• Define cmd-list-file and cmd-prefix to generate a file with commands that can be submitted to a cluster or run manually. These jobs will write per-ROI base counts in a subdirectory roi_covgs.

  • After all the parallelized calcRoiCovg jobs are completed, run this script again to add them up and generate the final per-gene base counts in a subdirectory gene_covgs. Remember to remove the cmd-list-file and cmd-prefix arguments or you will just be re-creating a list of commands.

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.
--bam-list
Provide a file containing sample names and normal/tumor BAM locations for each. Use the tab- delimited format [sample_name normal_bam tumor_bam] 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.
--cmd-list-file
Specify a file into which a list of calcRoiCovg jobs will be written to. These can be scheduled in parallel, and will write per-ROI covered base-counts into the output subdirectory roi_covgs. If cmd-list-file is left unspecified, this script runs calcRoiCovg per sample one after another, taking ~30 mins per sample, but it skips samples whose output is already in roi_covgs.
--cmd-prefix
Specify a job submission command that will be prefixed to each command in cmd-list-file. This makes batch submission easier. Just run the cmd-list-file file as a shell script to submit jobs. cmd-prefix is "bsub" if your cluster uses the LSF job scheduler, or "qsub" in Torque. Add arguments as necessary. For example, "bsub -M 4GB" sets a soft memory limit of 4GB.

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 $bam_list = $self->bam_list; my $output_dir = $self->output_dir; my $cmd_list_file = $self->cmd_list_file; my $cmd_prefix = $self->cmd_prefix; my $normal_min_depth = $self->normal_min_depth; my $tumor_min_depth = $self->tumor_min_depth; my $min_mapq = $self->min_mapq;

  my $optional_params = "";

  if ($normal_min_depth) {
    $optional_params .= " --normal-min-depth $normal_min_depth";
  }
  if ($tumor_min_depth) {
    $optional_params .= " --tumor-min-depth $tumor_min_depth";
  }
  if ($min_mapq) {
    $optional_params .= " --min-mapq $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 "List of BAMs not found or is empty: $bam_list\n" unless( -s $bam_list );
  print STDERR "Output directory not found: $output_dir\n" unless( -e $output_dir );
  return undef unless( -s $roi_file && -e $ref_seq && -s $bam_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

  $self->gene_covg_dir($gene_covg_dir);

  # 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 );

  # 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 );

  my ( $cmdFh, $totCovgFh );
  if( defined $cmd_list_file )
  {
    $cmdFh = IO::File->new( $cmd_list_file, ">" );
    print "Creating a list of parallelizable jobs at $cmd_list_file.\n";
    print "After successfully running all the jobs in $cmd_list_file,\n",
          "be sure to run this script a second time (without defining the cmd-list-file argument) to merge results in roi_covgs.\n";
  }
  else
  {
    $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 BAM files provided and run calcRoiCovg as necessary
  my $bamFh = IO::File->new( $bam_list );
  while( my $line = $bamFh->getline )
  {
    next if( $line =~ m/^#/ );
    chomp( $line );
    my ( $sample, $normal_bam, $tumor_bam ) = split( /\t/, $line );
    $normal_bam = '' unless( defined $normal_bam );
    $tumor_bam = '' unless( defined $tumor_bam );
    print STDERR "Normal BAM for $sample not found: \"$normal_bam\"\n" unless( -e $normal_bam );
    print STDERR "Tumor BAM for $sample not found: \"$tumor_bam\"\n" unless( -e $tumor_bam );
    next unless( -e $normal_bam && -e $tumor_bam );

    # Construct the command that calculates coverage per ROI
    my $calcRoiCovg_cmd = "\'gmt music bmr calc-covg-helper --normal-tumor-bam-pair \"$line\" --roi-file \"$roi_file\" ".
    "--reference-sequence \"$ref_seq\" --output-file \"$roi_covg_dir/$sample.covg\"$optional_params\'";

    # If user only wants the calcRoiCovg commands, write them to file and skip running calcRoiCovg
    if( defined $cmd_list_file )
    {
      $calcRoiCovg_cmd = $cmd_prefix . " $calcRoiCovg_cmd" if( defined $cmd_prefix );
      $cmdFh->print( "$calcRoiCovg_cmd\n" );
      next;
    }

    # If the calcRoiCovg output was already generated, then don't rerun it
    if( -s "$roi_covg_dir/$sample.covg" )
    {
      print "$sample.covg found in $roi_covg_dir. Skipping re-calculation.\n";
    }
    # Run the calcRoiCovg command on this tumor-normal pair. This could take a while
    else {
      my %params = (
        normal_tumor_bam_pair => $line,
        roi_file => $roi_file,
        reference_sequence => $ref_seq, 
        output_file => $roi_covg_dir."/".$sample.".covg",
      );
      if ($normal_min_depth) {
        $params{"normal_min_depth"} = $normal_min_depth;
      }
      if ($tumor_min_depth) {
        $params{"tumor_min_depth"} = $tumor_min_depth;
      }
      if ($min_mapq) {
        $params{"min_mapq"} = $min_mapq;
      }
      my $cmd = Genome::Model::Tools::Music::Bmr::CalcCovgHelper->create(%params);
      my $rv = $cmd->execute;
      if(!$rv)
      {
        print STDERR "Failed to execute: $calcRoiCovg_cmd\n";
        next;
      }
      else
      {
        print "$sample.covg generated and stored to $roi_covg_dir.\n";
      }
    }

    # Read the calcRoiCovg output and count covered bases per gene
    my %geneCovg = ();
    my ( $tot_covd, $tot_at_covd, $tot_cg_covg, $tot_cpg_covd );
    my $roiCovgFh = IO::File->new( "$roi_covg_dir/$sample.covg" );
    while( my $line = $roiCovgFh->getline )
    {
      chomp( $line );
      if( $line =~ m/^#NonOverlappingTotals/ )
      {
        ( undef, undef, undef, $tot_covd, $tot_at_covd, $tot_cg_covg, $tot_cpg_covd ) = split( /\t/, $line );
      }
      elsif( $line !~ m/^#/ )
      {
        my ( $gene, undef, $length, $covd, $at_covd, $cg_covd, $cpg_covd ) = split( /\t/, $line );
        $geneCovg{$gene}{len} += $length;
        $geneCovg{$gene}{covd_len} += $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 %geneCovg )
    {
      $geneCovgFh->print( join( "\t", $gene, $geneCovg{$gene}{len}, $geneCovg{$gene}{covd_len},
         $geneCovg{$gene}{at}, $geneCovg{$gene}{cg}, $geneCovg{$gene}{cpg} ), "\n" );
    }
    $geneCovgFh->close;

    # Write total coverages for this sample to a file
    $totCovgFh->print( "$sample\t$tot_covd\t$tot_at_covd\t$tot_cg_covg\t$tot_cpg_covd\n" );
  }
  $bamFh->close;
  $cmdFh->close if( defined $cmd_list_file );
  $totCovgFh->close unless( defined $cmd_list_file );

  return 1;
}

1;

1 POD Error

The following errors were encountered while parsing the POD:

Around line 85:

=back doesn't take any parameters, but you said =back HELP }