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#!/usr/bin/env perl
#
# Copyright (C) 2012-2014,2018, 2024 Genome Research Ltd.
#
# Author: Petr Danecek <pd3@sanger.ac.uk>
#
# Permission is hereby granted, free of charge, to any person obtaining a copy
# of this software and associated documentation files (the "Software"), to deal
# in the Software without restriction, including without limitation the rights
# to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
# copies of the Software, and to permit persons to whom the Software is
# furnished to do so, subject to the following conditions:
#
# The above copyright notice and this permission notice shall be included in
# all copies or substantial portions of the Software.
#
# THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
# IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
# FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL
# THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
# LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING
# FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER
# DEALINGS IN THE SOFTWARE.
use strict;
use warnings;
use Carp;
use POSIX qw/floor ceil/;
use URI::Escape qw(uri_escape);
use List::Util qw(max);
my $opts = parse_params();
parse_bamcheck($opts);
if ( @{$$opts{bamcheck}} > 1 ) { merge_bamcheck($opts); exit; }
plot_qualities($opts);
plot_acgt_cycles($opts);
plot_gc($opts);
plot_gc_depth($opts);
plot_isize($opts);
plot_coverage($opts);
plot_mismatches_per_cycle($opts);
plot_indel_dist($opts);
plot_indel_cycles($opts);
plot_read_len_hist($opts);
create_html($opts);
exit;
#--------------------------------
sub error
{
my (@msg) = @_;
if ( scalar @msg ) { confess @msg; }
die
"About: Parses output of samtools stats (former bamcheck) and calls gnuplot to create graphs.\n",
"Usage: plot-bamstats [OPTIONS] file.bam.bc\n",
" plot-bamstats -p outdir/ file.bam.bc\n",
"Options:\n",
" -k, --keep-files Do not remove temporary files.\n",
" -l, --log-y Set the Y axis scale of the Insert Size plot to log 10.\n",
" -m, --merge Merge multiple bamstats files and output to STDOUT.\n",
" -p, --prefix <path> The output files prefix, add a slash to create new directory.\n",
" -r, --ref-stats <file.fa.gc> Optional reference stats file with expected GC content (created with -s).\n",
" -s, --do-ref-stats <file.fa> Calculate reference sequence GC for later use with -r\n",
" -t, --targets <file.tab> Restrict -s to the listed regions (tab-delimited chr,from,to. 1-based, inclusive)\n",
" -q, --alt-qual-avg-method Calculate average base quality using average base error probabilities rather than average quality scores.\n",
" -h, -?, --help This help message.\n",
"\n";
}
sub parse_params
{
$0 =~ s{^.+/}{};
my $opts =
{
args => join(' ',$0,@ARGV),
# for merging
sections =>
[
{
id=>'SN',
header=>
"# Summary Numbers. Use `grep ^SN | cut -f 2-` to extract this part.\n",
},
{
id=>'FFQ',
header=>
"# First Fragment Qualities. Use `grep ^FFQ | cut -f 2-` to extract this part.\n" .
"# Columns correspond to qualities and rows to cycles. First column is the cycle number.\n",
},
{
id=>'LFQ',
header=>
"# Last Fragment Qualities. Use `grep ^LFQ | cut -f 2-` to extract this part.\n" .
"# Columns correspond to qualities and rows to cycles. First column is the cycle number.\n",
},
{
id=>'MPC',
header=>
"# Mismatches per cycle and quality. Use `grep ^MPC | cut -f 2-` to extract this part.\n" .
"# Columns correspond to qualities, rows to cycles. First column is the cycle number, second\n" .
"# is the number of N's and the rest is the number of mismatches\n",
},
{
id=>'GCF',
header=>"# GC Content of first fragments. Use `grep ^GCF | cut -f 2-` to extract this part.\n",
},
{
id=>'GCL',
header=>"# GC Content of last fragments. Use `grep ^GCL | cut -f 2-` to extract this part.\n",
},
{
id=>'GCC',
header=>"# ACGT content per cycle. Use `grep ^GCC | cut -f 2-` to extract this part. The columns are: cycle; A,C,G,T base counts as a percentage of all A/C/G/T bases [\%]; and N and O counts as a percentage of all A/C/G/T bases [\%]\n",
},
{
id => 'FBC',
header=>"# ACGT content per cycle for first fragments. Use `grep ^FBC | cut -f 2-` to extract this part. The columns are: cycle; A,C,G,T base counts as a percentage of all A/C/G/T bases [\%]; and N and O counts as a percentage of all A/C/G/T bases [\%]\n",
},
{
id => 'LBC',
header=>"# ACGT content per cycle for last fragments. Use `grep ^LBC | cut -f 2-` to extract this part. The columns are: cycle; A,C,G,T base counts as a percentage of all A/C/G/T bases [\%]; and N and O counts as a percentage of all A/C/G/T bases [\%]\n",
},
{
id=>'IS',
header=>"# Insert sizes. Use `grep ^IS | cut -f 2-` to extract this part. The columns are: insert size, pairs total, inward oriented pairs, outward oriented pairs, other pairs\n",
},
{
id=>'RL',
header=>"# Read lengths. Use `grep ^RL | cut -f 2-` to extract this part. The columns are: read length, count\n",
},
{
id=>'FRL',
header=>"# Read lengths - first fragments. Use `grep ^FRL | cut -f 2-` to extract this part. The columns are: read length, count\n",
},
{
id=>'LRL',
header=>"# Read lengths - last fragments. Use `grep ^LRL | cut -f 2-` to extract this part. The columns are: read length, count\n",
},
{
id=>'ID',
header=>"# Indel distribution. Use `grep ^ID | cut -f 2-` to extract this part. The columns are: length, number of insertions, number of deletions\n",
},
{
id=>'IC',
header=>"# Indels per cycle. Use `grep ^IC | cut -f 2-` to extract this part. The columns are: cycle, number of insertions (fwd), .. (rev) , number of deletions (fwd), .. (rev)\n",
},
{
id=>'COV',
header=>"# Coverage distribution. Use `grep ^COV | cut -f 2-` to extract this part.\n",
},
# {
# id=>'GCD',
# header=>"# GC-depth. Use `grep ^GCD | cut -f 2-` to extract this part. The columns are: GC%, unique sequence percentiles, 10th, 25th, 50th, 75th and 90th depth percentile\n",
# },
],
# for merging
merge_keys =>
{
'sum' =>
[
'raw total sequences:',
'filtered sequences:',
'sequences:',
'1st fragments:',
'last fragments:',
