#!/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 The output files prefix, add a slash to create new directory.\n", " -r, --ref-stats Optional reference stats file with expected GC content (created with -s).\n", " -s, --do-ref-stats Calculate reference sequence GC for later use with -r\n", " -t, --targets 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 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,$$cov[2]; } my $p99 = percentile(99.8,@vals); my $args = get_defaults($opts,"$$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}); }