sub _additional_help_sections { return ( "ARGUMENTS", <<EOS

--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). ROIs from the same chromosome must be listed adjacent to each other in this file. This allows the underlying C-based code to run much more efficiently and avoid re-counting bases seen in overlapping ROIs (for overall covered base counts). 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.

--normal-tumor-bam-pair

"sample-name path/to/normal_bam path/to/tumor_bam"

--output-file

Specify an output file where the per-ROI covered base counts will be written

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 $tumor_bam = $self->tumor_bam; my $normal_bam = $self->normal_bam; my $output_file = $self->final_output_file; my $normal_min_depth = $self->normal_min_depth; my $tumor_min_depth = $self->tumor_min_depth; my $min_mapq = $self->min_mapq;

# 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 "Normal BAM file not found or is empty: $normal_bam\n" unless( -s $normal_bam );
print STDERR "Tumor BAM file not found or is empty: $tumor_bam\n" unless( -s $tumor_bam );
return undef unless( -s $roi_file && -e $ref_seq && -s $normal_bam && -s $tumor_bam );

# Check whether the annotated regions of interest are clumped together by chromosome
my $roiFh = IO::File->new( $roi_file ) or die "ROI file could not be opened. $!\n";
my @chroms = ( "" );
while( my $line = $roiFh->getline ) # Emulate Unix's uniq command on the chromosome column
{
  my ( $chrom ) = ( $line =~ m/^(\S+)/ );
  push( @chroms, $chrom ) if( $chrom ne $chroms[-1] );
}
$roiFh->close;
my %chroms = map { $_ => 1 } @chroms; # Get the actual number of unique chromosomes
if( scalar( @chroms ) != scalar( keys %chroms ))
{
  print STDERR "ROIs from the same chromosome must be listed adjacent to each other in file. ";
  print STDERR "If in UNIX, try:\nsort -k 1,1 $roi_file\n";
  return undef;
}

# If the reference sequence FASTA file hasn't been indexed, do it
my $ref_seq_idx = "$ref_seq.fai";
system( "samtools faidx $ref_seq" ) unless( -e $ref_seq_idx );

$normal_bam = '' unless( defined $normal_bam );
$tumor_bam = '' unless( defined $tumor_bam );
print STDERR "Normal BAM not found: \"$normal_bam\"\n" unless( -e $normal_bam );
print STDERR "Tumor BAM not found: \"$tumor_bam\"\n" unless( -e $tumor_bam );
next unless( -e $normal_bam && -e $tumor_bam );

# Construct the command that calculates coverage per ROI
my $calcRoiCovg_cmd = "calcRoiCovg $normal_bam $tumor_bam $roi_file $ref_seq $output_file $normal_min_depth $tumor_min_depth $min_mapq";

# If the calcRoiCovg output was already generated, then don't rerun it
if( -s $output_file )
{
  print "Output file $output_file found. Skipping re-calculation.\n";
}
# Run the calcRoiCovg command on this tumor-normal pair. This could take a while
elsif( system( "$calcRoiCovg_cmd" ) != 0 )
{
  print STDERR "Failed to execute: $calcRoiCovg_cmd\n";
  return;
}
else
{
  print "$output_file generated and stored.\n";
  return 1;
}

}

1;