--roi-file

The regions of interest (ROIs) of each gene are typically regions targeted for sequencing or are merged exon loci (from multiple transcripts) of genes with 2-bp flanks (splice junctions). For per-gene base counts, an overlapping base will be counted each time it appears in an ROI of the same gene. To avoid this, be sure to merge together overlapping ROIs of the same gene. BEDtools' mergeBed can help if used per gene.

--reference-sequence

The reference sequence in FASTA format. If a reference sequence index is not found next to this file (a .fai file), it will be created.

--wig-list

Provide a file containing sample names and the wiggle track format file locations for each. Use the tab-delimited format [sample_name wig_file] per line. Additional columns like clinical data are allowed, but ignored. The sample_name must be the same as the tumor sample names used in the MAF file (16th column, with the header Tumor_Sample_Barcode).

--output-dir

Specify an output directory where the following will be created/written: roi_covgs: Subdirectory containing per-ROI covered base counts for each sample. gene_covgs: Subdirectory containing per-gene covered base counts for each sample. total_covgs: File containing the overall non-overlapping coverages per sample.

EOS ); }

sub _doc_authors { return " Cyriac Kandoth, Ph.D."; }

sub _doc_see_also { return <<EOS genome-music-bmr(1), genome-music(1), genome(1) EOS }

sub execute { my $self = shift; my $roi_file = $self->roi_file; my $ref_seq = $self->reference_sequence; my $wig_list = $self->wig_list; my $output_dir = $self->output_dir;

# Check on all the input data before starting work
print STDERR "ROI file not found or is empty: $roi_file\n" unless( -s $roi_file );
print STDERR "Reference sequence file not found: $ref_seq\n" unless( -e $ref_seq );
print STDERR "List of WIGs not found or is empty: $wig_list\n" unless( -s $wig_list );
print STDERR "Output directory not found: $output_dir\n" unless( -e $output_dir );
return undef unless( -s $roi_file && -e $ref_seq && -s $wig_list && -e $output_dir );

# Outputs of this script will be written to these locations in the output directory
$output_dir =~ s/(\/)+$//; # Remove trailing forward slashes if any
my $roi_covg_dir = "$output_dir/roi_covgs"; # Stores output from calcRoiCovg per sample
my $gene_covg_dir = "$output_dir/gene_covgs"; # Stores per-gene coverages per sample
my $tot_covg_file = "$output_dir/total_covgs"; # Stores total coverages per sample

# If the reference sequence FASTA file hasn't been indexed, do it
my $ref_seq_idx = "$ref_seq.fai";
unless( -e $ref_seq_idx ) {
    print "Reference fasta index not found. Creating one at: $ref_seq.fai\n";
    system( "samtools faidx $ref_seq" ) or die "Failed to run samtools! $!\n";
}

# Create a temporary 0-based ROI BED-file that we can use with joinx, and also measure gene lengths
my %geneLen = ();
my $roi_bed = Genome::Sys->create_temp_file_path();
my $roiBedFh = IO::File->new( $roi_bed, ">" ) or die "Temporary ROI BED file could not be created. $!\n";
my $roiFh = IO::File->new( $roi_file ) or die "ROI file could not be opened. $!\n";
while( my $line = $roiFh->getline ) {
    chomp( $line );
    my ( $chr, $start, $stop, $gene ) = split( /\t/, $line );
    --$start;
    unless( $start >= 0 && $start < $stop ) {
        print STDERR "Invalid ROI: $line\nPlease use 1-based loci and ensure that start <= stop\n";
        return undef;
    }
    $geneLen{$gene} += ( $stop - $start );
    $roiBedFh->print( "$chr\t$start\t$stop\t$gene\n" );
}
$roiFh->close;
$roiBedFh->close;

# Also create a merged BED file where overlapping ROIs are joined together into contiguous regions
# ::TODO:: Use joinx instead of mergeBed, because we'd rather add an in-house dependency
my $merged_roi_bed = Genome::Sys->create_temp_file_path();
system( "mergeBed -i $roi_bed | joinx sort -s - -o $merged_roi_bed" );# or die "Failed to run mergeBed or joinx!\n$roi_bed\n$merged_roi_bed\n $!\n";

# Create the output directories unless they already exist
mkdir $roi_covg_dir unless( -e $roi_covg_dir );
mkdir $gene_covg_dir unless( -e $gene_covg_dir );

