispositive($mediancov));
die "\n [!] Error: Parameter -x requires peaks refinement (-r) to be active\n\n" if ($relaxed && !$refine);
die "\n [!] Error: Parameter -eo requires meta-plot generation (-mp) to be active\n\n" if ($metaonlyenriched && (!$metaplot && !$metacoding));
die "\n [!] Error: Provided whitelist file does not exist\n\n" if (defined $whitelist && !-e $whitelist);
warn "\n [!] Warning: No control sample RC file provided. Background will be estimated on IP sample only.\n" if (!defined $control);
if (!defined $output) {
my ($cid, $iid);
$cid = fileparse($control, qr/\.[^.]*/) if (defined $control);
$iid = fileparse($ip, qr/\.[^.]*/);
$output = (defined $control ? $iid . "_vs_" . $cid : $iid) . "/";
}
else { $output =~ s/\/?$/\//; }
$SIG{__DIE__} = \&cleanup;
$tmpDir = "${output}tmp";
print "\n[+] Making output directory...";
if (-e $output) {
if ($overwrite) {
my $error = rmtree($output);
die "\n\n [!] Error: " . $error . "\n\n" if ($error);
}
else { die "\n\n [!] Error: Output directory already exists." .
"\n Please use -ow (or --overwrite) to overwrite output directory\n\n"; }
}
mkpath($tmpDir, { mode => 0755,
error => \$error });
mkpath($output . "plots", { mode => 0755,
error => \$error }) if ($metaplot || $metacoding);
mkpath($output . "plots/transcripts", { mode => 0755,
error => \$error }) if ($img);
# In case no index has been provided, we rebuild the index before generating the working threads
# The new index will be generated in the same path of the rc file, with extension .rci
$ctrlIndex = defined $index ? $index : (defined $control && -e "$control.rci" ? "$control.rci" : undef);
$ipIndex = defined $index ? $index : (-e "$ip.rci" ? "$ip.rci" : undef);
$ctrlrc = RF::Data::IO::RC->new( file => $control,
index => $ctrlIndex,
buildindex => defined $ctrlIndex ? 1 : 0 ) if ($control);
$iprc = RF::Data::IO::RC->new( file => $ip,
index => $ipIndex,
buildindex => defined $ipIndex ? 1 : 0 );
print "\n[+] Loading transcript IDs... ";
@qcaller = $iprc->ids();
if ($whitelist) {
my %whitelist;
open(my $fh, "<", $whitelist) or die "\n\n [!] Error: Unable to read from whitelist file ($!)\n\n";
while(<$fh>) {
chomp();
$whitelist{$_} = 1;
}
close($fh);
@qcaller = grep { exists $whitelist{$_} } @qcaller;
if (!@qcaller) { die "\n\n [!] Error: No transcript matched provided whitelist\n\n"; }
}
print scalar(@qcaller) . " transcripts loaded.";
if ($orfFile) {
print "\n[+] Importing ORF coordinates...";
open(my $fh, "<", $orfFile) or die "\n\n [!] Error: Unable to read from ORF file ($!)\n\n";
while(<$fh>) {
chomp();
my @row = split /\t/;
next if (@row < 3);
if (my $length = $iprc->length($row[0])) {
if (isint(@row[1, 2]) && ispositive(@row[1, 2]) && $row[1] < $row[2]) {
next if ($row[2] > $length);
$orfs{$row[0]} = [$row[1], $row[2] - 1];
}
else { die "\n\n [!] Error: Malformed ORF file\n\n"; }
}
}
close($fh);
print " " . scalar(keys %orfs) . " imported.";
}
$progressBar = shared_clone(Term::Progress->new( max => scalar(@qcaller),
width => 50,
colored => 1 ));
print "\n[+] Calling peaks...\n\n";
$progressBar->init();
@pool = map{ threads->create(\&call) } 1 .. $threads;
$_->join() for(@pool);
print "\n";
if ($results{peaks}) {
print "\n[+] Sorting peaks...";
