Bio::Tools::Run::Bowtie - Run wrapper for the Bowtie short-read assembler *BETA*


BioPerl-Run documentation Contained in the BioPerl-Run distribution.

Index


Code Index:

NAME

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Bio::Tools::Run::Bowtie - Run wrapper for the Bowtie short-read assembler *BETA*

SYNOPSIS

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 # create an index
 $bowtie_build = Bio::Tools::Run::Bowtie->new();
 $index = $bowtie_fac->run( 'reference.fasta', 'index_base' );

 # or with named args...

 $index = $bowtie_fac->run( -ref => 'reference.fasta', -ind => 'index_base' );

 # get the base name of the last index from an index builder
 $index = $bowtie_fac->result;

 # create an assembly
 $bowtie_fac = Bio::Tools::Run::Bowtie->new();
 $bowtie_fac->want('Bio::Assembly::Scaffold');
 $bowtie_assy = $bowtie_fac->run( 'reads.fastq', 'index_base' );

 # if IO::Uncompress::Gunzip is available and with named args...
 $bowtie_assy = $bowtie_fac->run( -seq => 'reads.fastq.gz', -ind => 'index_base' );

 # paired-end
 $bowtie_fac = Bio::Tools::Run::Bowtie->new(-command => 'paired',
                                            -want => 'Bio::Assembly::Scaffold');
 $bowtie_assy = $bowtie_fac->run( 'reads.fastq', 'index_base', 'paired-reads.fastq' );

 # be more strict
 $bowtie_fac->set_parameters( -max_qual_mismatch => 50 );

 # create a Bio::Assembly::Scaffold object
 $bowtie_assy = $bowtie_fac->run( 'reads.fastq', 'index_base', 'paired-reads.fastq'  );

 # print consensus sequences from assembly object
 for $contig ($bowtie_assy->all_contigs) {
    print $contig->get_consensus_sequence->seq,"\n";
 }

 # get the file object of the last assembly
 $io = $bowtie_fac->result( -want => 'Bio::Root::IO' );

 # get a merged SeqFeature::Collection of all hits
 #  - currently only available with SAM format 
 $io = $bowtie_fac->result( -want => 'Bio::SeqFeature::Collection' );

 #... or the file name directly
 $filename = $bowtie_fac->result( -want => 'raw' );

DESCRIPTION

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This module provides a wrapper interface for Ben Langmead and Col Trapnell's ultrafast memory-efficient short read aligner bowtie (see http://bowtie-bio.sourceforge.net/ for manuals and downloads).

OPTIONS

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bowtie is complex, with many command-line options. This module attempts to provide and options comprehensively. You can browse the choices like so:

 $bowtiefac = Bio::Tools::Run::Bowtie->new( -command => 'single' );
 # all bowtie commands
 @all_commands = $bowtiefac->available_parameters('commands');
 @all_commands = $bowtiefac->available_commands; # alias
 # just for single
 @assemble_params = $bowtiefac->available_parameters('params');
 @assemble_switches = $bowtiefac->available_parameters('switches');
 @assemble_all_options = $bowtiefac->available_parameters();

Reasonably mnemonic names have been assigned to the single-letter command line options. These are the names returned by available_parameters, and can be used in the factory constructor like typical BioPerl named parameters.

As a number of options are mutually exclusive, and the interpretation of intent is based on last-pass option reaching bowtie with potentially unpredicted results. This module will prevent inconsistent switches and parameters from being passed.

See http://bowtie.sourceforge.net/bowtie-manpage.shtml for details of bowtie options.

FILES

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When a command requires filenames, these are provided to the run method, not the constructor (new()). To see the set of files required by a command, use available_parameters('filespec') or the alias filespec():

  $bowtiefac = Bio::Tools::Run::Bowtie->new( -command => 'paired' );
  @filespec = $bowtiefac->filespec;

This example returns the following array:

 ind
 seq
 seq2
 #out

This indicates that ind (bowtie index file base name), seq (fasta/fastq),and seq2 (fasta/fastq) files MUST be specified, and that the out file MAY be specified. Use these in the run call like so:

 $bowtiefac->run( -ind => 'index_base', -seq => 'seq-a.fq',
                  -seq2 => 'seq-b.fq', -out => 'align.out' );

Note that named parameters in this form allow you to specify the location of the outfile; without named parameters, the outfile is located in a tempdir and does not persist beyond the life of the object - with the exception of index creation.

