changeset 26:859999cb135b draft

revised routines. Better handling of collapsing, isoforms and mutiple mapppings. Barplots revised to visualise average base coverage in exon instead of total number of reads in exon

--- a/CoverageReport.pl	Thu Feb 12 09:54:03 2015 -0500
+++ b/CoverageReport.pl	Wed Nov 29 08:12:27 2017 -0500
@@ -26,18 +26,20 @@
 # m : (m)inimal Coverage threshold
 # f : fraction of average as threshold
 # n : sample (n)ame.
- 
+# T : collapse overlapping Target regions. 
 
 getopts('b:t:o:z:rsSALm:n:f:T', \%opts) ;
 
 # make output directory in (tmp) working dir
 our $wd = "/tmp/Coverage.".int(rand(1000));
+
 while (-d $wd) {
 	$wd = "/tmp/Coverage.".int(rand(1000));
 
 }
 system("mkdir $wd");
-
+$wd = "/tmp/Coverage.993";
+print "wd : $wd\n";
 ## variables
 our %commandsrun = ();
 
@@ -89,38 +91,103 @@
 	my $tmptargets = "$wd/collapsedtargets.bed";
 	system("sort -k1,1 -k2,2n $targets > $wd/sorted.targets.bed");
 	system("bedtools merge -s -scores max -nms -i $wd/sorted.targets.bed > $tmptargets");
-	$opts{'t'} = $tmptargets;
+	open IN, $tmptargets;
+	open OUT, ">$wd/collapsed.targets.renamed.bed";
+	# we assume that overlapping fragments come from isoforms, not from different genes.
+	my %counters = ();
+	my @genes = ();
+	while (<IN>) {
+		chomp;
+		my @p = split(/\t/,$_);
+		my @g = split(/,/,$p[3]);
+		$g[0] =~ m/(\S+)\(.*/;
+		my $gene = $1;
+		if (!defined($counters{$gene})) {
+			push(@genes,$gene);
+			$counters{$gene}{'lines'} = ();
+			$counters{$gene}{'orient'} = $p[5];
+		}
+		$p[3] = $gene."(COLLAPSED)";
+		push(@{$counters{$gene}{'lines'}},\@p);
+	}
+	close IN;
+	foreach my $gene (@genes) {
+		if ($counters{$gene}{'orient'} eq '-') {
+			my $idx = scalar(@{$counters{$gene}{'lines'}}) + 1;
+			foreach my $line (@{$counters{$gene}{'lines'}}) {
+				$idx--;
+				$line->[3] .= "|Region_$idx";
+				print OUT join("\t",@$line)."\n";
+			}
+		}
+		else {
+			my $idx = 0;
+                        foreach my $line (@{$counters{$gene}{'lines'}}) {
+                                $idx++;
+                                $line->[3] .= "|Region_$idx";
+                                print OUT join("\t",@$line)."\n";
+                        }
+		}
+	}
+	close OUT;
+	$opts{'t'} = "$wd/collapsed.targets.renamed.bed";
 }
-
-# 1. Global Summary => default
-&GlobalSummary($opts{'b'}, $opts{'t'});
+# 1. Coverage per exon
+# included in 2.
 