# Sort lexicographically by transcript ID and numerically by start position
@qresults = sort {$a->[0] cmp $b->[0] ||
$a->[1] <=> $b->[1]} @qresults;
print "\n[+] Writing output BED file...";
open(my $wh, ">", $output . "/peaks.bed") or die "\n\n [!] Error: Unable to write peaks BED file (" . $! . ")\n\n";
select((select($wh), $|=1)[0]);
print $wh join("\t", @{$_}[0 .. 4]) . "\n" for (@qresults);
close($wh);
if ($summit) {
open(my $wh, ">", $output . "/summits.bed") or die "\n\n [!] Error: Unable to write summits BED file (" . $! . ")\n\n";
select((select($wh), $|=1)[0]);
print $wh join("\t", @{$_}[0, 5, 6]) . "\n" for (@qresults);
close($wh);
}
}
if ($metaplot &&
(($metaonlyenriched && @qresults) ||
!$metaonlyenriched)) {
my (@icov, @ccov, @pcov);
@icov = map { ($_ / $results{cov}) / ($metaonlyenriched ? $ipnreads : $iprc->mappedreads()) * (10 ** 6) } @metaip;
@ccov = map { ($_ / $results{cov}) / ($metaonlyenriched ? $ctrlnreads : $ctrlrc->mappedreads()) * (10 ** 6) } @metactrl if ($control);
@pcov = map { $_ / @qresults * 100 } @metapeaks if (@qresults);
plotcoverage($output . "plots/meta-gene.pdf", \@icov, \@ccov, \@pcov);
}
if ($metacoding &&
(($metaonlyenriched && @qresults) ||
!$metaonlyenriched)) {
my (@icov, @ccov, @pcov);
@icov = map { ($_ / $results{cov}) / (($metaonlyenriched ? $ipnreads : $iprc->mappedreads()) || 1) * (10 ** 6) } @metaipcod;
@ccov = map { ($_ / $results{cov}) / ($metaonlyenriched ? $ctrlnreads : $ctrlrc->mappedreads()) * (10 ** 6) } @metactrlcod if ($control);
@pcov = map { $_ / $results{codpeaks} * 100 } @metapeakscod if ($results{codpeaks});
plotcoverage($output . "plots/meta-coding-gene.pdf", \@icov, \@ccov, \@pcov);
}
cleanup();
print "\n[+] Calling statistics:\n" .
"\n [*] Covered transcripts: " . $results{cov} . " total" .
"\n " . $results{pass} . " transcripts with detected enrichment" .
"\n " . $results{peaks} . " called peaks" .
"\n [*] Discarded transcripts: " . ($results{incov} + $results{diffcseq} + $results{nocov} + $results{nocid}) . " total" .
"\n " . $results{incov} . " insufficient coverage" .
"\n " . $results{diffcseq} . " mismatch between IP and control sample sequence" .
"\n " . $results{nocid} . " absent in control sample reference";
print "\n\n[+] All done.\n\n";
sub call {
my ($crc, $irc, $attributes, $scale,
@cmeta, @imeta, @cmetacod, @imetacod,
@pmeta, @pmetacod);
$crc = RF::Data::IO::RC->new( file => $control,
index => $ctrlIndex ) if (defined $control);
$irc = RF::Data::IO::RC->new( file => $ip,
index => $ipIndex );
while (1) {
my ($id, $initial);
{ lock(@qcaller);
$id = shift(@qcaller) if (@qcaller);
if ($id) {
$initial = uc(substr($id, 0, 1));
mkpath($output . "plots/transcripts/" . $initial, { mode => 0755,
error => \$error }) if ($img && !-e $output . "plots/transcripts/" . $initial);
} }
last unless($id);
{ lock($progressBar);
$progressBar->update(1); }
my ($ientry, $sequence, $imedian, $cmedian,
$last, $length, $ssummit, $esummit,
$cnreads, $orfStart, $orfEnd, @icov,
@ccov, @pvalues, @wins, @enrich,
@peaks);
$ientry = $irc->read($id);
$imedian = $ientry->mediancoverage();
$sequence = $ientry->sequence();
$length = length($sequence);
@icov = $ientry->coverage();
if (defined $control) {
