[Bioperl-l] not able to use Bio::Root::IO method

[Dan Kortschak]

Thanks Mark, I'm not sure about that since @ISA still includes
Bio::Root:IO when it's at the call, but it might be.

cheers
Dan

Here is the entirety of the code (it reasonably short):

package Bio::Assembly::IO::bowtie;
use strict;
use warnings;

# Object preamble - inherits from Bio::Root::Root

use Bio::SeqIO;
use Bio::Tools::Run::Samtools;
use Bio::Assembly::IO;
use Carp;
use Bio::Root::Root;
use Bio::Root::IO;
use base qw( Bio::Root::Root Bio::Root::IO Bio::Assembly::IO );

our $HD = "\@HD\tVN:1.0\tSO:unsorted\n";
our $PG = "\@PG\tID=Bowtie\n";

our $HAVE_IO_UNCOMPRESS;
BEGIN {
# check requirements
    unless ( eval "require Bio::Tools::Run::Bowtie;") {
	Bio::Root::Root->throw("Bio::Tools::Run::Bowtie is not available - cannot extract refdb from index.");
    }
    unless ( eval "require IO::Uncompress::Gunzip; \$HAVE_IO_UNCOMPRESS = 1") {
	Bio::Root::Root->warn("IO::Uncompress::Gunzip is not available; you'll have to do your decompression by hand.");
    }
}

sub new {
	my $class = shift;
	my @args = @_;
	my $self = $class->SUPER::new(@args);
	my ($file, $index, $no_head, $no_sq) = $self->_rearrange([qw(FILE INDEX NO_HEAD NO_SQ)], @args);
	$file =~ s/^<//;
	$self->{'_no_head'} = $no_head;
	$self->{'_no_sq'} = $no_sq;
	# get the sequence so samtools can work with it
	my $inspector = Bio::Tools::Run::Bowtie->new( -command => 'inspect' );
	my $refdb = $inspector->run($index);
	my $bam_file = $self->_make_bam($self->_bowtie_to_sam($file, $refdb));
	my $sam = Bio::Assembly::IO->new( -file => "<$bam_file", -refdb => $refdb , -format => 'sam' );
	return $sam;
}

sub _bowtie_to_sam {
	my ($self, $file, $refdb) = @_;

	$self->throw("'$file' does not exist or is not readable.")
		unless ( -e $file && -r $file );
	my $guesser = Bio::Tools::GuessSeqFormat->new(-file=>$file);
	$self->throw("'$file' is not a bowtie formatted file.") unless $guesser->guess =~ m/^bowtie$/;

	my %SQ;
	my $mapq = 255;
	my $in_pair;
	my @mate_line;
	my $mlen;

	if ($file =~ m/\.gz[^.]*$/) {
		unless ($HAVE_IO_UNCOMPRESS) {
			croak( "IO::Uncompress::Gunzip not available, can't expand '$_'" );
		}
		my ($tfh, $tf) = $self->io->tempfile;
		my $z = IO::Uncompress::Gunzip->new($_);
		while (<$z>) { print $tfh $_ }
		close $tfh;
		$file = $tf;
	}

        open(my $fh, $file) or
		$self->throw("Can not open '$file' for reading: $!");
            
	# create temp file for working
	my ($sam_tmp_h, $sam_tmp_f) = $self->io->tempfile( -dir => $self->tempdir(), -suffix => '.sam' );
	
	while ($fh) {
		chomp;
		my ($qname,$strand,$rname,$pos,$seq,$qual,$m,$details)=split("\t",$_);
		$SQ{$rname} = 1;
		
		my $paired_f =  ($qname =~ m#/[12]#) ? 0x03 : 0;
		my $strand_f = ($strand eq '-') ? 0x10 : 0;
		my $op_strand_f = ($strand eq '+' && $paired_f) ? 0x20 : 0;
		my $first_f =  ($qname =~ m#/1#) ? 0x40 : 0;
		my $second_f = ($qname =~ m#/2#) ? 0x80 : 0;
		my $flag = $paired_f | $strand_f | $op_strand_f | $first_f | $second_f;