 # 2. Coverage per position
 &SubRegionCoverage($opts{'b'}, $opts{'t'});
 our %filehash;
+our $tcov;
 if (exists($opts{'s'}) || exists($opts{'S'}) || exists($opts{'A'}) || exists($opts{'L'})) {
-	system("mkdir $wd/SplitFiles");
+	system("mkdir -p $wd/SplitFiles");
+	system("rm $wd/SplitFiles/*");
 	## get position coverages
 	## split input files
 	open IN, "$wd/Targets.Position.Coverage";
+	open BCOVSUM, ">$wd/Results/".$opts{'n'}.".Average_Region_Coverage.txt";
 	my $fileidx = 0;
 	my $currreg = '';
+	my $elength = 0;
+	my $esum = 0;
+	my $eline = "";
+	my %out = ();
 	while (<IN>) {
 		my $line = $_;
 		chomp($line);
 		my @p = split(/\t/,$line);
-		my $reg = $p[0].'-'.$p[1].'-'.$p[2]; #.$p[3];
+		my $reg = $p[0].'-'.$p[1].'-'.$p[2]. ": $p[3]";
 		my $ex = $p[3];
+		my $epos = $p[1];
+		# average exon coverage calculation.
+		if (!defined($out{$ex})) {
+			$out{$ex} = ();
+		}
+		if (!defined($out{$ex}{$p[0]})) {
+			$out{$ex}{$p[0]} = ();
+		}
+		# needs to be transcript specific ($ex) and position specific ($epos) to handle both isoforms and PAR/multiple mapping situations.
+		if (!defined($out{$ex}{$p[0]}{$epos})) {
+			$out{$ex}{$p[0]}{$epos}{'r'} = "$p[0]\t$p[1]\t$p[2]\t$p[3]\t$p[4]\t$p[5]";
+			$out{$ex}{$p[0]}{$epos}{'c'} = ();
+
+		}
+		push(@{$out{$ex}{$p[0]}{$epos}{'c'}},$p[-1]);
+		# splitting files 
 		if ($reg ne $currreg) {
 			## new exon open new outfile
 			if ($currreg ne '') {
+				print BCOVSUM "$eline\t".($esum/$elength)."\n";
 				## filehandle is open. close it
 				close OUT; 
 			}
+			$eline = "$p[0]\t$p[1]\t$p[2]\t$p[3]\t$p[4]\t$p[5]"; #\t$p[6]\t$p[7]\t$p[8]";
+			$esum = 0;
+			$elength = 0;
 			if (!exists($filehash{$reg})) {
 				$fileidx++;
 				$filehash{$reg}{'idx'} = $fileidx;
-				$filehash{$reg}{'exon'} = $ex;
+				$filehash{$reg}{'exon'} = $reg;
 				open OUT, ">> $wd/SplitFiles/File_$fileidx.txt";
 				$currreg = $reg;
 			}
@@ -131,30 +198,49 @@
 		}
 		## print the line to the open filehandle. 	
 		print OUT "$line\n";
+		$esum += $p[-1];
+		$elength++;
 	}
 	close OUT;
 	close IN;
+	if ($esum > 0) {
+		print BCOVSUM "$eline\t".($esum/$elength)."\n";
+	}
+	close BCOVSUM;
+	open OUT, ">$wd/avg.tcov.txt";
+
+	foreach my $tr_ex (sort {$a cmp $b} keys(%out)) {
+	   foreach my $chr (sort {$a cmp $b} keys(%{$out{$tr_ex}})) {	
+		foreach(sort {$a <=> $b} keys(%{$out{$tr_ex}{$chr}})) {
+			my ($avg,$nr,$nrcov) = GetStats(\@{$out{$tr_ex}{$chr}{$_}{'c'}});
+			my $frac = 0;
+			if ($nr > 0) {
+				$frac = ($nrcov / $nr);
+			}
+			print OUT $out{$tr_ex}{$chr}{$_}{'r'}."\t".$avg."\t".$nrcov."\t".$nr."\t".$frac."\n";
+		}
+	   }
+	}
+	close OUT;
+	$tcov = "$wd/avg.tcov.txt";
 
 }
-
 ## sort output files according to targets file
-if (exists($opts{'r'}) ) {
-	my %hash = ();
-	open IN, "$wd/Targets.Global.Coverage";
-	while (<IN>) {
-		my @p = split(/\t/,$_) ;
-		$hash{$p[3]} = $_;
-	}
-	close IN;
-	open OUT, ">$wd/Targets.Global.Coverage";
-	open IN, $opts{'t'};
-	while (<IN>) {
-		my @p = split(/\t/,$_) ;
-		print OUT $hash{$p[3]};
-	}
-	close IN;
-	close OUT;
+my %hash = ();
+open IN, $tcov;
+while (<IN>) {
+	my @p = split(/\t/,$_) ;
+	$hash{$p[3].':'.$p[1]} = $_;
 }
+close IN;
+open OUT, ">$tcov";
+open IN, $opts{'t'};
+while (<IN>) {
+	my @p = split(/\t/,$_) ;
+	print OUT $hash{$p[3].':'.$p[1]};
+}
+close IN;
+close OUT;
 