if (my $centry = $crc->read($id)) {
$cmedian = $centry->mediancoverage();
if ($centry->sequence() ne $sequence) {
lock(%results);
$results{diffcseq}++;
next;
}
if ($centry->meancoverage() < $meancov ||
$cmedian < $mediancov) {
lock(%results);
$results{incov}++;
next;
}
$cnreads = $centry->readscount();
@ccov = $centry->coverage();
}
else {
lock(%results);
$results{nocid}++;
next;
}
}
if ($metacoding) {
if ($orfFile) { if (exists $orfs{$id}) { ($orfStart, $orfEnd) = @{$orfs{$id}}; } }
else {
my ($orf);
($orf, $orfStart) = longestorf($sequence, { gencode => $gencode,
altstart => $altstart,
ignorestart => $anystart,
minlength => $minorflen });
$orfEnd = $orfStart + length($orf) - 1 if (defined $orf);
}
}
# Calculates per-base enrichment
@enrich = $control ? map { logarithm((($icov[$_] + $pseudocount) / ($imedian + $pseudocount)) / (($ccov[$_] + $pseudocount) / ($cmedian + $pseudocount)), 2) } 0 .. $length - 1 :
map { logarithm((($icov[$_] + $pseudocount) / ($imedian + $pseudocount)), 2) } 0 .. $length - 1;
if (!$metaonlyenriched) {
if ($metaplot) {
my (@metacoverage);
if ($control) {
@metacoverage = metacoverage(\@ccov, SIZERNA);
@cmeta = map { $cmeta[$_] + $metacoverage[$_] } 0 .. SIZERNA - 1;
undef(@metacoverage);
}
@metacoverage = metacoverage(\@icov, SIZERNA);
@imeta = map { $imeta[$_] + $metacoverage[$_] } 0 .. SIZERNA - 1;
}
if ($metacoding && $orfEnd) {
my (@cmeta5, @cmeta3, @cmetac, @imeta5,
@imeta3, @imetac, @metacod);
@cmeta5 = (0) x SIZE5UTR;
@cmeta3 = (0) x SIZE3UTR;
@imeta5 = @cmeta5;
@imeta3 = @cmeta3;
if ($orfStart > 0) {
@cmeta5 = metacoverage([@ccov[0 .. $orfStart - 1]], SIZE5UTR) if ($control);
@imeta5 = metacoverage([@icov[0 .. $orfStart - 1]], SIZE5UTR);
}
@cmetac = metacoverage([@ccov[$orfStart .. $orfEnd]], SIZECDS) if ($control);
@imetac = metacoverage([@icov[$orfStart .. $orfEnd]], SIZECDS);
if ($orfEnd < $length - 1) {
@cmeta3 = metacoverage([@ccov[$orfEnd + 1 .. $length - 1]], SIZE3UTR) if ($control);
@imeta3 = metacoverage([@icov[$orfEnd + 1 .. $length - 1]], SIZE3UTR);
}
if ($control) {
@metacod = (@cmeta5, @cmetac, @cmeta3);
$cmetacod[$_] += ($metacod[$_] ) for (0 .. $#metacod);
undef(@metacod);
}
@metacod = (@imeta5, @imetac, @imeta3);
$imetacod[$_] += ($metacod[$_]) for (0 .. $#metacod);
}
}
if ($img) {
my ($icov, $ccov);
@{$icov} = map { $_ / $irc->mappedreads() * (10 ** 6) } @icov;
@{$ccov} = map { $_ / $crc->mappedreads() * (10 ** 6) } @ccov if ($control);
plotcoverage($output . "/plots/transcripts/" . $initial . "/" . $id . ".pdf", $icov, $ccov);
}
for (my $i = 0; $i < $#icov; $i += $offset) {
my ($start, $end, $score);
$start = $i;
if ($start + $win > $#icov) {
$start = $#icov - $win >= 0 ? $#icov - $win : 0;
$end = $#icov;
}
else { $end = $start + $win; }
$score = mean(@enrich[$start .. $end]);
push(@wins, { coords => [$start, $end],
score => [$score],
pvalue => undef });
}
{ lock(%results);
$results{cov}++; }
@imeans = map { mean(@icov[$_->{coords}->[0] .. $_->{coords}->[1]]) } @wins;
foreach my $win (@wins) {
my $p = defined $control ? fisher(round(mean(@icov[$win->{coords}->[0] .. $win->{coords}->[1]])), round($imedian), round(mean(@ccov[$win->{coords}->[0] .. $win->{coords}->[1]])), round($cmedian)) :