		$pos++;
		my $len = length $seq;
		die unless $len == length $qual;
		my $cigar = $len.'M';
		my @detail = split(',',$details);
		my $dist = 'NM:i:'.scalar @detail;
		
		my @mismatch;
		my $last_pos = 0;
		for (@detail) {
			m/(\d+):(\w)>\w/;
			my $err = ($1-$last_pos);
			$last_pos = $1+1;
			push @mismatch,($err,$2);
		}
		push @mismatch, $len-$last_pos;
		@mismatch = reverse @mismatch if $strand eq '-';
		my $mismatch = join('',('MD:Z:', at mismatch));

		if ($paired_f) {
			my $mrnm = '=';
			if ($in_pair) {
				my $mpos = $mate_line[3];
				$mate_line[7] = $pos;
				my $isize = $mpos-$pos-$len;
				$mate_line[8] = -$isize;
				print $sam_tmp_h join("\t", at mate_line),"\n";
				print $sam_tmp_h join("\t",$qname, $flag, $rname, $pos, $mapq, $cigar, $mrnm, $mpos, $isize, $seq, $qual, $mismatch, $dist),"\n";
				$in_pair = 0;
			} else {
				$mlen = $len;
				@mate_line = ($qname, $flag, $rname, $pos, $mapq, $cigar, $mrnm, undef, undef, $seq, $qual, $mismatch, $dist);
				$in_pair = 1;
			}
		} else {
			my $mrnm = '*';
			my $mpos = 0;
			my $isize = 0;
			print $sam_tmp_h join("\t",$qname, $flag, $rname, $pos, $mapq, $cigar, $mrnm, $mpos, $isize, $seq, $qual, $mismatch, $dist),"\n";
		}
	}

	close($fh);
	$sam_tmp_h->close;
	
	return $sam_tmp_f if $self->{'_no_head'};

	my ($samh, $samf) = $self->io->tempfile( -dir => $self->tempdir(), -suffix => '.sam' );

	# print header
	print $samh $HD;
	
	# print sequence dictionary
	unless ($self->{'_no_sq'}) {
		my $db  = Bio::SeqIO->new( -file => $refdb, -format => 'fasta' );
		while ( my $seq = $db->next_seq() ) {
			$SQ{$seq->id} = $seq->length if $SQ{$seq->id};
		}
	
		map { print $samh join("\t", ('@SQ', "SN:$_", "LN:$SQ{$_}")), "\n" } keys %SQ;
	}
	
	# print program
	print $samh $PG;
	
	open($sam_tmp_h, $sam_tmp_f) or
		$self->throw("Can not open '$sam_tmp_f' for reading: $!");

	print $samh $_ while ($sam_tmp_h);
	
	close($sam_tmp_h);
	$samh->close;
	
	return $samf;
}

sub _make_bam {
	my ($self, $file) = @_;
	
	$self->throw("'$file' does not exist or is not readable")
		unless ( -e $file && -r $file );

	# make a sorted bam file from a sam file input
	my ($bamh, $bamf) = $self->io->tempfile( -dir => $self->tempdir(), -suffix => '.bam' );
	my ($srth, $srtf) = $self->io->tempfile( -dir => $self->tempdir(), -suffix => '.srt' );
	$_->close for ($bamh, $srth);
	
	my $samt = Bio::Tools::Run::Samtools->new( -command => 'view',
						   -sam_input => 1,
						   -bam_output => 1 );

	$samt->run( -bam => $file, -out => $bamf );

	$samt = Bio::Tools::Run::Samtools->new( -command => 'sort' );

	$samt->run( -bam => $bamf, -pfx => $srtf);

	return $srtf.'.bam'
}

1;


On Thu, 2010-01-14 at 00:11 -0500, Mark A. Jensen wrote:
> Hey Dan-- what does your constructor look like? I wonder if
> something's getting 
> lost in new() and _initialize() chaining spaghetti- MAJ
>