The object will store the programs STDOUT and STDERR output for you in the stdout() and stderr() attributes:

 handle_map_warning($bowtiefac) if ($bowtiefac->stderr =~ /warning/);

FEEDBACK

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Mailing Lists

User feedback is an integral part of the evolution of this and other Bioperl modules. Send your comments and suggestions preferably to the Bioperl mailing list. Your participation is much appreciated.

  bioperl-l@bioperl.org                  - General discussion
  http://bioperl.org/wiki/Mailing_lists  - About the mailing lists

Support

Please direct usage questions or support issues to the mailing list:

bioperl-l@bioperl.org

Rather than to the module maintainer directly. Many experienced and reponsive experts will be able look at the problem and quickly address it. Please include a thorough description of the problem with code and data examples if at all possible.

Reporting Bugs

Report bugs to the Bioperl bug tracking system to help us keep track of the bugs and their resolution. Bug reports can be submitted via the web:

  http://redmine.open-bio.org/projects/bioperl/

AUTHOR - Dan Kortschak

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 Email dan.kortschak adelaide.edu.au

CONTRIBUTORS

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 Mark A. Jensen (maj -at- fortinbras -dot- us)

APPENDIX

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The rest of the documentation details each of the object methods. Internal methods are usually preceded with a _

new()

 Title   : new
 Usage   : my $obj = new Bio::Tools::Run::Bowtie();
 Function: Builds a new Bio::Tools::Run::Bowtie object
 Returns : an instance of Bio::Tools::Run::Bowtie
 Args    :

run()

 Title   : run
 Usage   : $assembly = $bowtie_assembler->run($read1_fastq_file,
                                           $index_location,
                                           $read2_fastq_file);
           $assembly = $bowtie_assembler->run(%params);
 Function: Run the bowtie assembly pipeline.
 Returns : Assembly results (file, IO object or Assembly object)
 Args    : - fastq file containing single-end reads
           - name of the base of the bowtie index
           - [optional] fastq file containing paired-end reads
           Named params are also available with args:
           -seq, -seq2, -ind (bowtie index), -ref (fasta reference) and -out
 Note    : gzipped inputs are allowed if IO::Uncompress::Gunzip
           is available
           The behaviour for locating indexes follows the definition in
           the bowtie manual - you may use the environment variable
           BOWTIE_INDEXES to specify the index path or use an 'indexes'
           directory under the directory where the bowtie executable
           is located

want()

 Title   : want
 Usage   : $bowtiefac->want( $class )
 Function: make factory return $class, or raw (scalar) results in file
 Returns : return wanted type
 Args    : [optional] string indicating class or raw of wanted result

result()

 Title   : result
 Usage   : $bowtiefac->result( [-want => $type|$format] )
 Function: return result in wanted format
 Returns : results
 Args    : [optional] hashref of wanted type

_determine_format()

 Title   : _determine_format
 Usage   : $bowtiefac->_determine_format
 Function: determine the format of output for current options
 Returns : format of bowtie output
 Args    :

_make_bam()

 Title   : _make_bam
 Usage   : $bowtiefac->_make_bam( $file, $sort )
 Function: make a sorted BAM format file from SAM file
 Returns : sorted BAM file name
 Args    : SAM file name and boolean flag to select sorted BAM format

_validate_file_input()

 Title   : _validate_file_input
 Usage   : $bowtiefac->_validate_file_input( -type => $file )
 Function: validate file type for file spec
 Returns : file type if valid type for file spec
 Args    : hash of filespec => file_name

Bio::Tools::Run::AssemblerBase overrides

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_assembly_format()

 Title   : _assembly_format
 Usage   : $bowtiefac->_determine_format
 Function: set the format of output for current options
 Returns : format of bowtie output
 Args    :

_check_sequence_input()

 No-op.