'reads mapped:',
'reads mapped and paired:',
'reads unmapped:',
'reads properly paired:',
'reads paired:',
'reads duplicated:',
'reads MQ0:',
'reads QC failed:',
'non-primary alignments:',
'total length:',
'total first fragment length:',
'total last fragment length:',
'bases mapped:',
'bases mapped (cigar):',
'bases trimmed:',
'bases duplicated:',
'mismatches:',
'inward oriented pairs:',
'outward oriented pairs:',
'pairs with other orientation:',
'pairs on different chromosomes:',
],
'min' =>
[
'is sorted:',
],
'max' =>
[
'maximum length:',
],
},
};
for my $key (keys %{$$opts{merge_keys}})
{
for my $val (@{$$opts{merge_keys}{$key}}) { $$opts{hmerge}{$val} = $key; }
}
while (defined(my $arg=shift(@ARGV)))
{
if ( $arg eq '-k' || $arg eq '--keep-files' ) { $$opts{keep_files}=1; next; }
if ( $arg eq '-l' || $arg eq '--log-y' ) { $$opts{y_axis_log10}=1; next; }
if ( $arg eq '-m' || $arg eq '--merge' ) { $$opts{merge}=1; next; }
if ( $arg eq '-r' || $arg eq '--ref-stats' ) { $$opts{ref_stats}=shift(@ARGV); next; }
if ( $arg eq '-s' || $arg eq '--do-ref-stats' ) { $$opts{do_ref_stats}=shift(@ARGV); next; }
if ( $arg eq '-t' || $arg eq '--targets' ) { $$opts{targets}=shift(@ARGV); next; }
if ( $arg eq '-p' || $arg eq '--prefix' ) { $$opts{prefix}=shift(@ARGV); next; }
if ( $arg eq '-q' || $arg eq '--alt-qual-avg-method' ) { $$opts{alt_qual_avg_method}=1; next; }
if ( $arg eq '-?' || $arg eq '-h' || $arg eq '--help' ) { error(); }
if ( $arg eq '-' || -e $arg ) { push @{$$opts{bamcheck}},$arg; next; }
error("Unknown parameter or non-existent file \"$arg\". Run -h for help.\n");
}
if ( exists($$opts{do_ref_stats }) ) { do_ref_stats($opts); exit; }
if ( !exists($$opts{bamcheck}) ) { error("No samtools stats file?\n") }
if ( !exists($$opts{prefix}) )
{
if ( !$$opts{merge} ) { error("Expected -p parameter.\n") }
$$opts{prefix} = './';
}
elsif ( $$opts{merge} ) { error("Only one of -p or -m should be given.\n"); }
if ( $$opts{merge} )
{
if ( @{$$opts{bamcheck}} < 2 ) { error("Nothing to merge\n") }
}
else
{
if ( !exists($$opts{prefix}) ) { error("Expected -p parameter.\n") }
if ( $$opts{prefix}=~m{/$} ) { `mkdir -p $$opts{prefix}`; }
elsif ( !($$opts{prefix}=~/-$/) ) { $$opts{prefix} .= '-'; }
}
return $opts;
}
# Creates GC stats for either the whole reference or only on target regions for exome QC
sub do_ref_stats
{
my ($opts) = @_;
my %targets = ();
if ( exists($$opts{targets}) )
{
my ($prev_chr,$prev_pos);
open(my $fh,'<',$$opts{targets}) or error("$$opts{targets}: $!");
while (my $line=<$fh>)
{
if ( $line=~/^#/ ) { next; }
my ($chr,$from,$to) = split(/\s+/,$line);
chomp($to);
push @{$targets{$chr}}, $from,$to;
if ( !defined $prev_chr or $chr ne $prev_chr ) { $prev_chr=$chr; $prev_pos=$from }
if ( $prev_pos > $from ) { error("The file must be sorted: $$opts{targets}\n"); }
$prev_pos = $from;
}
close($fh);
}
my $nlen = 0;
my %lens = ();
my %gc_counts = ();
my ($skip_chr,$pos,$ireg,$regions);
open(my $fh,'<',$$opts{do_ref_stats}) or error("$$opts{do_ref_stats}: $!");
while (my $line=<$fh>)
{
if ( $line=~/^>/ )
{
if ( !scalar %targets ) { next; }
if ( !($line=~/>(\S+)/) ) { error("FIXME: could not determine chromosome name: $line"); }
if ( !exists($targets{$1}) ) { $skip_chr=$1; next; }
undef $skip_chr;
$pos = 0;
$ireg = 0;
$regions = $targets{$1};
next;
}
if ( defined $skip_chr ) { next; }
# Only $_len sized lines are considered and no chopping for target regions.
chomp($line);
my $len = length($line);
$lens{$len}++;
$nlen++;
if ( scalar %targets )
{
while ( $ireg<@$regions && $$regions[$ireg+1]<=$pos ) { $ireg += 2; }
$pos += $len;
if ( $ireg==@$regions ) { next; }
if ( $pos < $$regions[$ireg] ) { next; }
}
my $gc_count = 0;
for (my $i=0; $i<$len; $i++)
{
my $base = substr($line,$i,1);
if ( $base eq 'g' || $base eq 'G' || $base eq 'c' || $base eq 'C' ) { $gc_count++; }
}
$gc_counts{$gc_count}++;
}
# Calculate median length
my $n = 0;
my $median_len = 0;
for my $len (sort { $a<=>$b } keys %lens)
{
$n += $lens{$len};
if ( $n >= $nlen ) { $median_len = $len; last; }
}
if ( !$median_len ) { error("FIXME: median_len=$median_len??\n"); }
print "# Generated by $$opts{args}\n";
print "# The columns are: GC content bin, normalized frequency\n";
my $max;
for my $count (values %gc_counts)
{
if ( !defined $max or $count>$max ) { $max=$count; }
}
for my $gc (sort {$a<=>$b} keys %gc_counts)
{
if ( $gc==0 ) { next; }
printf "%f\t%f\n", $gc*100./$median_len, $gc_counts{$gc}/$max;
}
}
sub plot
{
my ($cmdfile) = @_;
my $cmd = "gnuplot $cmdfile";
system($cmd);
if ( $? ) { error("The command exited with non-zero status $?:\n\t$cmd\n\n"); }
}
sub open_file
{
my ($file) = @_;
my $fh;
if ( $file eq '-' ) { $fh = *STDIN; }
elsif ( $file=~/\.gz$/i ) { open($fh, "gunzip -c $file |") or error("gunzip -c $file: $!"); }
else { open($fh,'<',$file) or error("$file: $!"); }
return $fh;
}
sub parse_bamcheck1
{
my ($opts, $i) = @_;
my $file = $$opts{bamcheck}[$i];
print STDERR "Parsing samtools stats output: $file\n" unless !$$opts{verbose};
my $fh = open_file($file);
my $line = <$fh>;
if ( !($line=~/^# This file was produced by (\S+)/) ) { error("Sanity check failed: was this file generated by samtools stats or plot-bamstats?"); }
if ( $1 ne 'plot-bamstats' && $1 ne 'samtools' ) { error("Sanity check failed: was this file generated by samtools stats or plot-bamstats?"); }
my %dat;
my $sn_order = $$opts{sn_order} || [];
my %sn_seen = map { $_ => 1 } @$sn_order;
while ($line=<$fh>)
{
if ( $line=~/^#/ ) { next; }
my @items = split(/\t/,$line);
chomp($items[-1]);
if ( $items[0] eq 'SN' )
{
$dat{SN}{$items[1]} = $items[2];
if (!exists($sn_seen{$items[1]})) {
push(@$sn_order, $items[1]);
$sn_seen{$items[1]} = 1;
}
next;
}
push @{$dat{$items[0]}}, [splice(@items,1)];
}
close($fh) unless $file eq '-';
$$opts{sn_order} = $sn_order;
# Check sanity
if ( !exists($dat{'SN'}{'sequences:'}) or !$dat{'SN'}{'sequences:'} )
{
error("Sanity check failed: no sequences found by samtools stats??\n");
}
my $nseq_new = $dat{'SN'}{'sequences:'};
my $nseq_ori = exists($$opts{dat}{'SN'}) ? $$opts{dat}{'SN'}{'sequences:'} : 0;
