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author lsong10
date Fri, 26 Mar 2021 16:52:45 +0000
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#!/usr/bin/env perl
#
# Author: petr.danecek@sanger
#

use strict;
use warnings;
use Carp;

my $opts = parse_params();
parse_bamcheck($opts);
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);

exit;

#--------------------------------

sub error
{
    my (@msg) = @_;
    if ( scalar @msg ) { confess @msg; }
    die
        "Usage: plot-bamcheck [OPTIONS] file.bam.bc\n",
        "       plot-bamcheck -p outdir/ file.bam.bc\n",
        "Options:\n",
        "   -k, --keep-files                    Do not remove temporary files.\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",
        "   -h, -?, --help                      This help message.\n",
        "\n";
}


sub parse_params
{
    $0 =~ s{^.+/}{};
    my $opts = { args=>join(' ',$0,@ARGV) };
    while (defined(my $arg=shift(@ARGV)))
    {
        if ( $arg eq '-k' || $arg eq '--keep-files' ) { $$opts{keep_files}=1; next; }
        if ( $arg eq '-r' || $arg eq '--ref-stats' ) { $$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' ) { $$opts{prefix}=shift(@ARGV); next; }
        if ( $arg eq '-?' || $arg eq '-h' || $arg eq '--help' ) { error(); }
        if ( -e $arg ) { $$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 bamcheck file?\n") }
    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 $_len = 60;  # for now do only standard fasta's with 60 bases per line
    my %gc_counts = ();
    my ($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};
        }
        if ( defined $skip_chr ) { next; }

        # Only $_len sized lines are considered and no chopping for target regions.
        chomp($line);
        my $len = length($line);
        if ( $len ne $_len ) { next; }

        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}++;
    }

    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", $gc*100./$_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 parse_bamcheck
{
    my ($opts) = @_;
    open(my $fh,'<',$$opts{bamcheck}) or error("$$opts{bamcheck}: $!");
    my $line = <$fh>;
    if ( !($line=~/^# This file was produced by bamcheck (\S+)/) ) { error("Sanity check failed: was this file generated by bamcheck?"); }
    $$opts{dat}{version} = $1;
    while ($line=<$fh>)
    {
        if ( $line=~/^#/ ) { next; }
        my @items = split(/\t/,$line);
        chomp($items[-1]);
        if ( $items[0] eq 'SN' )
        {
            $$opts{dat}{$items[1]} = splice(@items,2);
            next;
        }
        push @{$$opts{dat}{$items[0]}}, [splice(@items,1)];
    }
    close($fh);

    # Check sanity
    if ( !exists($$opts{dat}{'sequences:'}) or !$$opts{dat}{'sequences:'} )
    {
        error("Sanity check failed: no sequences found by bamcheck??\n");
    }
}

sub older_than
{
    my ($opts,$version) = @_;
    my ($year,$month,$day) = split(/-/,$version);
    $version = $$opts{dat}{version};
    if ( !($version=~/\((\d+)-(\d+)-(\d+)\)$/) ) { return 1; }
    if ( $1<$year ) { return 1; }
    elsif ( $1>$year ) { return 0; }
    if ( $2<$month ) { return 1; }
    elsif ( $2>$month ) { return 0; }
    if ( $3<$day ) { return 1; }
    return 0;
}

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'; }

    # Determine the default title:
    #       5446_6/5446_6.bam.bc.gp -> 5446_6
    #       test.aaa.png -> test.aaa
    if ( !($$opts{bamcheck}=~m{([^/]+?)(?:\.bam)?(?:\.bc)?$}i) ) { error("FIXME: Could not determine the title from [$img_fname]\n"); }
    my $title = $1;