_check_optional_quality_input()

 No-op.

_prepare_input_sequences()

 Prepare and check input sequences for bowtie.

set_parameters()

 Title   : set_parameters
 Usage   : $bowtiefac->set_parameters(%params);
 Function: sets the parameters listed in the hash or array,
           maintaining sane options.
 Returns : true on success
 Args    : [optional] hash or array of parameter/values.
 Note    : This will unset conflicts and set required options,
           but will not prevent non-sane requests in the arguments

version()

 Title   : version
 Usage   : $version = $bowtiefac->version()
 Function: Returns the program version (if available)
 Returns : string representing location and version of the program


BioPerl-Run documentation Contained in the BioPerl-Run distribution.
# $Id: Bowtie.pm kortsch $
#
# BioPerl module for Bio::Tools::Run::Bowtie
#
# Please direct questions and support issues to <bioperl-l@bioperl.org>
#
# Cared for by Dan Kortschak <dan.kortschak@adelaide.edu.au>
#
# Copyright Dan Kortschak and Mark A. Jensen
#
# You may distribute this module under the same terms as perl itself

# POD documentation - main docs before the code

# Let the code begin...


package Bio::Tools::Run::Bowtie;
use strict;
our $HAVE_IO_UNCOMPRESS;

BEGIN {
    eval 'require IO::Uncompress::Gunzip; $HAVE_IO_UNCOMPRESS = 1';
}

use IPC::Run;

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

use lib '../../..';
use Bio::Tools::Run::Bowtie::Config;
use Bio::Tools::Run::WrapperBase;
use Bio::Tools::Run::WrapperBase::CommandExts;
use Bio::Tools::GuessSeqFormat;
use Bio::Tools::Run::Samtools;
use Bio::Seq;
use File::Basename;

use base qw( Bio::Tools::Run::WrapperBase Bio::Tools::Run::AssemblerBase );

## bowtie
our $program_name = '*bowtie';
our $default_cmd = 'single';

our $asm_format; # this is determined dynamically

# Note:
#  other globals required by Bio::Tools::Run::AssemblerBase are
#  imported from Bio::Tools::Run::Bowtie::Config

our $qual_param = undef;
our $use_dash = 'mixed';
our $join = ' ';

sub new {
	my ($class,@args) = @_;
	unless (grep /command/, @args) {
		push @args, '-command', $default_cmd;
	}
	#default to SAM output if no other format specified and we are running an alignment
	my %args=@args;
	if ($args{'-command'} =~ m/(?:single|paired|crossbow)/) {
		unless (grep /(?:sam_format|concise|quiet|refout|refidx)/, @args) {
			push @args, ('-sam_format', 1);
		}
	}
	my $self = $class->SUPER::new(@args);
	foreach (keys %command_executables) {
		$self->executables($_, $self->_find_executable($command_executables{$_}));
	}
	my ($want) = $self->_rearrange([qw(WANT)],@args);
	$self->want($want);
	$asm_format = $self->_assembly_format;
	$self->parameters_changed(1); # set on instantiation, per Bio::ParameterBaseI
	return $self;
}

sub run {
	my $self = shift;

	my ($arg1, $arg2, $arg3);                # these are useless names because the different
	                                         # programs take very different arguments
	my ($index, $seq, $seq2, $ref, $out); # these are the meaningful names that are used
	                                         # with named args

	if (!(@_ % 2)) {
		my %args = @_;
		if ((grep /^-\w+/, keys %args) == keys %args) {
			($index, $seq, $seq2, $ref, $out) =
				$self->_rearrange([qw( IND SEQ SEQ2 REF OUT )], @_);
		} elsif (grep /^-\w+/, keys %args) {
			$self->throw("Badly formed named args: ".join(' ',@_));
		} else {
			($arg1, $arg2) = @_;
		}
	} else {
		if (grep /^-\w+/, @_) {
			$self->throw("Badly formed named args: ".join(' ',@_));
		} else {
			($arg1, $arg2, $arg3) = @_;
		}
	}