fisher(round(mean(@icov[$win->{coords}->[0] .. $win->{coords}->[1]])), round($imedian), round(mean(@imeans)), round($imedian));
$win->{pvalue} = $p;
}
@pvalues = grep { isnumeric($_) } (map { $_->{pvalue} } @wins);
next if (!@pvalues);
@pvalues = padjust(\@pvalues); # Benjamini-Hochberg adjusted p-values
foreach my $win (@wins) { $win->{pvalue} = [ shift(@pvalues) ] if (isnumeric($win->{pvalue})); } # Update p-values with BH adjusted p-values
# Delete peaks whose corrected p-value is greater than cutoff
# or whose enrichment is lower than cutoff
for (my $i = 0; $i < @wins; $i++) {
if (!isnumeric($wins[$i]->{pvalue}->[0]) ||
$wins[$i]->{pvalue}->[0] >= $pvalue ||
$wins[$i]->{score}->[0] < $enrich) {
splice(@wins, $i, 1);
$i--;
}
}
next if (!@wins);
if ($refine) {
$_ = refinepeak( peak => $_,
imedian => $imedian,
cmedian => $cmedian,
length => $length,
icov => \@icov,
ccov => \@ccov,
enrichment => \@enrich ) for (@wins);
}
$last = shift(@wins);
while(my $current = shift(@wins)) {
if (intersect($current->{coords}, [$last->{coords}->[0], $last->{coords}->[1] + $mergedist])) {
$last->{coords}->[1] = $current->{coords}->[1];
push(@{$last->{score}}, $current->{score}->[0]);
push(@{$last->{pvalue}}, $current->{pvalue}->[0]);
}
else {
push(@peaks, $last);
$last = $current;
}
}
push(@peaks, $last);
foreach my $peak (@peaks) {
{ lock(@qresults);
push(@qresults, shared_clone([$id, $peak->{coords}->[0], $peak->{coords}->[1] + 1, sprintf("%." . $decimals . "f", mean(@{$peak->{score}})), sprintf("%." . $decimals . "e", pcombine($peak->{pvalue}, "S")), summit($peak->{coords}->[0], $peak->{coords}->[1], \@icov)])); }
if ($metaplot) {
my @metapeak = metacoverage([(0) x $peak->{coords}->[0], (1) x ($peak->{coords}->[1] - $peak->{coords}->[0] + 1), (0) x ($length - 1 - $peak->{coords}->[1])], SIZERNA);
@pmeta = map { $pmeta[$_] + $metapeak[$_] } 0 .. SIZERNA - 1;
}
if ($metacoding) {
my (@peakcov, @cov5utr, @cov3utr, @covcds);
@peakcov = (0) x $length;
$peakcov[$_] = 1 for ($peak->{coords}->[0] .. $peak->{coords}->[1]);
@cov5utr = (0) x SIZE5UTR;
@cov3utr = (0) x SIZE3UTR;
@cov5utr = metacoverage([@peakcov[0 .. $orfStart - 1]], SIZE5UTR) if ($orfStart > 0);
@covcds = metacoverage([@peakcov[$orfStart .. $orfEnd]], SIZECDS);
@cov3utr = metacoverage([@peakcov[$orfEnd + 1 .. $length - 1]], SIZE3UTR) if ($orfEnd < $length - 1);
undef(@peakcov);
@peakcov = (@cov5utr, @covcds, @cov3utr);
@pmetacod = map { $pmetacod[$_] + $peakcov[$_] } 0 .. $#peakcov;
{ lock(%results);
$results{codpeaks}++; }
}
}
{ lock(%results);
$results{pass}++;
$results{peaks} += @peaks; }
if ($metaonlyenriched) {
if ($control) {
lock($ctrlnreads);
$ctrlnreads += $cnreads;
}
{ lock($ipnreads);
$ipnreads += $ientry->readscount(); }
if ($metaplot) {
my (@metacoverage);
if ($control) {
@metacoverage = metacoverage(\@ccov, SIZERNA);
@cmeta = map { $cmeta[$_] + $metacoverage[$_] } 0 .. SIZERNA - 1;
undef(@metacoverage);
}
@metacoverage = metacoverage(\@icov, SIZERNA);
@imeta = map { $imeta[$_] + $metacoverage[$_] } 0 .. SIZERNA - 1;
}
if ($metacoding && $orfEnd) {
my (@cmeta5, @cmeta3, @cmetac, @imeta5,
@imeta3, @imetac, @metacod);
@cmeta5 = (0) x SIZE5UTR;
@cmeta3 = (0) x SIZE3UTR;
@imeta5 = @cmeta5;
@imeta3 = @cmeta3;
if ($orfStart > 0) {