    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) = @_;

    if ( !exists($$opts{dat}{FFQ}) or !@{$$opts{dat}{FFQ}} ) { return; }

    my $yrange = @{$$opts{dat}{FFQ}[0]} > 50 ? @{$$opts{dat}{FFQ}[0]} : 50;
    my $is_paired = $$opts{dat}{'is 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}"
            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 (@{$$opts{dat}{FFQ}})
    {
        my $sum=0; my $n=0; 
        for (my $iqual=1; $iqual<@$cycle; $iqual++) 
        { 
            $sum += $$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";
        push @fmean, sprintf "%d\t%.2f\n", $$cycle[0],$sum/$n;
        printf $fh $fmean[-1];
    }
    print $fh "end\n";
    if ( $is_paired )
    {
        for my $cycle (@{$$opts{dat}{LFQ}})
        {
            my $sum=0; my $n=0; 
            for (my $iqual=1; $iqual<@$cycle; $iqual++) 
            { 
                $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, "$$cycle[0]\t$p50\n";
            push @lmean, sprintf "%d\t%.2f\n", $$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,"$$opts{prefix}quals2.png",wh=>'700,500');
    $fh = $$args{fh};
    print $fh qq[
            $$args{terminal}
            set output "$$args{img}"
            $$args{grid}
            set multiplot
            set rmargin 0 
            set lmargin 0 
            set tmargin 0 
            set bmargin 0 
            set origin 0.1,0.1
            set size 0.4,0.8
            set yrange [0:$yrange]
            set ylabel "Quality"
            set xlabel "Cycle (fwd reads)"
            plot '-' using 1:2:3 with filledcurve lt 1 lc rgb "#cccccc" t '25-75th percentile' , '-' using 1:2 with lines lc rgb "#000000" t 'Median', '-' using 1:2 with lines lt 1 t 'Mean'
        ];
    print $fh join('',@fp75),"end\n";
    print $fh join('',@fp50),"end\n";
    print $fh join('',@fmean),"end\n";
    if ( $is_paired )
    {
        print $fh qq[
                set origin 0.55,0.1
                set size 0.4,0.8
                unset ytics
                set y2tics mirror
				set yrange [0:$yrange]
                unset ylabel
                set xlabel "Cycle (rev reads)"
                set label "$$args{title}" at screen 0.5,0.95 center
                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,"$$opts{prefix}quals3.png",wh=>'600,600');
    $fh = $$args{fh};
    my $nquals = @{$$opts{dat}{FFQ}[0]}-1;
    my $ncycles = @{$$opts{dat}{FFQ}};
    print $fh qq[
            $$args{terminal}
            set output "$$args{img}"
            $$args{grid}
            set multiplot
            set rmargin 0
            set lmargin 0
            set tmargin 0
            set bmargin 0
            set origin 0.15,0.52
            set size 0.8,0.4
            set title "$$args{title}"
            set ylabel "Frequency (fwd reads)"
            set label "Cycle $fmax_cycle" at $fmax_qual+1,$fmax
            unset xlabel
            set xrange [0:$nquals]
            set format x ""
        ];
    my @plots;
    for (my $i=0; $i<$ncycles; $i++) { push @plots, q['-' using 1:2 with lines t ''] }
    print $fh "plot ", join(",", @plots), "\n";
    for my $cycle (@{$$opts{dat}{FFQ}})
    {
        for (my $iqual=1; $iqual<$nquals; $iqual++) { print $fh "$iqual\t$$cycle[$iqual]\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)" 
            ];
        print $fh "plot ", join(",", @plots), "\n";
        for my $cycle (@{$$opts{dat}{LFQ}})
        {
            for (my $iqual=1; $iqual<$nquals; $iqual++) 
            { 
                print $fh "$iqual\t$$cycle[$iqual]\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;
    for my $cycle (@{$$opts{dat}{FFQ}}) { if ( $$cycle[0]%10==0 ) { push @ytics,qq["$$cycle[0]" $$cycle[0]]; } }
    my $ytics = join(',', @ytics);
    print $fh qq[
            $$args{terminal}
            set output "$$args{img}"
            unset key
            unset colorbox
            set palette defined (0 0 0 0, 1 0 0 1, 3 0 1 0, 4 1 0 0, 6 1 1 1)
            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 
            set origin 0,0.46
            set size 0.95,0.6
            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)
            unset xtics
            set title "$$args{title}"
            splot '-' matrix with image
        ];
    for my $cycle (@{$$opts{dat}{FFQ}})
    {
        for (my $iqual=1; $iqual<@$cycle; $iqual++) { print $fh "\t$$cycle[$iqual]"; }
        print $fh "\n";
    }
    print $fh "end\nend\n";
    @ytics = ();
    for my $cycle (@{$$opts{dat}{LFQ}}) { if ( $$cycle[0]%10==0 ) { push @ytics,qq["$$cycle[0]" $$cycle[0]]; } }
    $ytics = join(',', @ytics);
    print $fh qq[
            set origin 0,0.03
            set size 0.95,0.6
            set ylabel "Cycle (rev reads)" offset character -1,0
            set xlabel "Base Quality"
            unset title
            unset ytics
            set ytics ($ytics)
            set xrange  [0:$nquals]
            set xtics
            set colorbox vertical user origin first ($nquals+1),0 size screen 0.025,0.812
            set cblabel "Number of bases"
            splot '-' matrix with image
        ];
    for my $cycle (@{$$opts{dat}{LFQ}})
    {
        for (my $iqual=1; $iqual<@$cycle; $iqual++) { print $fh "\t$$cycle[$iqual]"; }
        print $fh "\n";
    }
    print $fh "end\nend\n";
    close($fh);
    plot($$args{gp});
}