	# Sanity checks
	$self->_check_executable();
	my $cmd = $self->command if $self->can('command');

	for ($cmd) {
		m/(?:single|paired|crossbow)/ && do {
			$seq ||= $arg1;
			$index ||= $arg2;
			$seq2 ||= $arg3;
			$seq or $self->throw("Fasta/fastq/raw read(s) file/Bio::Seq required at arg 1/-seq");
			$index or $self->throw("Bowtie index base required at arg 2/-index");

			# expand gzipped files as nec.
			for ($seq, $seq2) {
				next unless $_;
				if (/\.gz[^.]*$/) {
					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;
					$_ = $tf;
				}
			}

			# confirm index files exist
			$self->_validate_file_input( -ind => $index ) or
			($self->_validate_file_input( -ind => $self->io->catfile(dirname($self->executable),'indexes',$index)) and 
				$index = $self->io->catfile(dirname($self->executable),'indexes',$index)) or
			($self->_validate_file_input( -ind => $self->io->catfile($ENV{BOWTIE_INDEXES},$index)) and
			        $index = $self->io->catfile($ENV{BOWTIE_INDEXES},$index)) or
					$self->throw("Incorrect filetype (expecting bowtie index) or absent file arg 2/-index");
		
			# bowtie prepare the multiple input types
			$seq = $self->_prepare_input_sequences($seq);
			if ($cmd =~ m/^p/) {
				$seq2 && ($seq2 = $self->_prepare_input_sequences($seq2));				
			} else {
				$seq2 && $self->throw("Second sequence input not wanted for command: $cmd");
			}
			
			# Assemble
			my $format = $self->_assembly_format;
			my $suffix = '.'.$format;
			
			if ($out) {
				$out .= $suffix;
			} else {
				my ($bowtieh, $bowtief) = $self->io->tempfile( -dir => $self->tempdir(), -suffix => $suffix );
				$bowtieh->close;
				$out = $bowtief;
			}

			my %params = ( -ind => $index, -seq => $seq, -seq2 => $seq2, -out => $out );
			map {
				delete $params{$_} unless defined $params{$_}
			} keys %params;
			$self->_run(%params);
			
			$self->{'_result'}->{'index'} = $index;
			$self->{'_result'}->{'file_name'} = $out;
			$self->{'_result'}->{'format'} = $format;
			$self->{'_result'}->{'file'} = Bio::Root::IO->new( -file => $out );
			
			return $self->result;
		};
		
		m/build/ && do {
			$ref ||= $arg1;
			$index ||= $arg2;
			$ref or $self->throw("Fasta read(s) file/Bio::Seq required at arg 1/-ref");
			$index ||= $self->io->tempdir(CLEANUP => 1).'/index'; # we want a new one each time
			$arg3 && $self->throw("Second sequence input not wanted for command: $cmd");

			my $format = $self->_assembly_format;
			
			# expand gzipped file as nec.
			if ($ref =~ (m/\.gz[^.]*$/)) {
				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;
				$ref = $tf;
			}

			# bowtie prepare the two input types for the first argument
			$ref = $self->_prepare_input_sequences($ref);
		
			# Build index
			$self->_run( -ref => $ref, -out => $index );
			$self->{'_result'}->{'format'} = $format;
			$self->{'_result'}->{'file_name'} = $index;
			
			return $index;
		};
		
		m/inspect/ && do {
			$index ||= $arg1;
			$out ||= $arg2;
			$index or $self->throw("Bowtie index required at arg 1");

			$self->_validate_file_input( -ind => $index ) or
			($self->_validate_file_input( -ind => $self->io->catfile(dirname($self->executable),'indexes',$index)) and 
				$index = $self->io->catfile(dirname($self->executable),'indexes',$index)) or
			($self->_validate_file_input( -ind => $self->io->catfile($ENV{BOWTIE_INDEXES},$index)) and
			        $index = $self->io->catfile($ENV{BOWTIE_INDEXES},$index)) or
					$self->throw("'$index' doesn't look like a bowtie index or index component is missing at arg 1/-ind");
			$arg3 && $self->throw("Second sequence input not wanted for command: $cmd");