my %add = map { $_ => 1 } (qw(FFQ LFQ MPC GCF GCL IS ID IC RL FRL LRL));
for my $a (keys %dat)
{
if ( !exists($$opts{dat}{$a}) ) { $$opts{dat}{$a} = $dat{$a}; next; } # first bamcheck file
if ( $a eq 'SN' )
{
for my $b (keys %{$dat{$a}})
{
if ( exists($$opts{hmerge}{$b}) )
{
if ( $$opts{hmerge}{$b} eq 'sum' ) { $$opts{dat}{$a}{$b} += $dat{$a}{$b}; next; }
if ( $$opts{hmerge}{$b} eq 'min' ) { $$opts{dat}{$a}{$b} = $$opts{dat}{$a}{$b} < $dat{$a}{$b} ? $$opts{dat}{$a}{$b} : $dat{$a}{$b}; }
if ( $$opts{hmerge}{$b} eq 'max' ) { $$opts{dat}{$a}{$b} = $$opts{dat}{$a}{$b} > $dat{$a}{$b} ? $$opts{dat}{$a}{$b} : $dat{$a}{$b}; }
}
}
next;
}
if ( $add{$a} ) { add_to_matrix($$opts{dat}{$a}, $dat{$a}, 0); next; }
if ( $a eq 'COV' ) { merge_coverage($$opts{dat}{$a}, $dat{$a}); next; }
if ( $a eq 'GCC' || $a eq 'FBC' || $a eq 'LBC' ) {
merge_gcc($nseq_ori, $$opts{dat}{$a}, $nseq_new, $dat{$a});
next;
}
warn("Not processed: $a\n");
}
if ( $i==0 ) { return; }
my $bases_mapped_cigar = get_value($opts,'SN','bases mapped (cigar):');
$$opts{dat}{SN}{'error rate:'} = $bases_mapped_cigar ? sprintf "%e", get_value($opts,'SN','mismatches:') / $bases_mapped_cigar : 0;
update_average_length($opts);
update_average_quality($opts);
update_average_isize($opts);
}
sub parse_bamcheck
{
my ($opts) = @_;
for (my $i=0; $i<@{$$opts{bamcheck}}; $i++) { parse_bamcheck1($opts,$i); }
# Determine the default title, for now use the first BAM name:
# 5446_6/5446_6.bam.bc.gp -> 5446_6
# test.aaa.png -> test.aaa
if ( !($$opts{bamcheck}[0]=~m{([^/]+?)(?:\.bam)?(?:\.bc)?$}i) ) { error("FIXME: Could not determine the title from [$$opts{bamcheck}[0]]\n"); }
$$opts{title} = $1;
}
sub add_to_matrix
{
my ($dst,$src,$key) = @_;
my $id = 0;
my $is = 0;
while ($is<@$src)
{
while ( $id<@$dst && $$src[$is][$key] > $$dst[$id][$key] ) { $id++; }
if ( $id<@$dst && $$src[$is][$key] == $$dst[$id][$key] )
{
for (my $j=0; $j<@{$$src[$is]}; $j++)
{
if ( $j==$key ) { next; }
$$dst[$id][$j] += $$src[$is][$j];
}
}
else { splice(@$dst,$id,0,$$src[$is]); }
$is++;
}
}
sub rebin_values
{
my ($vals,$bin_size,$col) = @_;
error("pd3\@sanger todo: rebin_values\n");
}
sub merge_coverage
{
my ($dst,$src) = @_;
if ( !($$dst[0][0]=~/^\[(\d+)-(\d+)\]$/) ) { error("Could not determine bin size in COV: $$dst[0][0]\n"); }
my $dst_bin = $2 - $1 + 1;
if ( !($$src[0][0]=~/^\[(\d+)-(\d+)\]$/) ) { error("Could not determine bin size in COV: $$src[0][0]\n"); }
my $src_bin = $2 - $1 + 1;
my (@dst, @src);
for my $row (@$dst) { splice(@$row,0,1); push @dst,$row; }
for my $row (@$src) { splice(@$row,0,1); push @src,$row; }
my $dst_out = pop(@dst);
my $src_out = pop(@src);
my $bin_size = $dst_bin;
if ( $src_bin > $dst_bin ) { my $vals = rebin_values(\@dst,$src_bin,0); @dst = @$vals; $bin_size = $src_bin; }
elsif ( $src_bin < $dst_bin ) { my $vals = rebin_values(\@src, $dst_bin, 0); @src = @$vals; }
add_to_matrix(\@dst, \@src, 0);
for my $row (@dst) { unshift(@$row, sprintf("[%d-%d]", $$row[0],$$row[0]+$bin_size-1)); }
push @dst, [sprintf("[%d<]", $dst[-1][1]), $dst[-1][1], $$dst_out[1]+$$src_out[1] ];
@$dst = @dst;
}
sub merge_gcc
{
my ($ndst,$dst,$nsrc,$src) = @_;
if ( @$dst != @$src ) { error("todo: improve me\n"); }
for (my $i=0; $i<@$dst; $i++)
{
if ( $$dst[$i][0] ne $$src[$i][0] ) { error("todo: improve me\n"); }
for (my $j=1; $j<@{$$dst[$i]}; $j++)
{
$$dst[$i][$j] = sprintf("%.2f", ($$dst[$i][$j]*$ndst + $$src[$i][$j]*$nsrc) / ($ndst + $nsrc));
}
}
}
sub merge_gcd
{
# todo
}
sub get_value
{
my ($opts, $id, $key) = @_;
if ( !exists($$opts{dat}{$id}) ) { return undef; }
if ( !exists($$opts{dat}{$id}{$key}) ) { return undef}
return $$opts{dat}{$id}{$key};
}
sub get_values
{
my ($opts, $id) = @_;
if ( !exists($$opts{dat}{$id}) ) { return (); }
# todo: add version sanity check here
return (@{$$opts{dat}{$id}});
}
sub update_average_length
{
my ($opts) = @_;
my @vals = get_values($opts,'RL');
my $sum = 0;
my $avg = 0;
for my $val (@vals) { $sum += $$val[1]; }
for my $val (@vals) { $avg += $$val[0]*$$val[1] / $sum; }
$$opts{dat}{SN}{'average length:'} = sprintf "%.1f", $avg;
}
sub update_average_quality
{
my ($opts) = @_;
my @vals;
push @vals, get_values($opts, 'FFQ'), get_values($opts,'LFQ');
my $qsum = 0;
for my $row (@vals)
{
for (my $i=1; $i<@$row; $i++) { $qsum += $$row[$i] }
}
my $qavg = 0;
for my $row (@vals)
{
for (my $i=1; $i<@$row; $i++) { $qavg += ($i-1)*$$row[$i] / $qsum; }
}
$$opts{dat}{SN}{'average quality:'} = sprintf "%.1f", $qavg;
}
sub update_average_isize
{
my ($opts) = @_;
my @vals = get_values($opts, 'IS');
my $sum = 0;
for my $row (@vals) { $sum += $$row[1]; }
my $avg = 0;
for my $row (@vals) { $avg += $$row[0]*$$row[1] / $sum; }
my $dev = 0;
for my $row (@vals) { $dev += ($avg - $$row[0])*($avg - $$row[0]) * $$row[1] / $sum; }
$$opts{dat}{SN}{'insert size average:'} = sprintf "%.1f", $avg;
$$opts{dat}{SN}{'insert size standard deviation:'} = sprintf "%.1f", sqrt($dev);
}
sub get_defaults
{
my ($opts,$img_fname,%args) = @_;
if ( !($img_fname=~/\.png$/i) ) { error("FIXME: currently only PNG supported. (Easy to extend.)\n"); }
# Determine the gnuplot script file name
my $gp_file = $img_fname;
$gp_file =~ s{\.[^.]+$}{.gp};
if ( !($gp_file=~/.gp$/) ) { $gp_file .= '.gp'; }
my $title = $$opts{title};
my $dir = $gp_file;
$dir =~ s{/[^/]+$}{};
if ( $dir && $dir ne $gp_file ) { `mkdir -p $dir`; }
my $wh = exists($args{wh}) ? $args{wh} : '600,400';
open(my $fh,'>',$gp_file) or error("$gp_file: $!");
return {
title => $title,
gp => $gp_file,
img => $img_fname,
fh => $fh,
terminal => qq[set terminal png size $wh truecolor],
grid => 'set grid xtics ytics y2tics back lc rgb "#cccccc"',
};
}
sub percentile
{
my ($p,@vals) = @_;
my $N = 0;
for my $val (@vals) { $N += $val; }
my $n = $p*($N+1)/100.;
my $k = int($n);
my $d = $n-$k;
if ( $k<=0 ) { return 0; }
if ( $k>=$N ) { return scalar @vals-1; }
my $cnt;
for (my $i=0; $i<@vals; $i++)
{
$cnt += $vals[$i];
if ( $cnt>=$k ) { return $i; }
}
error("FIXME: this should not happen [percentile]\n");
}
sub plot_qualities
{
my ($opts) = @_;