sub plot_acgt_cycles
{
    my ($opts) = @_;

    if ( !exists($$opts{dat}{GCC}) or !@{$$opts{dat}{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}"
            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 (@{$$opts{dat}{GCC}})
        {
            print $fh $$cycle[0]+1,"\t",$$cycle[$base],"\n";
        }
        print $fh "end\n";
    }
    close($fh);
    plot($$args{gp});
}


sub plot_gc
{
    my ($opts) = @_;

    my $is_paired = $$opts{dat}{'is paired:'};
    my $args = get_defaults($opts,"$$opts{prefix}gc-content.png");
    my $fh = $$args{fh};
    my ($gcl_max,$gcf_max,$lmax,$fmax);
    for my $gc (@{$$opts{dat}{GCF}}) { if ( !defined $gcf_max or $gcf_max<$$gc[1] ) { $gcf_max=$$gc[1]; $fmax=$$gc[0]; } }
    for my $gc (@{$$opts{dat}{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 "$$args{title}"
            set ylabel "Normalized Frequency"
            set xlabel "GC Content [%]"
            set yrange [0:1.1]
            set label sprintf("%.1f",$gcmax) at $gcmax,1 front offset 1,0
            plot ] 
                 . (exists($$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("$$opts{ref_stats}: $!");
        while (my $line=<$ref>) { print $fh $line }
        close($ref);
        print $fh "end\n";
    }
    for my $cycle (@{$$opts{dat}{GCF}}) { printf $fh "%d\t%f\n", $$cycle[0],$$cycle[1]/$gcf_max; }
    print $fh "end\n";
    if ( $is_paired )
    {
        for my $cycle (@{$$opts{dat}{GCL}}) { printf $fh "%d\t%f\n", $$cycle[0],$$cycle[1]/$gcl_max; }
        print $fh "end\n";
    }
    close($fh);
    plot($$args{gp});
}


sub plot_gc_depth
{
    my ($opts) = @_;

    if ( !exists($$opts{dat}{GCD}) or !@{$$opts{dat}{GCD}} ) { 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 (@{$$opts{dat}{GCD}})
    {
        for my $tic (@tics)
        {
            my $diff = abs($$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}"
            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 (@{$$opts{dat}{GCD}}) { print $fh "$$gc[1]\t$$gc[2]\t$$gc[6]\n"; } print $fh "end\n";
    for my $gc (@{$$opts{dat}{GCD}}) { print $fh "$$gc[1]\t$$gc[3]\t$$gc[5]\n"; } print $fh "end\n";
    for my $gc (@{$$opts{dat}{GCD}}) { print $fh "$$gc[1]\t$$gc[4]\n"; } print $fh "end\n";
    close($fh);
    plot($$args{gp});
}