 
 ####################################
@@ -211,24 +297,28 @@
 $totalmapped = $s[2];
 $totalmapped =~ s/^(\d+)(\s.+)/$1/;
 # count columns
-my $head = `head -n 1 $wd/Targets.Global.Coverage`;
+my $head = `head -n 1 $tcov`;
 chomp($head);
 my @cols = split(/\t/,$head);
 my $nrcols = scalar(@cols);
 my $covcol = $nrcols - 3;
 # get min/max/median/average coverage => values
-my $covs = `cut -f $covcol $wd/Targets.Global.Coverage`;
+my $covs = `cut -f $covcol $tcov`;
 my @coverages = split(/\n/,$covs);
 my ($eavg,$med,$min,$max,$first,$third,$ontarget) = arraystats(@coverages);
-my $spec = sprintf("%.1f",($ontarget / $totalmapped)*100);
+my $spec = '';
+if ($totalmapped != 0 && $totalmapped ne '') {
+	$spec = sprintf("%.1f",($ontarget / $totalmapped)*100);
+}
 # get min/max/median/average coverage => boxplot in R
 open OUT, ">$wd/Rout/boxplot.R";
-print OUT 'coverage <- read.table("../Targets.Global.Coverage",as.is=TRUE,sep="\t",header=FALSE)'."\n";
+print OUT 'coverage <- read.table("'.$tcov.'",as.is=TRUE,sep="\t",header=FALSE)'."\n";
 print OUT 'coverage <- coverage[,'.$covcol.']'."\n";
 print OUT 'png(file="../Plots/CoverageBoxPlot.png", bg="white", width=240, height=480,type=c("cairo"))'."\n";
 print OUT 'boxplot(coverage,range=1.5,main="Target Region Coverage")'."\n";
 print OUT 'graphics.off()'."\n";
 close OUT;
+print "Running boxplot.R : \n";
 system("cd $wd/Rout && Rscript boxplot.R");
 
 ## global nt coverage plot
@@ -323,6 +413,7 @@
 print OUT 'graphics.off()'."\n";
 
 close OUT;
+print "Running ntplot.r\n";
 system("cd $wd/Rout && Rscript ntplot.R");
 ## PRINT TO .TEX FILE
 open OUT, ">>$wd/Report/Report.tex";
@@ -359,6 +450,8 @@
 	$two =~ s/>=/\$\\ge\$/g;
 	$two = ucfirst($two);
 	print OUT '\textbf{'.$two.'} & '.$one.' \\\\'."\n";
+	
+
 }
 print OUT '\end{tabular}\end{minipage}'."\n";
 print OUT '\hspace{1.5cm}'."\n";
@@ -388,15 +481,18 @@
 @allexons;
 @allregions;
 @failedregions;
-if (exists($opts{'r'}) || exists($opts{'s'}) || exists($opts{'S'})) {
+%failednames;
+%allnames;
+
+if (exists($opts{'r'}) || exists($opts{'s'}) || exists($opts{'S'})|| exists($opts{'A'})) {
 	# count columns
-	my $head = `head -n 1 $wd/Targets.Global.Coverage`;
+	my $head = `head -n 1 '$tcov'`;
 	chomp($head);
 	my @cols = split(/\t/,$head);
 	my $nrcols = scalar(@cols);
 	my $covcol = $nrcols - 3;
 	# Coverage Plots for each gene => barplots in R, table here.
-	open IN, "$wd/Targets.Global.Coverage";
+	open IN, "$tcov";
 	my $currgroup = '';
 	my $startline = 0;
 	my $stopline = 0;
@@ -405,22 +501,31 @@
 		$linecounter++;
 		chomp($_);
 		my @c = split(/\t/,$_);
-		push(@allregions,$c[0].'-'.$c[1].'-'.$c[2]);
-		my $group = $c[3];
+		my $reg = $c[0].'-'.$c[1].'-'.$c[2];
+		push(@allregions,$reg);
+		my $group = $reg .": ".$c[3];
+		#my $gene = $c[3];
 		## coverage failure?
 		if ($c[$nrcol-1] < 1 || $c[$covcol-1] < $thresh) {
 			push(@failedexons,$group);
 			push(@failedregions,$c[0].'-'.$c[1].'-'.$c[2]);
+			$failednames{$group} = $c[0].'-'.$c[1].'-'.$c[2];
 		}
 		## store exon
 		push(@allexons,$group);
+		$allnames{$group} = $c[0].'-'.$c[1].'-'.$c[2];
+		if (!exists($opts{'r'}) && !exists($opts{'s'}) && !exists($opts{'S'}) && exists($opts{'A'})) {
+			## no need for barplots
+			next;
+		}
 		## extract and check gene
-		$group =~ s/^(\S+)[\|\s](.+)/$1/;
-		if ($group ne $currgroup ) {
+		my $gene = $group;
+		$gene =~ s/^chr\S+: (\S+)[\|\s](.+)/$1/;
+		if ($gene ne $currgroup ) {
 		    if ($currgroup ne '') {
 			# new gene, make plot. 
 			open OUT, ">$wd/Rout/barplot.R";
-			print OUT 'coveragetable <- read.table("../Targets.Global.Coverage",as.is=TRUE,sep="\t",header=FALSE)'."\n";
+			print OUT 'coveragetable <- read.table("'.$tcov.'",as.is=TRUE,sep="\t",header=FALSE)'."\n";
 			print OUT 'coverage <- coveragetable[c('.$startline.':'.$stopline.'),'.$covcol.']'."\n";
 			print OUT 'entries <- coveragetable[c('.$startline.':'.$stopline.'),4]'."\n";
 			print OUT 'entries <- sub("\\\\S+\\\\|","",entries,perl=TRUE)'."\n";
@@ -457,7 +562,7 @@
 			}
 