my @ffq = get_values($opts, 'FFQ');
if ( !@ffq ) { return; }
my $yrange = @{$ffq[0]} > 50 ? @{$ffq[0]} : 50;
my $is_paired = get_value($opts,'SN','reads paired:');
# Average quality per cycle, forward and reverse reads in one plot
my $args = get_defaults($opts,"$$opts{prefix}quals.png");
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set ylabel "Average Quality"
set xlabel "Cycle"
set yrange [0:$yrange]
set title "$$args{title}" noenhanced
plot '-' using 1:2 with lines title 'Forward reads' ] . ($is_paired ? q[, '-' using 1:2 with lines title 'Reverse reads'] : '') . q[
];
my (@fp75,@fp50,@fmean);
my (@lp75,@lp50,@lmean);
my ($fmax,$fmax_qual,$fmax_cycle);
my ($lmax,$lmax_qual,$lmax_cycle);
for my $cycle (@ffq)
{
my $sum=0; my $n=0;
for (my $iqual=1; $iqual<@$cycle; $iqual++)
{
if ( exists($$opts{alt_qual_avg_method }) ) {
my $prob_base_error = 10 ** (-$iqual/10);
$sum += $$cycle[$iqual]*$prob_base_error;
} else {
$sum += $$cycle[$iqual]*$iqual;
}
$n += $$cycle[$iqual];
if ( !defined $fmax or $fmax<$$cycle[$iqual] ) { $fmax=$$cycle[$iqual]; $fmax_qual=$iqual; $fmax_cycle=$$cycle[0]; }
}
my $p25 = percentile(25,(@$cycle)[1..$#$cycle]);
my $p50 = percentile(50,(@$cycle)[1..$#$cycle]);
my $p75 = percentile(75,(@$cycle)[1..$#$cycle]);
if ( !$n ) { next; }
push @fp75, "$$cycle[0]\t$p25\t$p75\n";
push @fp50, "$$cycle[0]\t$p50\n";
if ( exists($$opts{alt_qual_avg_method }) ) {
my $mean_prob_base_error = $sum/$n;
my $mean_error_quality_score = -10 * ( log($mean_prob_base_error) / log(10) );
push @fmean, sprintf "%d\t%.2f\n", $$cycle[0],$mean_error_quality_score;
} else {
push @fmean, sprintf "%d\t%.2f\n", $$cycle[0],$sum/$n;
}
printf $fh $fmean[-1];
}
print $fh "end\n";
my @lfq = ();
if ( $is_paired )
{
@lfq = get_values($opts, 'LFQ');
for my $cycle (@lfq)
{
my $sum=0; my $n=0;
for (my $iqual=1; $iqual<@$cycle; $iqual++)
{
if ( exists($$opts{alt_qual_avg_method }) ) {
my $prob_base_error = 10 ** (-$iqual/10);
$sum += $$cycle[$iqual]*$prob_base_error;
} else {
$sum += $$cycle[$iqual]*$iqual;
}
$n += $$cycle[$iqual];
if ( !defined $lmax or $lmax<$$cycle[$iqual] ) { $lmax=$$cycle[$iqual]; $lmax_qual=$iqual; $lmax_cycle=$$cycle[0]; }
}
my $p25 = percentile(25,(@$cycle)[1..$#$cycle]);
my $p50 = percentile(50,(@$cycle)[1..$#$cycle]);
my $p75 = percentile(75,(@$cycle)[1..$#$cycle]);
if ( !$n ) { next; }
push @lp75, "$$cycle[0]\t$p25\t$p75\n";
push @lp50, "$$cycle[0]\t$p50\n";
if ( exists($$opts{alt_qual_avg_method }) ) {
my $mean_prob_base_error = $sum/$n;
my $mean_error_quality_score = -10 * ( log($mean_prob_base_error) / log(10) );
push @lmean, sprintf "%d\t%.2f\n", $$cycle[0],$mean_error_quality_score;
} else {
push @lmean, sprintf "%d\t%.2f\n", $$cycle[0],$sum/$n;
}
printf $fh $lmean[-1];
}
print $fh "end\n";
}
close($fh);
plot($$args{gp});
# Average, mean and quality percentiles per cycle, forward and reverse reads in separate plots
$args = get_defaults($opts,"$$opts{prefix}quals2.png",wh=>$is_paired ? '700,500' : '600,400');
$fh = $$args{fh};
my $pos_size = " set rmargin 0; set lmargin 0; set tmargin 0; set bmargin 0; set origin 0.1,0.1; set size 0.4,0.8";
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set multiplot
$pos_size
set yrange [0:$yrange]
set ylabel "Quality"
set xlabel "Cycle (fwd reads)"
plot '-' using 1:2:3 with filledcurve lt 1 lc rgb "#cccccc" t '25-75th percentile' , '-' using 1:2 with lines lc rgb "#000000" t 'Median', '-' using 1:2 with lines lt 1 t 'Mean'
];
print $fh join('',@fp75),"end\n";
print $fh join('',@fp50),"end\n";
print $fh join('',@fmean),"end\n";
if ( $is_paired )
{
print $fh qq[
set origin 0.55,0.1
set size 0.4,0.8
unset ytics
set y2tics mirror
set y2range [0:$yrange]
unset ylabel
set xlabel "Cycle (rev reads)"
set label "$$args{title}" at screen 0.5,0.95 center noenhanced
plot '-' using 1:2:3 with filledcurve lt 1 lc rgb "#cccccc" t '25-75th percentile' , '-' using 1:2 with lines lc rgb "#000000" t 'Median', '-' using 1:2 with lines lt 2 t 'Mean'
];
print $fh join('',@lp75),"end\n";
print $fh join('',@lp50),"end\n";
print $fh join('',@lmean),"end\n";
}
close($fh);
plot($$args{gp});
# Quality distribution per cycle, the distribution is for each cycle plotted as a separate curve
$args = get_defaults($opts,"$$opts{prefix}quals3.png",wh=>$is_paired ? '600,600' : '600,400');
$fh = $$args{fh};
my $nquals = @{$ffq[0]}-1;
my $ncycles = @ffq;
$pos_size = $is_paired ? " set rmargin 0; set lmargin 0; set tmargin 0; set bmargin 0; set origin 0.15,0.52; set size 0.8,0.4" : '';
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set multiplot
$pos_size
set title "$$args{title}" noenhanced
set ylabel "Frequency (fwd reads)"
set label "Cycle $fmax_cycle" at $fmax_qual+1,$fmax
unset xlabel
set xrange [0:$nquals]
set format x ""
plot '-' using 1:2:3 with lines linecolor variable title ''
];
for my $cycle (0..$#ffq)
{
for (my $iqual=1; $iqual<$nquals; $iqual++) {
printf $fh "%d\t%d\t%d\n", $iqual, $ffq[$cycle]->[$iqual], $cycle + 1;
}
print $fh "\n";
}
print $fh "end\n";
if ( $is_paired )
{
print $fh qq[
set origin 0.15,0.1
set size 0.8,0.4
unset title
unset format
set xtics
set xlabel "Quality"
unset label
set label "Cycle $lmax_cycle" at $lmax_qual+1,$lmax
set ylabel "Frequency (rev reads)"
plot '-' using 1:2:3 with lines linecolor variable title ''
];
for my $cycle (0..$#lfq)
{
for (my $iqual=1; $iqual<$nquals; $iqual++)
{
printf $fh "%d\t%d\t%d\n", $iqual, $lfq[$cycle]->[$iqual], $cycle + 1;
}
print $fh "\n";
}
print $fh "end\n";
}
close($fh);
plot($$args{gp});
# Heatmap qualitites
$args = get_defaults($opts,"$$opts{prefix}quals-hm.png", wh=>'600,500');
$fh = $$args{fh};
my $max = defined $lmax && $lmax > $fmax ? $lmax : $fmax;
my $ytics = generate_ticks([ map { $_->[0] } @ffq ], $is_paired);
my $colorbox = "set colorbox vertical user origin first ($nquals+1),0 size screen 0.025,0.812";
$pos_size = $is_paired
? " set origin 0.05,0.5\n set size 0.85,0.5" :
" set origin 0.05,0.05\n set size 0.85,1.0\n$colorbox" ;
my $set_xtics = $is_paired ? "unset" : "set";
print $fh qq[
$$args{terminal}
set output "$$args{img}"