			# Inspect index
			my $format = $self->_assembly_format;
			my $suffix = '.'.$format;

			if ($out) {
				$out .= $suffix;
			} else {
				my ($desch, $descf) = $self->io->tempfile( -dir => $self->tempdir(), -suffix => $suffix );
				$desch->close;
				$out = $descf;
			}

			$self->_run( -ind => $index, -out => $out );
			
			$self->{'_result'}->{'file_name'} = $out;
			$self->{'_result'}->{'format'} = $format;
			$self->{'_result'}->{'file'} = Bio::Root::IO->new( -file => $out );
			
			return $self->result;
		}
	}
}

sub want {
	my $self = shift;
	return $self->{'_want'} = shift if @_;
	return $self->{'_want'};
}

sub result {
	my ($self, @args) = @_;
	
	my $want = $self->want ? $self->want : $self->want($self->_rearrange([qw(WANT)],@args));
	my $cmd = $self->command if $self->can('command');
	my $format = $self->{'_result'}->{'format'};

	return $self->{'_result'}->{'format'} if (defined $want && $want eq 'format');
	return $self->{'_result'}->{'file_name'} if (!$want || $want eq 'raw' || $cmd eq 'build');
	return $self->{'_result'}->{'file'} if ($want =~ m/^Bio::Root::IO/);
	
	for ($cmd) {
		m/(?:single|paired|crossbow)/ && do {
			my $scaffold;
			for ($format) {
				m/^bowtie/i && $want =~ m/^Bio::Assembly::Scaffold/ && do {
					unless (defined $self->{'_result'}->{'object'} &&
						ref($self->{'_result'}->{'object'}) =~ m/^Bio::Assembly::Scaffold/) {
							$self->{'_result'}->{'object'} =
								$self->_export_results( $self->{'_result'}->{'file_name'},
								                       -index => $self->{'_result'}->{'index'},
								                       -keep_asm => 1 );
					}
					last;
				};
				m/^bowtie/i && $want =~ m/^Bio::SeqFeature::Collection/ && do {
					$self->warn("Don't know how to create a $want object for $cmd with bowtie format - try SAM format.");
					last;
				};
				m/^sam/i && $want =~ m/^Bio::Assembly::Scaffold/ && do {
					unless (defined $self->{'_result'}->{'object'} &&
						ref($self->{'_result'}->{'object'}) =~ m/^Bio::Assembly::Scaffold/) {
							my $bamf = $self->_make_bam($self->{'_result'}->{'file_name'}, 1);
							my $inspector = Bio::Tools::Run::Bowtie->new( -command => 'inspect' );
							my $refdb = $inspector->run($self->{'_result'}->{'index'});
							$self->{'_result'}->{'object'} =
								$self->_export_results($bamf, -refdb => $refdb, -keep_asm => 1 );
					}
					last;
				};
				m/^sam/i && $want =~ m/^Bio::SeqFeature::Collection/ && do {
					unless (defined $self->{'_result'}->{'object'} &&
						ref($self->{'_result'}->{'object'}) =~ m/^Bio::Assembly::Scaffold/) {
							my $bamf = $self->_make_bam($self->{'_result'}->{'file_name'}, 0);
							my $convert = Bio::Tools::Run::BEDTools->new( -command => 'bam_to_bed' );
							my $bedf = $convert->run( -bed => $bamf );
							my $merge = Bio::Tools::Run::BEDTools->new( -command => 'merge' );
							$merge->run($self->{'_result'}->{'index'});
							$self->{'_result'}->{'object'} = $merge->result( -want => $want );
					}
					last;
				};
				do {
					$self->warn("Don't know how to create a $want object for $cmd.");
					return;
				}
			};
			last;
		};
		m/inspect/ && do {
			for ($want) {
				m/^Bio::SeqIO/ && $format eq 'fasta' && do {
					unless (defined $self->{'_result'}->{'object'} &&
						ref($self->{'_result'}->{'object'}) =~ m/^Bio::SeqIO/) {
							$self->{'_result'}->{'object'} =
								Bio::SeqIO->new(-file => $self->{'_result'}->{'file'},
								                -format => 'fasta');
					}
					last;
				};
				m/^Bio::SeqIO/ && $format ne 'fasta' && do {
					$self->warn("Don't know how to create a $want object for names only - try -want => 'Bio::Root::IO'.");
					return;
				};
				do {
					$self->warn("Don't know how to create a $want object for $cmd.");
					return;
				}
			}
		}
	}
	