sub plot_isize
{
    my ($opts) = @_;

    if ( !$$opts{dat}{'is paired:'} or !exists($$opts{dat}{IS}) or !@{$$opts{dat}{IS}} ) { return; }

    my ($isize_max,$isize_cnt);
    for my $isize (@{$$opts{dat}{IS}})
    {
        if ( !defined $isize_max or $isize_cnt<$$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}"
            plot \\
                '-' with lines lc rgb 'black' title 'All pairs', \\
                '-' with lines title 'Inward', \\
                '-' with lines title 'Outward', \\
                '-' with lines title 'Other'
        ];
    for my $isize (@{$$opts{dat}{IS}}) { print $fh "$$isize[0]\t$$isize[1]\n"; } print $fh "end\n";
    for my $isize (@{$$opts{dat}{IS}}) { print $fh "$$isize[0]\t$$isize[2]\n"; } print $fh "end\n";
    for my $isize (@{$$opts{dat}{IS}}) { print $fh "$$isize[0]\t$$isize[3]\n"; } print $fh "end\n";
    for my $isize (@{$$opts{dat}{IS}}) { print $fh "$$isize[0]\t$$isize[4]\n"; } print $fh "end\n";
    close($fh);
    plot($$args{gp});
}


sub plot_coverage
{
    my ($opts) = @_;

    if ( !exists($$opts{dat}{COV}) or !@{$$opts{dat}{COV}} ) { return; }

    my @vals;
    for my $cov (@{$$opts{dat}{COV}}) { push @vals,$$cov[2]; }
    my $i = percentile(99.8,@vals);
    my $p99 = $$opts{dat}{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}"
            $$args{grid}
            set ylabel "Number of mapped bases" 
            set xlabel "Coverage"
            set style fill solid border -1
            set title "$$args{title}"
            set xrange [:$p99]
            plot '-' with lines notitle
        ];
    for my $cov (@{$$opts{dat}{COV}}) 
    { 
        if ( $$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) = @_;

    if ( !exists($$opts{dat}{MPC}) or !@{$$opts{dat}{MPC}} ) { return; }
    if ( older_than($opts,'2012-02-06') ) { plot_mismatches_per_cycle_old($opts); }

    my $nquals = @{$$opts{dat}{MPC}[0]} - 2;
    my $ncycles = @{$$opts{dat}{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}"
            $$args{grid}
            set style line 1 linecolor rgb "#e40000"
            set style line 2 linecolor rgb "#ff9f00"
            set style line 3 linecolor rgb "#eeee00"
            set style line 4 linecolor rgb "#4ebd68"
            set style line 5 linecolor rgb "#0061ff"
            set style increment user
            set key left top
            $style
            set ylabel "Number of mismatches" 
            set xlabel "Read Cycle"
            set style fill solid border -1
            set title "$$args{title}"
            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 (@{$$opts{dat}{MPC}}) 
    { 
        my $sum; for my $idx (31..$#$cycle) { $sum += $$cycle[$idx]; } 
        print $fh "$sum\n"; 
    }
    print $fh "end\n";
    for my $cycle (@{$$opts{dat}{MPC}}) 
    { 
        my $sum; for my $idx (22..31) { $sum += $$cycle[$idx]; } 
        print $fh "$sum\n"; 
    }
    print $fh "end\n";
    for my $cycle (@{$$opts{dat}{MPC}}) 
    { 
        my $sum; for my $idx (12..21) { $sum += $$cycle[$idx]; } 
        print $fh "$sum\n"; 
    }
    print $fh "end\n";
    for my $cycle (@{$$opts{dat}{MPC}}) 
    { 
        my $sum; for my $idx (2..11) { $sum += $$cycle[$idx]; } 
        print $fh "$sum\n"; 
    }
    print $fh "end\n";
    for my $cycle (@{$$opts{dat}{MPC}}) { print $fh "$$cycle[1]\n"; }
    print $fh "end\n";
    close($fh);
    plot($$args{gp});
}