 		    }
-		    $currgroup = $group;
+		    $currgroup = $gene;
 		    $startline = $linecounter;
 		}
 		$stopline = $linecounter;
@@ -466,7 +571,7 @@
 	if ($currgroup ne '') {
 		# last gene, make plot. 
 		open OUT, ">$wd/Rout/barplot.R";
-		print OUT 'coveragetable <- read.table("../Targets.Global.Coverage",as.is=TRUE,sep="\t",header=FALSE)'."\n";
+		print OUT 'coveragetable <- read.table("'.$tcov.'",as.is=TRUE,sep="\t",header=FALSE)'."\n";
 		print OUT 'coverage <- coveragetable[c('.$startline.':'.$stopline.'),'.$covcol.']'."\n";
 		print OUT 'entries <- coveragetable[c('.$startline.':'.$stopline.'),4]'."\n";
 		print OUT 'entries <- sub("\\\\S+\\\\|","",entries,perl=TRUE)'."\n";
@@ -502,8 +607,8 @@
 	open OUT, ">>$wd/Report/Report.tex";
 	print OUT '\subsection*{Gene Summaries}'."\n";
 	print OUT '\underline{Legend:} \\\\'."\n";
-	print OUT '{\color{red}\textbf{RED:} Coverage did not reach set threshold of '.$thresh.'} \\\\'."\n";
-	print OUT '{\color{orange}\textbf{ORANGE:} Coverage was incomplete for the exon. Overruled by red.} \\\\' ."\n";
+	print OUT '{\color{red}\textbf{RED:} Average coverage did not reach set threshold of '.$thresh.'} \\\\'."\n";
+	print OUT '{\color{orange}\textbf{ORANGE:} Coverage was incomplete for the exon (section with zero coverage found). Overruled by red.} \\\\' ."\n";
 	$col = 1;
 	foreach (@small) {
 		if ($col > 2) {
@@ -540,16 +645,19 @@
 	print TEX '\subsection*{Failed Exon Plots}'."\n";
 	$col = 1;
 	print TEX '\underline{NOTE:} Only exons with global coverage $<$'.$thresh.' or incomplete coverage were plotted \\\\'."\n";
-	foreach(@failedregions) {
+	foreach(sort(keys(%failednames)) ) {
+	#foreach(@failedregions) {
 		if ($col > 2) {
 			$col = 1;
 			print TEX "\n";
 		}
 		# which exon
-		my $region = $_;
-		my $exon = $filehash{$region}{'exon'};
+		my $group = $_;
+		my ($region,$name) = split(/: /,$group);
+		#my $region = $failednames{$_};
+		my $exon = $filehash{$group}{'exon'};
 		# link exon to tmp file
-		my $exonfile = "$wd/SplitFiles/File_".$filehash{$region}{'idx'}.".txt";
+		my $exonfile = "$wd/SplitFiles/File_".$filehash{$group}{'idx'}.".txt";
 		## determine transcript orientation and location
 		my $firstline = `head -n 1 $exonfile`;
 		my @firstcols = split(/\t/,$firstline);
@@ -576,8 +684,10 @@
 		my $height = 240 ;
 		my $exonstr = $exon;
 		$exonstr =~ s/\s/_/g;
+		$exonstr =~ s/:/_/g;
 		$exon =~ s/_/ /g;
 		$exon =~ s/\|/ /g;
+		$exon =~ s/chr.*: (.*)$/$1/;
 		print OUT 'png(file="../Plots/Coverage_'.$exonstr.'.png", bg="white", width='.$width.', height='.$height.',type=c("cairo"))'."\n";
 		print OUT 'ylim = c(0,log10(max(max(coverage),'.($thresh+10).')))'."\n";
 		if ($orient eq '-') {
@@ -624,16 +734,21 @@
 		print TEX '\underline{NOTE:} ALL exons are plotted, regardless of coverage \\\\'."\n";
 	}
 	$col = 1;
-	foreach(@allregions) {
+	foreach(sort(keys(%allnames))) {
+
+	#foreach(@allregions) {
 		if ($col > 2) {
 			$col = 1;
 			print TEX "\n";
 		}
+		my $group = $_;
+		my ($region,$name) = split(/: /,$group);
 		# which exon
-		my $region = $_;
-		my $exon = $filehash{$region}{'exon'};
+		#my $region = $_;
+		#my $region = $allnames{$_};
+		my $exon = $filehash{$group}{'exon'};
 		# grep exon to tmp file
-		my $exonfile = "$wd/SplitFiles/File_".$filehash{$region}{'idx'}.".txt";
+		my $exonfile = "$wd/SplitFiles/File_".$filehash{$group}{'idx'}.".txt";
 		## determine transcript orientation.
                 my $firstline = `head -n 1 $exonfile`;
                 my @firstcols = split(/\t/,$firstline);
@@ -673,8 +788,10 @@
 		my $height = 240 ;
 		my $exonstr = $exon;
 		$exonstr =~ s/\s/_/g;
+		$exonstr =~ s/:/_/g;
 		$exon =~ s/_/ /g;
 		$exon =~ s/\|/ /g;
+		$exon =~ s/^chr.*: (.*)$/$1/;
 		print OUT 'png(file="../Plots/Coverage_'.$exonstr.'.png", bg="white", width='.$width.', height='.$height.',type=c("cairo"))'."\n";
 		print OUT 'ylim = c(0,log10(max(max(coverage),'.($thresh+10).')))'."\n";
 		if ($orient eq '-') {
@@ -739,12 +856,12 @@
 		my $genomicstop = $firstcols[2];
 