unset key
unset colorbox
# Perceptually uniform heatmap "plasma" by Nathaniel J. Smith & Stefan van der Walt
# used in Python's matplotlib: https://github.com/BIDS/colormap/blob/master/colormaps.py
# Values from: https://github.com/Gnuplotting/gnuplot-palettes/blob/master/plasma.pal
# Dark end squashed to make low values more distinguishable.
set palette model RGB
set palette defined ( \\
0 '#000000' \\
, .7 '#0c0847' \\
, 4 '#4b0381' \\
, 10 '#7d03a8' \\
, 20 '#a82296' \\
, 30 '#cb4679' \\
, 40 '#e56b5d' \\
, 50 '#f89441' \\
, 60 '#fdc328' \\
, 70 '#f0f921' )
set cbrange [0:$max]
set yrange [0:$ncycles]
set xrange [0:$nquals]
set view map
set multiplot
set rmargin 0
set lmargin 0
set tmargin 0
set bmargin 0
$pos_size
set obj 1 rectangle behind from first 0,0 to first $nquals,$ncycles
set obj 1 fillstyle solid 1.0 fillcolor rgbcolor "black"
set ylabel "Cycle (fwd reads)" offset character -1,0
unset ytics
set ytics ($ytics)
$set_xtics xtics
set title "$$args{title}" noenhanced
splot '-' matrix with image
];
for my $cycle (@ffq)
{
for (my $iqual=1; $iqual<@$cycle; $iqual++) { print $fh "\t$$cycle[$iqual]"; }
print $fh "\n";
}
print $fh "\nend\n";
if ( $is_paired )
{
my $ytics = generate_ticks([ map { $_->[0] } @lfq ], $is_paired);
print $fh qq[
set origin 0.05,0.05
set size 0.85,0.5
set ylabel "Cycle (rev reads)" offset character -1,0
set xlabel "Base Quality"
unset title
unset ytics
set ytics ($ytics)
set xrange [0:$nquals]
set xtics
$colorbox
set cblabel "Number of bases"
splot '-' matrix with image
];
for my $cycle (@lfq)
{
for (my $iqual=1; $iqual<@$cycle; $iqual++) { print $fh "\t$$cycle[$iqual]"; }
print $fh "\n";
}
print $fh "\nend\n";
}
close($fh);
plot($$args{gp});
}
sub generate_ticks {
my ($cycles, $skip_even) = @_;
my @marks;
my $div = 10;
while (scalar(@$cycles)/$div > 20) {
$div *= 2;
}
$div = nice_num($div);
foreach my $v (@$cycles) {
if ($v % $div == 0) {
if ($skip_even) {
my $label = $v % 20 ? $v : '';
push @marks, qq{"$label" $v};
}
else {
push @marks, qq{"$v" $v};
}
}
}
return join(', ', @marks);
}
sub plot_acgt_cycles
{
my ($opts) = @_;
my @gcc = get_values($opts, 'GCC');
if ( !@gcc ) { return; }
my $args = get_defaults($opts,"$$opts{prefix}acgt-cycles.png");
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set style line 1 linecolor rgb "green"
set style line 2 linecolor rgb "red"
set style line 3 linecolor rgb "black"
set style line 4 linecolor rgb "blue"
set style increment user
set ylabel "Base content [%]"
set xlabel "Read Cycle"
set yrange [0:100]
set title "$$args{title}" noenhanced
plot '-' w l ti 'A', '-' w l ti 'C', '-' w l ti 'G', '-' w l ti 'T'
];
for my $base (1..4)
{
for my $cycle (@gcc)
{
print $fh $$cycle[0]+1,"\t",$$cycle[$base],"\n";
}
print $fh "end\n";
}
close($fh);
plot($$args{gp});
}
sub plot_gc
{
my ($opts) = @_;
my $is_paired = get_value($opts,'SN','reads paired:');
my $args = get_defaults($opts,"$$opts{prefix}gc-content.png");
my $fh = $$args{fh};
my ($gcl_max,$gcf_max,$lmax,$fmax);
my @gcf = get_values($opts, 'GCF');
my @gcl = get_values($opts, 'GCL');
for my $gc (@gcf) { if ( !defined $gcf_max or $gcf_max<$$gc[1] ) { $gcf_max=$$gc[1]; $fmax=$$gc[0]; } }
for my $gc (@gcl) { if ( !defined $gcl_max or $gcl_max<$$gc[1] ) { $gcl_max=$$gc[1]; $lmax=$$gc[0]; } }
my $gcmax = $is_paired && $gcl_max > $gcf_max ? $lmax : $fmax;
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set title "$$args{title}" noenhanced
set ylabel "Normalized Frequency"
set xlabel "GC Content [%]"
set yrange [0:1.1]
set label sprintf("%.1f",$gcmax) at $gcmax,1 front offset 1,0
plot ]
. (exists($$opts{ref_stats}) ? q['-' smooth csplines with lines lt 0 title 'Reference', ] : '')
. q['-' smooth csplines with lines lc 1 title 'First fragments' ]
. ($is_paired ? q[, '-' smooth csplines with lines lc 2 title 'Last fragments'] : '')
. q[
];
if ( exists($$opts{ref_stats}) )
{
open(my $ref,'<',$$opts{ref_stats}) or error("$$opts{ref_stats}: $!");
while (my $line=<$ref>) { print $fh $line }
close($ref);
print $fh "end\n";
}
for my $cycle (@gcf) { printf $fh "%d\t%f\n", $$cycle[0],$$cycle[1]/$gcf_max; }
print $fh "end\n";
if ( $is_paired )
{
for my $cycle (@gcl) { printf $fh "%d\t%f\n", $$cycle[0],$$cycle[1]/$gcl_max; }
print $fh "end\n";
}
close($fh);
plot($$args{gp});
}
sub plot_gc_depth
{
my ($opts) = @_;
my @gcd = get_values($opts,'GCD');