sub plot_indel_dist
{
    my ($opts) = @_;

    if ( !exists($$opts{dat}{ID}) or !@{$$opts{dat}{ID}} ) { 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}"
        plot '-' w l ti 'Insertions', '-' w l ti 'Deletions', '-' axes x1y2 w l ti "Ins/Dels ratio"
    ];
    for my $len (@{$$opts{dat}{ID}}) { print $fh "$$len[0]\t$$len[1]\n"; } print $fh "end\n";
    for my $len (@{$$opts{dat}{ID}}) { print $fh "$$len[0]\t$$len[2]\n"; } print $fh "end\n";
    for my $len (@{$$opts{dat}{ID}}) { printf $fh "%d\t%f\n", $$len[0],$$len[2]?$$len[1]/$$len[2]:0; } print $fh "end\n";
    close($fh);
    plot($$args{gp});
}

sub plot_indel_cycles
{
    my ($opts) = @_;

    if ( !exists($$opts{dat}{IC}) or !@{$$opts{dat}{IC}} ) { return; }

    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}"
        plot '-' w l ti 'Insertions (fwd)', '' w l ti 'Insertions (rev)', '' w l ti 'Deletions (fwd)', '' w l ti 'Deletions (rev)'
    ];
    for my $len (@{$$opts{dat}{IC}}) { print $fh "$$len[0]\t$$len[1]\n"; } print $fh "end\n";
    for my $len (@{$$opts{dat}{IC}}) { print $fh "$$len[0]\t$$len[2]\n"; } print $fh "end\n";
    for my $len (@{$$opts{dat}{IC}}) { print $fh "$$len[0]\t$$len[3]\n"; } print $fh "end\n";
    for my $len (@{$$opts{dat}{IC}}) { print $fh "$$len[0]\t$$len[4]\n"; } print $fh "end\n";
    close($fh);
    plot($$args{gp});
}







sub has_values
{
    my ($opts,@tags) = @_;
    for my $tag (@tags)
    {
        my (@lines) = `cat $$opts{bamcheck} | grep ^$tag | wc -l`;
        chomp($lines[0]);
        if ( $lines[0]<2 ) { return 0; }
    }
    return 1;
}

sub plot_mismatches_per_cycle_old
{
    my ($opts) = @_;

    my $args = get_defaults($opts,"$$opts{prefix}mism-per-cycle.png");
    my ($nquals)  = `grep ^MPC $$opts{bamcheck} | awk '\$2==1' | sed 's,\\t,\\n,g' | wc -l`;
    my ($ncycles) = `grep ^MPC $$opts{bamcheck} | wc -l`;
    chomp($nquals);
    chomp($ncycles);
    $nquals--;
    $ncycles--;
    my @gr0_15  = (2..17);
    my @gr16_30 = (18..32);
    my @gr31_n  = (33..$nquals);
    my $gr0_15  = '$'. join('+$',@gr0_15); 
    my $gr16_30 = '$'. join('+$',@gr16_30); 
    my $gr31_n  = '$'. join('+$',@gr31_n); 

    open(my $fh,'>',$$args{gp}) or error("$$args{gp}: $!");
    print $fh q[
        set terminal png size 600,400 truecolor font "DejaVuSansMono,9"
        set output "] . $$args{img} . q["
        
        set key left top
        set style data histogram
        set style histogram rowstacked

        set grid back lc rgb "#aaaaaa"
        set ylabel "Number of mismatches" 
        set xlabel "Read Cycle"
        set style fill solid border -1
        set title "] . $$args{title} . qq["
        set xrange [-1:$ncycles]
        
        plot '< grep ^MPC $$opts{bamcheck} | cut -f 2-' using ($gr31_n) ti 'Base Quality>30', '' using ($gr16_30) ti '30>=Q>15', '' using ($gr0_15) ti '15>=Q'
    ];
    close($fh);

    plot($$args{gp});
}