# This is a file that will report the overall non-overlapping coverages per WIG
my $totCovgFh = IO::File->new( $tot_covg_file, ">" );
$totCovgFh->print( "#Sample\tCovered_Bases\tAT_Bases_Covered\tCG_Bases_Covered\tCpG_Bases_Covered\n" );

# Parse through each pair of WIG files provided and run calcRoiCovg as necessary
my $wigFh = IO::File->new( $wig_list );
while( my $line = $wigFh->getline ) {
    next if( $line =~ m/^#/ );
    chomp( $line );
    my ( $sample, $wig_file ) = split( /\t/, $line );
    $wig_file = '' unless( defined $wig_file );
    print STDERR "Wiggle track format file for $sample not found: \"$wig_file\"\n" unless( -e $wig_file );
    next unless( -e $wig_file );

    # Use joinx to parse the WIG file and return per-ROI coverages of AT, CG (non-CpG), and CpG
    system( "joinx wig2bed -Zc $wig_file | joinx sort -s | joinx intersect -F \"I A3\" $roi_bed - | joinx ref-stats - $ref_seq | cut -f 1-7 > $roi_covg_dir/$sample.covg" );# or die "Failed to run joinx to calculate per-gene coverages in $sample! $!\n";

    # Read the joinx formatted coverage file and count covered bases per gene
    my %geneCovg = ();
    my $roiCovgFh = IO::File->new( "$roi_covg_dir/$sample.covg" );
    while( my $line = $roiCovgFh->getline ) {
        chomp( $line );
        if( $line !~ m/^#/ ) {
            my ( undef, undef, undef, $gene, $at_covd, $cg_covd, $cpg_covd ) = split( /\t/, $line );
            $geneCovg{$gene}{covd} += ( $at_covd + $cg_covd + $cpg_covd );
            $geneCovg{$gene}{at} += $at_covd;
            $geneCovg{$gene}{cg} += $cg_covd;
            $geneCovg{$gene}{cpg} += $cpg_covd;
        }
    }
    $roiCovgFh->close;

    # Write the per-gene coverages to a file named after this sample_name
    my $geneCovgFh = IO::File->new( "$gene_covg_dir/$sample.covg", ">" );
    $geneCovgFh->print( "#Gene\tLength\tCovered\tAT_covd\tCG_covd\tCpG_covd\n" );
    foreach my $gene ( sort keys %geneLen ) {
        if( defined $geneCovg{$gene} ) {
            $geneCovgFh->print( join( "\t", $gene, $geneLen{$gene}, $geneCovg{$gene}{covd},
                                $geneCovg{$gene}{at}, $geneCovg{$gene}{cg}, $geneCovg{$gene}{cpg} ), "\n" );
        }
        else {
            $geneCovgFh->print( "$gene\t" . $geneLen{$gene} . "\t0\t0\t0\t0\n" );
        }
    }
    $geneCovgFh->close;

    # Measure coverage stats on the merged ROI file, so that bps across the genome are not counted twice
    my $merged_roi_bed_covg = Genome::Sys->create_temp_file_path();
    system( "joinx wig2bed -Zc $wig_file | joinx sort -s | joinx intersect $merged_roi_bed - | joinx ref-stats - $ref_seq | cut -f 1-6 > $merged_roi_bed_covg" );# or die "Failed to run joinx to calculate overall coverages in $sample! $!\n";

    # Read the joinx formatted coverage file and sum up the coverage stats per region
    my ( $tot_covd, $tot_at_covd, $tot_cg_covg, $tot_cpg_covd );
    my $totRoiCovgFh = IO::File->new( $merged_roi_bed_covg );
    while( my $line = $totRoiCovgFh->getline ) {
        chomp( $line );
        if( $line !~ m/^#/ ) {
            my ( $chr, $start, $stop, $at_covd, $cg_covd, $cpg_covd ) = split( /\t/, $line );
            $tot_covd += ( $at_covd + $cg_covd + $cpg_covd );
            $tot_at_covd += $at_covd;
            $tot_cg_covg += $cg_covd;
            $tot_cpg_covd += $cpg_covd;
        }
    }
    $totRoiCovgFh->close;
    $totCovgFh->print( "$sample\t$tot_covd\t$tot_at_covd\t$tot_cg_covg\t$tot_cpg_covd\n" );
}
$wigFh->close;
$totCovgFh->close;

return 1;
}

1;