	return $self->{'_result'}->{'object'};
}

sub _determine_format {
        my ($self) = shift;

	my $cmd = $self->command if $self->can('command');
	for ($cmd) {
		m/build/ && do {
			return 'ebwt';
		};
		m/inspect/ && do {
			$self->{'_summary'} && return 'text';
			return $self->{'_names_only'} ? 'text' : 'fasta';
		};
		m/(?:single|paired|crossbow)/ && do {
			my $format = 'bowtie'; # this is our default position
			for (keys %format_lookup) {
				$format = $format_lookup{$_} if $self->{'_'.$_};
			}
			return $format;
		}
	}
}

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

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

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

	if ($sort) {
		my ($srth, $srtf) = $self->io->tempfile( -dir => $self->io->tempdir(CLEANUP=>1), -suffix => '.srt' ); 
			# shared tempdir, so make new - otherwise it is scrubbed during Bio::DB::Sam
		$srth->close;
		
		$samt = Bio::Tools::Run::Samtools->new( -command => 'sort' );
		$samt->run( -bam => $bamf, -pfx => $srtf);
		
		return $srtf.'.bam';
	} else {
		return $bamf;
	}
}

sub _validate_file_input {
	my ($self, @args) = @_;
	my (%args);
	if (grep (/^-/, @args)) { # named parms
		$self->throw("Wrong number of args - requires one named arg") if (@args > 2);
		s/^-// for @args;
		%args = @args;
	} else {
		$self->throw("Must provide named filespec");
	}
	
	for (keys %args) {
		m/^seq|seq2|ref$/ && do {
			return unless ( -e $args{$_} && -r _ );
			my $guesser = Bio::Tools::GuessSeqFormat->new(-file=>$args{$_});
			return $guesser->guess if grep {$guesser->guess =~ m/$_/} @{$accepted_types{$_}};
		};
		m/^ind$/ && do {
			return 'ebwt' if
				(-e $args{$_}.'.1.ebwt' && -e $args{$_}.'.2.ebwt' && -e $args{$_}.'.3.ebwt' &&
				-e $args{$_}.'.4.ebwt' && -e $args{$_}.'.rev.1.ebwt' && -e $args{$_}.'.rev.2.ebwt');
		}
	}
	return;
}

sub _assembly_format {
	my $self = shift;

	my $format = $self->_determine_format;
	return $self->SUPER::_assembly_format($format);
}
	

sub _check_sequence_input {
	return 1;
}

sub _check_optional_quality_input {
	return 1;
}

sub _prepare_input_sequences {

	my ($self, @args) = @_;
	my (%args, $read);
	if (grep (/^-/, @args)) { # named parms
		$self->throw("Input args not an even number") unless !(@args % 2);
		%args = @args;
		($read) = @args{qw( -sequence )};
	} else {
		($read) = @args;
	}