@cmeta5 = metacoverage([@ccov[0 .. $orfStart - 1]], SIZE5UTR) if ($control);
@imeta5 = metacoverage([@icov[0 .. $orfStart - 1]], SIZE5UTR);
}
@cmetac = metacoverage([@ccov[$orfStart .. $orfEnd]], SIZECDS) if ($control);
@imetac = metacoverage([@icov[$orfStart .. $orfEnd]], SIZECDS);
if ($orfEnd < $length - 1) {
@cmeta3 = metacoverage([@ccov[$orfEnd + 1 .. $length - 1]], SIZE3UTR) if ($control);
@imeta3 = metacoverage([@icov[$orfEnd + 1 .. $length - 1]], SIZE3UTR);
}
if ($control) {
@metacod = (@cmeta5, @cmetac, @cmeta3);
$cmetacod[$_] += ($metacod[$_] ) for (0 .. $#metacod);
undef(@metacod);
}
@metacod = (@imeta5, @imetac, @imeta3);
$imetacod[$_] += ($metacod[$_] ) for (0 .. $#metacod);
}
}
}
if ($metaplot) {
if ($control) {
lock(@metactrl);
@metactrl = map { $metactrl[$_] + $cmeta[$_] } 0 .. SIZERNA - 1;
}
{ lock(@metaip);
@metaip = map { $metaip[$_] + $imeta[$_] } 0 .. SIZERNA - 1; }
{ lock(@metapeaks);
@metapeaks = map { $metapeaks[$_] + $pmeta[$_] } 0 .. SIZERNA - 1; }
}
if ($metacoding) {
if ($control) {
lock(@metactrlcod);
@metactrlcod = map { $metactrlcod[$_] + $cmetacod[$_] } 0 .. $codsize - 1;
}
{ lock(@metaipcod);
@metaipcod = map { $metaipcod[$_] + $imetacod[$_] } 0 .. $codsize - 1; }
{ lock(@metapeakscod);
@metapeakscod = map { $metapeakscod[$_] + $pmetacod[$_] } 0 .. $codsize - 1; }
}
}
sub cleanup {
unlink(glob($tmpDir . "*"));
rmtree($tmpDir);
}
sub refinepeak {
my %parameters = @_;
my ($start, $end, $imean, $cmean,
$peakicovmedian);
($start, $end) = ($parameters{peak}->{coords}->[0], $parameters{peak}->{coords}->[1]);
$peakicovmedian = median(@{$parameters{icov}});
for (my $i = $parameters{peak}->{coords}->[0]; $i <= $parameters{peak}->{coords}->[1]; $i++) {
my $enrichment = $relaxed ? mround($parameters{enrichment}->[$i], 0.5) : $parameters{enrichment}->[$i];
if ($enrichment >= $enrich &&
$parameters{icov}->[$i] >= $peakicovmedian) {
$start = $i;
last;
}
}
if ($start == $parameters{peak}->{coords}->[0]) {
for (my $i = $parameters{peak}->{coords}->[0]; $i >= 0; $i--) {
my $enrichment = $relaxed ? mround($parameters{enrichment}->[$i], 0.5) : $parameters{enrichment}->[$i];
last if ($enrichment < $enrich ||
$parameters{icov}->[$i] < $peakicovmedian);
$start = $i;
}
}
for (my $i = $parameters{peak}->{coords}->[1]; $i >= $parameters{peak}->{coords}->[0]; $i--) {
my $enrichment = $relaxed ? mround($parameters{enrichment}->[$i], 0.5) : $parameters{enrichment}->[$i];
if ($enrichment >= $enrich &&
$parameters{icov}->[$i] >= $peakicovmedian) {
$end = $i;
last;
}
}
if ($end == $parameters{peak}->{coords}->[1]) {
for (my $i = $parameters{peak}->{coords}->[1]; $i < $parameters{length}; $i++) {
my $enrichment = $relaxed ? mround($parameters{enrichment}->[$i], 0.5) : $parameters{enrichment}->[$i];
last if ($enrichment < $enrich ||
$parameters{icov}->[$i] < $peakicovmedian);
$end = $i;
}
}
($parameters{peak}->{coords}->[0], $parameters{peak}->{coords}->[1]) = ($start, $end);
$imean = mean(@{$parameters{icov}}[$start .. $end]);
if ($control) {
$cmean = mean(@{$parameters{ccov}}[$start .. $end]);
@{$parameters{peak}->{score}} = logarithm((($imean + $pseudocount) / ($parameters{imedian} + $pseudocount)) / (($cmean + $pseudocount) / ($parameters{cmedian} + $pseudocount)), 2);