if ( @gcd <= 1 ) { return; }
# Find unique sequence percentiles for 30,40, and 50% GC content, just to draw x2tics.
my @tics = ( {gc=>30},{gc=>40},{gc=>50} );
for my $gc (@gcd)
{
for my $tic (@tics)
{
my $diff = abs($$gc[0]-$$tic{gc});
if ( !exists($$tic{pr}) or $diff<$$tic{diff} ) { $$tic{pr}=$$gc[1]; $$tic{diff}=$diff; }
}
}
my @x2tics;
for my $tic (@tics) { push @x2tics, qq["$$tic{gc}" $$tic{pr}]; }
my $x2tics = join(',',@x2tics);
my $args = get_defaults($opts,"$$opts{prefix}gc-depth.png", wh=>'600,500');
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set ylabel "Mapped depth"
set xlabel "Percentile of mapped sequence ordered by GC content"
set x2label "GC Content [%]"
set title "$$args{title}" noenhanced
set x2tics ($x2tics)
set xtics nomirror
set xrange [0.1:99.9]
plot '-' using 1:2:3 with filledcurve lt 1 lc rgb "#dedede" t '10-90th percentile' , \\
'-' using 1:2:3 with filledcurve lt 1 lc rgb "#bbdeff" t '25-75th percentile' , \\
'-' using 1:2 with lines lc rgb "#0084ff" t 'Median'
];
for my $gc (@gcd) { print $fh "$$gc[1]\t$$gc[2]\t$$gc[6]\n"; } print $fh "end\n";
for my $gc (@gcd) { print $fh "$$gc[1]\t$$gc[3]\t$$gc[5]\n"; } print $fh "end\n";
for my $gc (@gcd) { print $fh "$$gc[1]\t$$gc[4]\n"; } print $fh "end\n";
close($fh);
plot($$args{gp});
}
sub plot_isize
{
my ($opts) = @_;
my $is_paired = get_value($opts,'SN','reads paired:');
my @isizes = get_values($opts, 'IS');
if ( !$is_paired or !@isizes ) { return; }
my ($isize_max,$isize_cnt);
for my $isize (@isizes)
{
if ( !defined $isize_max or $isize_cnt<$$isize[1] ) { $isize_cnt=$$isize[1]; $isize_max=$$isize[0]; }
}
my $args = get_defaults($opts,"$$opts{prefix}insert-size.png");
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set rmargin 5
set label sprintf("%d",$isize_max) at $isize_max+10,$isize_cnt
set ylabel "Number of pairs"
set xlabel "Insert Size"
set title "$$args{title}" noenhanced];
print $fh qq[
set logscale y 10] if exists $$opts{y_axis_log10};
print $fh qq[
plot \\
'-' with lines lc rgb 'black' title 'All pairs', \\
'-' with lines title 'Inward', \\
'-' with lines title 'Outward', \\
'-' with lines title 'Other'
];
for my $isize (@isizes) { print $fh "$$isize[0]\t$$isize[1]\n"; } print $fh "end\n";
for my $isize (@isizes) { print $fh "$$isize[0]\t$$isize[2]\n"; } print $fh "end\n";
for my $isize (@isizes) { print $fh "$$isize[0]\t$$isize[3]\n"; } print $fh "end\n";
for my $isize (@isizes) { print $fh "$$isize[0]\t$$isize[4]\n"; } print $fh "end\n";
close($fh);
plot($$args{gp});
}
sub plot_coverage
{
my ($opts) = @_;
my @cov = get_values($opts, 'COV');
if ( !@cov ) { return; }
my @vals;
for my $cov (@cov) { push @vals,$$cov[2]; }
my $i = percentile(99.8,@vals);
my $p99 = $cov[$i][1];
my $args = get_defaults($opts,"$$opts{prefix}coverage.png");
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set ylabel "Number of mapped bases"
set xlabel "Coverage"
set log y
set style fill solid border -1
set title "$$args{title}" noenhanced
set xrange [:$p99]
plot '-' with lines notitle
];
for my $cov (@cov)
{
if ( $$cov[2]==0 ) { next; }
print $fh "$$cov[1]\t$$cov[2]\n";
}
print $fh "end\n";
close($fh);
plot($$args{gp});
}
sub plot_mismatches_per_cycle
{
my ($opts) = @_;
my @mpc = get_values($opts, 'MPC');
if ( !@mpc ) { return; }
my $nquals = @{$mpc[0]} - 2;
my $ncycles = @mpc;
my ($style,$with);
if ( $ncycles>100 ) { $style = ''; $with = 'w l'; }
else { $style = 'set style data histogram; set style histogram rowstacked'; $with = ''; }
my $args = get_defaults($opts,"$$opts{prefix}mism-per-cycle.png");
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set style line 1 linecolor rgb "#e40000"
set style line 2 linecolor rgb "#ff9f00"
set style line 3 linecolor rgb "#bbbb00"
set style line 4 linecolor rgb "#4ebd68"
set style line 5 linecolor rgb "#0061ff"
set style increment user
set key left top
$style
set ylabel "Number of mismatches"
set xlabel "Read Cycle"
set style fill solid border -1
set title "$$args{title}" noenhanced
set xrange [-1:$ncycles]
plot '-' $with ti 'Base Quality>30', \\
'-' $with ti '30>=Q>20', \\
'-' $with ti '20>=Q>10', \\
'-' $with ti '10>=Q', \\
'-' $with ti "N's"
];
for my $cycle (@mpc)
{
my $sum; for my $idx (31..$#$cycle) { $sum += $$cycle[$idx]; }
print $fh "$sum\n";
}
print $fh "end\n";
for my $cycle (@mpc)
{
my $sum; for my $idx (22..31) { $sum += $$cycle[$idx]; }
print $fh "$sum\n";
}
print $fh "end\n";
for my $cycle (@mpc)
{
my $sum; for my $idx (12..21) { $sum += $$cycle[$idx]; }
print $fh "$sum\n";
}
print $fh "end\n";
for my $cycle (@mpc)
{
my $sum; for my $idx (2..11) { $sum += $$cycle[$idx]; }
print $fh "$sum\n";
}
print $fh "end\n";
for my $cycle (@mpc) { print $fh "$$cycle[1]\n"; }
print $fh "end\n";
close($fh);
plot($$args{gp});
}
sub plot_indel_dist
{
my ($opts) = @_;
my @indels = get_values($opts, 'ID');
if ( !@indels ) { return; }
my $args = get_defaults($opts,"$$opts{prefix}indel-dist.png");
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set style line 1 linetype 1 linecolor rgb "red"
set style line 2 linetype 2 linecolor rgb "black"
set style line 3 linetype 3 linecolor rgb "green"
set style increment user
set ylabel "Indel count [log]"
set xlabel "Indel length"
set y2label "Insertions/Deletions ratio"
set log y
set y2tics nomirror
set ytics nomirror
set title "$$args{title}" noenhanced
plot '-' w l ti 'Insertions', '-' w l ti 'Deletions', '-' axes x1y2 w l ti "Ins/Dels ratio"
];
for my $len (@indels) { print $fh "$$len[0]\t$$len[1]\n"; } print $fh "end\n";
for my $len (@indels) { print $fh "$$len[0]\t$$len[2]\n"; } print $fh "end\n";
for my $len (@indels) { printf $fh "%d\t%f\n", $$len[0],$$len[2]?$$len[1]/$$len[2]:0; } print $fh "end\n";
close($fh);
plot($$args{gp});
}
sub plot_indel_cycles
{
my ($opts) = @_;
my @indels = get_values($opts, 'IC');
if ( !@indels ) { return; }
my $is_paired = get_value($opts,'SN','reads paired:');
my $args = get_defaults($opts,"$$opts{prefix}indel-cycles.png");
my $fh = $$args{fh};
print $fh qq[
$$args{terminal}
set output "$$args{img}"
$$args{grid}
set style line 1 linetype 1 linecolor rgb "red"
set style line 2 linetype 2 linecolor rgb "black"
set style line 3 linetype 3 linecolor rgb "green"
set style line 4 linetype 4 linecolor rgb "blue"
set style increment user
set ylabel "Indel count"
set xlabel "Read Cycle"
set title "$$args{title}" noenhanced
];
if ( $is_paired )
{
print $fh qq[plot '-' w l ti 'Insertions (fwd)', '' w l ti 'Insertions (rev)', '' w l ti 'Deletions (fwd)', '' w l ti 'Deletions (rev)'\n];
for my $len (@indels) { print $fh "$$len[0]\t$$len[1]\n"; } print $fh "end\n";
for my $len (@indels) { print $fh "$$len[0]\t$$len[2]\n"; } print $fh "end\n";
for my $len (@indels) { print $fh "$$len[0]\t$$len[3]\n"; } print $fh "end\n";
for my $len (@indels) { print $fh "$$len[0]\t$$len[4]\n"; } print $fh "end\n";
}
else
{
print $fh qq[plot '-' w l ti 'Insertions', '' w l ti 'Deletions'\n];
for my $len (@indels) { print $fh "$$len[0]\t$$len[2]\n"; } print $fh "end\n";
for my $len (@indels) { print $fh "$$len[0]\t$$len[4]\n"; } print $fh "end\n";
}
close($fh);
plot($$args{gp});
}
sub nice_num
{
my ($num) = @_;
my $ret;
if ($num < 1) {
$ret = 1;
} else {
my $expon_num = floor(log($num)/log(10));
my $fract_num = ($num / (10.0 ** $expon_num));
my $nf = ceil($fract_num);
$ret = ($nf * (10.0 ** $expon_num));
}
return $ret;
}
sub plot_read_len_hist
{
my ($opts) = @_;
my @rl = get_values($opts, 'RL');