	# Could use the AssemblerBase routine for this, except that would not permit
	# an array of strings
	if ($self->inline) { # expect inline data
		if (UNIVERSAL::isa($read,'can') && $read->isa("Bio::PrimarySeqI")) { # we have a Bio::*Seq*
			$read=$read->seq();
		} else { # we have something else
			if (ref($read) =~ /ARRAY/i) {
				my @ts;
				foreach my $seq (@$read) {
					if ($seq->isa("Bio::PrimarySeqI")) {
						$seq=$seq->seq();
					} else {
						next if $read=~m/[[^:alpha:]]/;
					}
					push @ts,$seq;
				}
				$self->throw("bowtie requires at least one sequence read") unless (@ts);
				if (@ts>1) {
					$read="'".join(',',@ts)."'";
				} else {
					($read)=@ts;
				} 
			} else { #must be a string... fail if non-alpha
				$self->throw("bowtie requires at least one valid sequence read") if $read=~m/[[^:alpha:]]/;
			}
		}    	    
	} else { # expect file(s) - so test whether it's/they're appropriate
	         # and make a comma-separated list of filenames
		my @ts = (ref($read) =~ /ARRAY/i) ? @$read : ($read);
		for my $file (@ts) {
			if ( -e $file ) {
				my $cmd = $self->command if $self->can('command');
				my $guesser = Bio::Tools::GuessSeqFormat->new(-file=>$file);
				for ($guesser->guess) {
					m/^fasta$/ && do {
						$cmd =~ m/^b/ && last;
						($cmd =~ m/^c/ or $self->fastq or $self->raw) and $self->throw("Fasta reads file '$file' inappropriate");
						$self->fasta(1);
						last;
					};
					m/^fastq$/ && do {
						($cmd =~ m/^[cb]/ or $self->fasta or $self->raw) and $self->throw("Fastq reads file '$file' inappropriate");
						$self->fastq(1);
						last;
					};
					m/^tab$/ && do {
						$cmd =~ m/^c/ or $self->throw("Crossbow reads file '$file' inappropriate"); # this is unrecoverable since the object has default program defined
						last;
					};
					m/^raw$/ && do {
						($cmd =~ m/^[cb]/ or $self->fasta or $self->fastq) and $self->throw("Raw reads file '$file' inappropriate");
						$self->raw(1);
						last;
					};
					do {
						$self->throw("File '$file' not a recognised bowtie input filetype");
					}
				}
			} else {
				$self->throw("Sequence read file '$file' does not exist");
			}
		}
		if (@ts>1) {
			$read="'".join(',',@ts)."'";
		} else {
			($read)=@ts;
		} 
	}

	return $read;
}

sub set_parameters {
	my ($self, @args) = @_;

	# Mutually exclusive switches/params prevented from being set to
	# avoid confusion resulting from setting incompatible switches.

	$self->throw("Input args not an even number") if (@args % 2);
	my %args = @args;


	foreach (keys %args) {
		my @added;
		my @removed;
		s/^-//;
		foreach my $conflict (@{$incompat_params{$_}}) {
			return if grep /$conflict/, @added;
			delete $args{'-'.$conflict};
			$args{'-'.$conflict} = undef if $self->{'_'.$conflict};
			push @removed, $conflict;
		}
		foreach my $requirement (@{$corequisite_switches{$_}}) {
			return if grep /$requirement/, @removed;
			$args{'-'.$requirement}=1 if $args{$_};
			push @added, $requirement;
		}
	}

	return $self->SUPER::set_parameters(%args);
}

sub version{
	my ($self) = @_;

	my $cmd = $self->command if $self->can('command');

	defined $cmd or $self->throw("No command defined - cannot determine program executable");

	my ($in, $out, $err);
	my $dum;
	$in = \$dum;
	$out = \$self->{'stdout'};
	$err = \$self->{'stderr'};

	# Get program executable
	my $exe = $self->executable;
	# Get version switch from switches, translate and dash it
	my $version_switch = $param_translation{"$command_prefixes{$cmd}|version"};
	$version_switch = $self->_dash_switch( $version_switch );

	my @ipc_args = ( $exe, $version_switch );

	eval {
		IPC::Run::run(\@ipc_args, $in, $out, $err) or
			die ("There was a problem running $exe : $!");
	};
	if ($@) {
		$self->throw("$exe call crashed: $@");
	}

	my @details = split("\n",$self->stdout);
	(my $version) = grep /$exe version [[:graph:]]*$/, @details;
	$version =~ s/version //;
	(my $addressing) = grep /-bit$/, @details;

	return $version.' '.$addressing;
}

sub available_commands { shift->available_parameters('commands') };

sub filespec { shift->available_parameters('filespec') };

1;