 		if ($orient eq '+') {
-			$bps = $genomicstop - $genomicstart + 1;
+			#$bps = $genomicstop - $genomicstart + 1;
 			$subtitle = "$genomicchr:".$de->format_number($genomicstart)."+".$de->format_number($genomicstop);
 
 		}
 		else {
-			$bps = $genomicstop - $genomicstart + 1;
+			#$bps = $genomicstop - $genomicstart + 1;
 			$subtitle = "$genomicchr:".$de->format_number($genomicstart)."-".$de->format_number($genomicstop);
 		}
 
@@ -796,13 +913,16 @@
 
 system("cd $wd  && tar czf '$tarfile' Results/");
 ## clean up (galaxy stores outside wd)
-system("rm -Rf $wd");
+#system("rm -Rf $wd");
 ###############
 ## FUNCTIONS ##
 ###############
 sub arraystats{
 	my @array = @_;
 	my $count = scalar(@array);
+	if ($count == 0 ) {
+		return (0,0,0,0,0,0,0);
+	}
 	@array = sort { $a <=> $b } @array;
 	# median
 	my $median = 0;
@@ -824,26 +944,31 @@
 	return ($average,$median,$min,$max,$first,$third,$sum);
 }
 
-sub GlobalSummary {
-	my ($bam,$targets) = @_;
-
-	my $command = "cd $wd && coverageBed -abam $bam -b $targets > $wd/Targets.Global.Coverage";
-	if (exists($commandsrun{$command})) {
-		return;
+sub GetStats {
+	my ($aref) = @_;
+	if (scalar(@$aref) == 0) {
+		return qw/0 0/;
 	}
-	system($command);
-	$commandsrun{$command} = 1;
+	# median
+	my @s = sort {$a <=> $b } @$aref;
+	my $nrzero = 0;
+	my $len = scalar(@s);
+	for (my $i = 0; $i< $len;$i++) {
+		if ($s[$i] == 0) {
+			$nrzero++;
+		}
+		else {
+			last;
+		}
+	}
+	my $nrcov = $len - $nrzero;
+	# avg
+	my $avg = 0;
+    	foreach (@s) { $avg += $_ };
+	$avg = sprintf("%.1f",($avg / scalar(@s)));
+	return($avg,$len,$nrcov);
 }
 
-sub CoveragePerRegion {
-	my ($bam,$targets) = @_;
-	my $command = "cd $wd && coverageBed -abam $bam -b $targets > $wd/Targets.Global.Coverage";
-	if (exists($commandsrun{$command})) {
-		return;
-	}
-	system($command);
-	$commandsrun{$command} = 1;
-}
 
 sub SubRegionCoverage {
 	my ($bam,$targets) = @_;