if ( !@rl ) { return; }
# TODO: Add modified version for paired end reads.
# my $is_paired = get_value($opts,'SN','reads paired:');
my $args = get_defaults($opts,"$$opts{prefix}read-length.png");
my $fh = $$args{fh};
# anonymous subroutines for code readibility
my $log_bin = sub {
my $x = $_[0];
return floor((log($x) / log(10)) * 10);
};
my $inv_log_bin = sub {
my $x = $_[0];
return floor(10**($x/10));
};
# Open files to store data: this is more elegant than copying four sets of
# data into a single GNUplot file.
open(my $rd_fh, '>', "$$opts{prefix}read_len_rawdata.txt");
open(my $hd_fh, '>', "$$opts{prefix}read_len_histdata.txt");
# Create gnuplot command file options
my $min_x = $rl[0][0];
my $max_x = $rl[-1][0];
my $min_y = $rl[0][1];
my $max_y = $min_y;
my @logscale_data = (0)x&$log_bin($max_x);
my $log_x = 0;
my $start_i = 1;
for my $line (@rl)
{
my @deref_line = @{$line};
my $curr_x = $deref_line[0];
my $curr_y = $deref_line[1];
if ($min_y > $curr_y) {
$min_y = $curr_y;
}
if ($max_y < $curr_y) {
$max_y = $curr_y;
}
print $rd_fh "$curr_x $curr_y\n";
$log_x = max(&$log_bin($curr_x), 5);
@logscale_data[floor($log_x)] += $curr_y;
$start_i = $curr_x + 1;
}
# Copy binned data into its own file
# This is a special case to deal with the problem that reads of len < 5 do
# not cooperate with the log10(x)*10 transformation and cause small
# fragments to graph badly.
my $start = 1;
my $end = 3;
my $div_binw_val = $logscale_data[5]/($end - $start);
my $start_idx = -1;
for my $i (0..@logscale_data-1) {
if($logscale_data[$i] != 0)
{
$start_idx = $i;
if ($i == 5)
{
print $hd_fh "5 $logscale_data[5] $div_binw_val $start $end\n";
$start_idx += 1;
}
last;
}
}
my $bin_min_x = &$inv_log_bin($start_idx);
my $bin_max_x = &$inv_log_bin($start_idx + 1);
my $log_scale_max = $logscale_data[$start_idx];
my $log_bin_max = $logscale_data[$start_idx] / max(($bin_max_x - $bin_min_x), 1);
for my $i ($start_idx..@logscale_data-1) {
$start = &$inv_log_bin($i);
$end = &$inv_log_bin($i+1);
if ($bin_max_x < $end) {
$bin_max_x = $end;
}
if ($log_scale_max < $logscale_data[$i]) {
$log_scale_max = $logscale_data[$i];
}
$div_binw_val = $logscale_data[$i]/max(($end - $start), 1);
if ($log_bin_max < $div_binw_val) {
$log_bin_max = $div_binw_val;
}
print $hd_fh "$i $logscale_data[$i] $div_binw_val $start $end\n";
}
if ($max_x < $bin_max_x) {
$max_x = $bin_max_x;
}
if ($min_x > $bin_min_x) {
$min_x = $bin_min_x;
}
$max_y = nice_num($max_y);
$log_scale_max = nice_num($log_scale_max);
$log_bin_max = nice_num($log_bin_max);
my $logscale = "";
if (($max_x - $min_x) > 100) {
$logscale = "set logscale x\n";
}
# Gnuplot commands for normalized binned data
# The normalized plot bins reads by read length with bin widths on a
# logarithmic scale (i.e. bins 10-11 bp, 12-14 bp, 15-18 bp, etc.), then
# bin height is normalized by the width of the bins.
# The end goal is to smooth the distribution of reads compared to plotting
# each length without binning. Visualization of unbinned data is included
# in order to make single value spikes more visible, for example in the
# case that spike ins are used as a control.
print $fh qq[
set ytics nomirror
set y2tic nomirror
set terminal png size 800,1000 truecolor
set output "$$args{img}"
$$args{grid}
set grid noy2tics
set multiplot layout 2,1 columnsfirst scale 1,0.9
set yrange[0: $max_y]
set ylabel "Read Count"
$logscale
set xrange[$min_x: $max_x]
set xtics rotate by -45 out scale 1 nomirror
set xlabel "Read Length"
set boxwidth .8 relative
set title "$$args{title}" noenhanced
set title offset -15,-.5
set key at graph 1, 1.15
set y2range[0:$log_bin_max]
plot "$$opts{prefix}read_len_rawdata.txt" using 1:2 lt rgb "orange" with boxes fs solid .7 title 'unbinned', "$$opts{prefix}read_len_histdata.txt" using 4:3 lt rgb "blue" with boxes title 'normalized bins(count/bin width)' axis x1y2
];
print $fh qq[];