}
else { @{$parameters{peak}->{score}} = logarithm((($imean + $pseudocount) / ($parameters{imedian} + $pseudocount)), 2); }
return($parameters{peak});
}
sub summit {
my ($start, $end, $icov) = @_;
if ($summit &&
$icov) {
my ($max, $first, @max, @win, @last);
$max = max(@{$icov}[$start .. $end]);
@max = grep { $icov->[$_] == $max } $start .. $end;
$first = shift(@max);
@last = ($first, $first + 1);
@win = @last;
while(my $base = shift(@max)) {
if ($base == $win[1]) { $win[1] = $base + 1; }
else {
if (($win[1] - $win[0]) > ($last[1] - $last[0])) {
@last = @win;
@win = ($base, $base + 1);
}
}
}
@last = @win if (($win[1] - $win[0]) > ($last[1] - $last[0]));
return(@last);
}
}
sub metacoverage {
my ($coverage, $size) = @_;
$size--;
my @metacoverage = map { $coverage->[round($_ / $size * $#{$coverage})] } 0 .. $size;
return(@metacoverage);
}
sub plotcoverage {
my ($file, $ip, $ctrl, $peak) = @_;
my ($maxy, $metaprofile, $metacodprofile);
$metaprofile = $file =~ m/meta-gene/ ? 1 : 0;
$metacodprofile = $file =~ m/meta-coding/ ? 1 : 0;
$maxy = $control ? max(@{$ip}, @{$ctrl}) : max(@{$ip});
$ctrl ||= [];
$peak ||= [];
if ($maxy) {
my ($hasPeaks, $plot, $profilePlot, @plots, %legendColors, %xBreaks);
$hasPeaks = ($metacodprofile || $metaprofile) && @$peak && sum(@$peak) ? 1 : 0;
%legendColors = ("IP" => "#376eae");
$legendColors{"Control"} = "#006400" if (@$ctrl);
if ($metacodprofile) {
%xBreaks = ( "0" => "TSS",
"75" => "Start",
"725" => "Stop",
"1000" => "TES" );
}
elsif ($metaprofile) {
%xBreaks = ( "0" => "0",
"250" => "25",
"500" => "50",
"750" => "75",
"1000" => "100" );
}
$plot = Graphics::Image->new( file => $file,
width => 10,
height => $hasPeaks ? 8 : 4,
R => $R,
tmpdir => $tmpDir );
$profilePlot = Graphics::Chart::Linedot->new( data => [ @$ip, @$ctrl ],
dataLabels => { sample => [ ("IP") x scalar(@$ip), ("Control") x scalar(@$ctrl) ],
pos => [ (1 .. scalar(@$ip)), (1 .. scalar(@$ctrl))] },
dataLabelType => { pos => "numeric" },
x => "pos",
fill => "sample",
plotDataPoints => 0,
legendColors => \%legendColors,
xTitle => $metaprofile ? "Relative position (\%)" : ($metacodprofile ? undef : "Position (nt)"),
yTitle => "RPM",
axisTitleSize => 14,
labelTextSize => 12,
legendKeyWidth => 14,
legendTextSize => 12,
xBreaks => \%xBreaks );
push(@plots, $profilePlot);
if ($hasPeaks) {
my $peakPlot = Graphics::Chart::Linedot->new( data => $peak,
dataLabels => { sample => [ ("Peaks") x scalar(@$peak) ],
pos => [ (1 .. scalar(@$peak)) ] },
dataLabelType => { pos => "numeric" },
x => "pos",
fill => "sample",
plotDataPoints => 0,
legend => 0,
legendColors => { "Peaks" => "#000000" },
xTitle => $metaprofile ? "Relative position (\%)" : undef,
yTitle => "% Peaks",
axisTitleSize => 14,
legendKeyWidth => 14,
legendTextSize => 12,
labelTextSize => 12,
xBreaks => \%xBreaks );
push(@plots, $peakPlot);
}
$plot->plot([ map { [ $_ ] } @plots ]);
# if ($control) {
# my $ctrlPlot = Graphics::Chart::Linedot->new( x => "fp",
# legend => 1,
# data => \@tp,
# dataLabels => { fp => \@fp,
# id => \@id },
# dataLabelType => { fp => "numeric"},
# legendSort => [ (grep { $_ ne "Overall" } sort(uniq(@id))), "Overall" ],
# fill => "id",
# xLabelAngle => 90,
# plotDataPoints => 0,