# Gnuplot commands for non-normalized binned data
# The un-normalized plot has reads assigned to bins identically to the
# normalized plot, however, the bin height is not divided by bin width. The
# goal of this visualization is to more accurately reflect the distribution
# of reads by volume, and will generally collect around the mean read
# length.
# Inclusion of unbinned data is to give point of visual comparison to
# normalized histogram.
print $fh qq[
set boxwidth 1 relative
set y2range[0:$log_scale_max]
plot "$$opts{prefix}read_len_rawdata.txt" using 1:2 lt rgb "orange" with boxes fs solid .7 title 'unbinned', "$$opts{prefix}read_len_histdata.txt" using 4:2 lt rgb "blue" with boxes title 'unnormalized bins' axis x1y2
];
# Close file buffers and clean up workspace
close($fh);
close($hd_fh);
close($rd_fh);
plot($$args{gp});
unlink("$$opts{prefix}read_len_rawdata.txt");
unlink("$$opts{prefix}read_len_histdata.txt");
}
sub merge_bamcheck
{
my ($opts) = @_;
my $fh = *STDOUT;
if ( !$$opts{merge} )
{
open($fh,'>',"$$opts{prefix}merge.bchk") or error("$$opts{prefix}merge.bchk: $!\n");
}
print $fh "# This file was produced by plot-bamstats and can be plotted using plot-bamstats\n# The command line was $$opts{args}\n";
for my $sec (@{$$opts{sections}})
{
my $sid = $$sec{id};
if ( !exists($$opts{dat}{$sid}) ) { next; }
print $fh $$sec{header};
if ($sid eq 'SN') {
for my $key (@{$$opts{sn_order}}) {
if (exists($$opts{dat}{$sid}{$key})) {
print "$sid\t$key\t$$opts{dat}{$sid}{$key}\n";
}
}
next;
}
elsif ( ref($$opts{dat}{$sid}) eq 'HASH' )
{
for my $key (keys %{$$opts{dat}{$sid}})
{
print "$sid\t$key\t$$opts{dat}{$sid}{$key}\n";
}
next;
}
elsif ( ref($$opts{dat}{$sid}) eq 'ARRAY' )
{
for my $rec (@{$$opts{dat}{$sid}})
{
print $fh "$sid\t", join("\t",@$rec), "\n";
}
}
#
# if ( $sid eq 'ID' )
# {
# print $fh "# $$opts{id2sec}{SN}{header}\n$$opts{id2sec}{SN}{exp}";
# # output summary numbers here
# for my $id (keys %{$$opts{dat}})
# {
# if ( exists($$opts{exp}{$id}) ) { next; }
# for my $key (keys %{$$opts{dat}{$id}})
# {
# print $fh "SN\t$id\t$key\t$$opts{dat}{$id}{$key}\n";
# }
# }
# }
}
}
sub bignum
{
my ($num) = @_;
if ( !defined $num ) { return 0; }
my $out = '';
my $slen = length($num);
for (my $i=0; $i<$slen; $i++)
{
$out .= substr($num,$i,1);
if ( $i+1<$slen && ($slen-$i-1)%3==0 ) { $out .= ','; }
}
return $out;
}
sub create_html
{
my ($opts) = @_;
my ($fname,$prefix);
if ( $$opts{prefix}=~m{/$} )
{
$fname = "$$opts{prefix}index.html";
$prefix = '';
}
else
{
$prefix = $$opts{prefix};
$prefix =~ s{^.*/}{};
$fname = $$opts{prefix};
$fname =~ s/-$/.html/;
}
open(my $fh,'>',$fname) or error("$fname: $!");
print $fh q[
<html>
<head>
<style>
.thumbnail
{
text-decoration:none;
color:black;
font-weight:bold;
}
.thumbnail span /*CSS for enlarged image*/
{
visibility: hidden;
position: absolute;
padding: 5px;
border: 1px solid #000;
background-color: #e5e5e5;
}
.thumbnail:hover span /*CSS for enlarged image on hover*/
{
visibility: visible;
left: 550px;
top: 10px;
}
.imgs td
{
vertical-align:middle;
padding: 0.5em;
border: 1px solid black;
}
table.imgs
{
border-collapse:collapse;
margin-left:20px;
}
.nums th
{
text-align: left;
}
table.nums
{
margin-top: 1em;
margin-left:20px;
border: 1px dotted #83A4C3;
background-color: #F5F5F5;
padding: 0.5em;
}
.pad { padding-left:1em; vertical-align:top; }
.right { text-align:right; padding-left:1em; }
</style>
</head>
<body>
<table class="imgs">
];
my %imgs =
(
'insert-size' => 'Insert size',
'gc-content' => 'GC content',
'acgt-cycles' => 'Per-base sequence content',
'mism-per-cycle' => 'Mismatches per cycle',
'quals' => 'Quality per cycle',
'quals2' => 'Quality per cycle',
'quals3' => 'Quality per cycle',
'quals-hm' => 'Quality per cycle',
'indel-cycles' => 'Indels per cycle',
'indel-dist' => 'Indel lengths',
'gc-depth' => 'Mapped depth vs GC',
'read-length' => 'Read lengths',
# I think this may be broken: 'coverage' => '',
);
my @imgs = (qw(insert-size gc-content acgt-cycles mism-per-cycle quals quals2 quals3 quals-hm indel-cycles indel-dist gc-depth read-length));
for (my $i=0; $i<@imgs; $i++)
{
if ( $i % 3 == 0 )
{
# new row
if ( $i>0 ) { print $fh "</tr>\n"; }
print $fh "<tr>\n";
}
if ( -e "$$opts{prefix}$imgs[$i].png" )
{
my $desc = $imgs{$imgs[$i]};
my $link = uri_escape("$prefix$imgs[$i].png");
print $fh qq[ <td><a class="thumbnail" href="$link"><img src="$link" width="150px"><span>$desc<br><img src="$link"></span></a>\n];
}
else { print $fh " <td />\n"; }
}
print $fh "</tr></table>\n";
my $reads_total = get_value($opts,'SN','raw total sequences:');
my $reads_filtered = get_value($opts,'SN','filtered sequences:');
my $percent_filtered = sprintf "(%.1f%%)", $reads_filtered * 100. / $reads_total;
my $reads_mapped = get_value($opts,'SN','reads mapped:');
my $percent_mapped = sprintf "(%.1f%%)", $reads_mapped * 100. / ($reads_total - $reads_filtered);
my $reads_dup = get_value($opts,'SN','reads duplicated:');
my $percent_dup = sprintf "(%.1f%%)", $reads_dup * 100. / ($reads_total - $reads_filtered);
my $reads_mq0 = get_value($opts,'SN','reads MQ0:');
my $percent_mq0 = sprintf "(%.1f%%)", ($reads_mapped ? $reads_mq0 * 100. / $reads_mapped : 0);
my $reads_nonprim = get_value($opts,'SN','non-primary alignments:');
my $read_avglen = get_value($opts,'SN','average length:');
my $bases_total = get_value($opts,'SN','total length:');
my $bases_mapped = get_value($opts,'SN','bases mapped (cigar):');
my $bpercent_mapped = sprintf "(%.1f%%)", $bases_mapped * 100. / $bases_total;
my $error_rate = sprintf "%.2f%%", 100.*get_value($opts,'SN','error rate:');
$reads_total = bignum($reads_total);
$reads_filtered = bignum($reads_filtered);
$reads_mapped = bignum($reads_mapped);
$reads_dup = bignum($reads_dup);
$reads_mq0 = bignum($reads_mq0);
$reads_nonprim = bignum($reads_nonprim);
$bases_total = bignum($bases_total);
$bases_mapped = bignum($bases_mapped);
print $fh qq[
<table class="nums">
<tr><th>Reads</tr>
<tr>
<td class="pad"><table>
<tr><td>total: <td class="right"> $reads_total <td class="right"></tr>
<tr><td>filtered: <td class="right"> $reads_filtered <td class="right"> $percent_filtered</tr>
<tr><td>non-primary: <td class="right"> $reads_nonprim <td class="right"> </tr>
<tr><td>duplicated: <td class="right"> $reads_dup <td class="right"> $percent_dup</tr>
<tr><td>mapped: <td class="right"> $reads_mapped <td class="right"> $percent_mapped</tr>
<tr><td>zero MQ: <td class="right"> $reads_mq0 <td class="right"> $percent_mq0</tr>
<tr><td>avg read length: <td class="right"> $read_avglen <td class="right"></tr>
</table></tr>
<tr><th>Bases</tr>
<tr>
<td class="pad"><table>
<tr><td>total: <td class="right"> $bases_total <td class="right"></tr>
<tr><td>mapped: <td class="right"> $bases_mapped <td class="right"> $bpercent_mapped</tr>
<tr><td>error rate: <td class="right"> $error_rate <td class="right"></tr>
</table></tr>
</table>
</body>
</html>
];
close($fh);
}
|