package Bio::SCF; use strict; use vars qw($VERSION @ISA); require DynaLoader; use Bio::SCF::Arrays; use Carp 'croak'; @ISA = qw(DynaLoader); $VERSION = '1.01'; use constant KEYS => { index => 0, A => 1, C => 2, G => 3, T => 4, bases => 5, spare1 => 6, spare1 => 7, spare1 => 8, samplesA => 11, samplesC => 12, samplesG => 13, samplesT => 14 }; use constant HEADER_FIELDS => { samples_length => 0, bases_length => 1, version => 2, sample_size => 3, code_set => 4, }; bootstrap Bio::SCF $VERSION; sub new { my $class = shift; my $file_name = shift; my $sample_hash = shift || 0; defined $file_name or die "SCF :: Unable to tie hash to undefined file name\n"; my $scf_pointer; if ($sample_hash) { $scf_pointer = $file_name; # file name became scf pointer } else { if ( defined fileno($file_name)){ $scf_pointer = get_scf_fpointer($file_name); # file_name here is file handle }else{ $scf_pointer = get_scf_pointer($file_name); # actually reads scf file into memory } } my $scf_file = { file_name => $file_name, scf_pointer => $scf_pointer, sample_hash => $sample_hash, cache => {} }; return bless $scf_file, $class; } sub TIEHASH { shift->new(@_); } sub FETCH { my $self = shift; my $key = shift; my @array; if ($self->{sample_hash}) { my $k = "sample_$key"; return $self->{cache}{$k} if exists $self->{cache}{$k}; tie @array, 'Bio::SCF::Arrays', $self->{scf_pointer}, $k; return $self->{cache}{$k} = \@array; } else { if (defined( my $header_field = HEADER_FIELDS->{$key})) { return get_from_header($self->{scf_pointer}, $header_field); } elsif ($key eq "comments") { return get_comments($self->{scf_pointer}); } elsif ($key eq 'samples') { return $self->{cache}{$key} if exists $self->{cache}{$key}; my %sample; tie %sample, 'Bio::SCF', $self->{scf_pointer}, 1; $self->{cache}{key} = \%sample; return \%sample; } elsif (exists KEYS->{$key}) { return $self->{cache}{$key} if exists $self->{cache}{$key}; tie @array, 'Bio::SCF::Arrays', $self->{scf_pointer}, $key; $self->{cache}{$key} = \@array; return \@array; } } } sub bases_length { my $self = shift; get_from_header($self->{scf_pointer},HEADER_FIELDS->{bases_length}); } sub samples_length { my $self = shift; get_from_header($self->{scf_pointer},HEADER_FIELDS->{samples_length}); } sub sample_size { my $self = shift; get_from_header($self->{scf_pointer},HEADER_FIELDS->{sample_size}); } sub code_set { my $self = shift; get_from_header($self->{scf_pointer},HEADER_FIELDS->{code_set}); } sub index { my $self = shift; my $index = shift; my $d = $self->at('index',$index); $self->set('index',$index,shift) if @_; $d; } sub sample { my $self = shift; my $base = uc shift; my $index = shift; my $d = $self->at("samples${base}",$index); $self->set("samples${base}",$index,shift) if @_; $d; } sub base { my $self = shift; my $index = shift; my $d = $self->at('bases',$index); $self->set('bases',$index,shift) if @_; $d; } sub base_score { my $self = shift; my $base = uc shift; my $index = shift; my $d = $self->at($base,$index); $self->set($base,$index,shift) if @_; $d; } sub score { my $self = shift; my $index = shift; my $base = uc $self->base($index); my $d = exists KEYS->{$base} ? $self->base_score($base,$index) : 0; $self->set($base,$index,shift) if @_; $d; } sub comments { my $self = shift; get_comments($self->{scf_pointer}); } sub at { my $self = shift; # possible keys { bases, A, C, G, T, spare1/2/3, sampleA/C/G/T } my $key = shift; my $index = shift; return get_at($self->{scf_pointer}, $index, KEYS->{$key}); } sub set { my $self = shift; # possible keys { bases, A, C, G, T, spare1/2/3, sampleA/C/G/T } my $key = shift; my $index = shift; my $value = shift or die "Bio::SCF::set(...) value not defined\n"; if ( $key eq "bases" ){ set_base_at($self->{scf_pointer}, $index, KEYS->{$key}, $value); }else{ set_at($self->{scf_pointer}, $index, KEYS->{$key}, $value); } } sub write { my $self = shift; my $file_name = shift || $self->{file_name}; return scf_write($self->{scf_pointer}, $file_name); } sub fwrite { my $self = shift; my $file_handle = shift || die "Bio::SCF::fwrite(...) : file handle is not defined\n"; return scf_fwrite($self->{scf_pointer}, $file_handle); } sub STORE { my $self = shift; my $key = shift; my $value = shift; SWITCH: { $key eq "comments" && do { set_comments($self->{scf_pointer}, $value); last SWITCH; }; die "Bio::SCF::STORE field $key doesn't exist or not allowed to be modified\n"; } } sub FIRSTKEY { my $self = shift; my $a = keys %{KEYS()}; each %{KEYS()} } sub NEXTKEY { my $self = shift; each %{KEYS()}; } sub CLEAR { croak "The Bio::SCF module does not support this operation"; } sub DELETE { croak "The Bio::SCF module does not support this operation"; } sub DESTROY { my $self = shift; Bio::SCF::scf_free($self->{scf_pointer}) unless $self->{sample_hash}; } # Autoload methods go after =cut, and are processed by the autosplit program. 1; __END__ =head1 NAME Bio::SCF - Perl extension for reading and writting SCF sequence files =head1 SYNOPSIS use Bio::SCF; # tied interface tie %hash,'Bio::SCF','my_scf_file.scf'; my $sequence_length = $hash{bases_length}; my $chromatogram_sample_length = $hash{samples_length}; my $third_base = $hash{bases}[2]; my $quality_score = $hash{$third_base}[2]; my $sample_A_at_time_1400 = $hash{samples}{A}[1400]; # change the third base and write out new file $hash{bases}[2] = 'C'; tied (%hash)->write('new.scf'); # object-oriented interface my $scf = Bio::SCF->new('my_scf_file.scf'); my $sequence_length = $scf->bases_length; my $chromatogram_sample_length = $scf->samples_length; my $third_base = $scf->bases(2); my $quality_score = $scf->score(2); my $sample_A_at_time_1400 = $scf->sample('A',1400); # change the third base and write out new file $scf->bases(2,'C'); $scf->write('new.scf'); =head1 DESCRIPTION This module provides a perl interface to SCF DNA sequencing files. It has both tied hash and an object-oriented interfaces. It provides the ability to read fields from SCF files and limited ability to modify them and write them back. =head2 Tied Methods =over 4 =item $obj = tie %hash,'Bio::SCF',$filename_or_handle Tie the Bio::SCF module to a filename or filehandle. If successful, tie() will return the object. =item $value = $hash{'key'} Fetch a field from the SCF file. Valid keys are as follows: Key Value --- ----- bases_length Number of called bases in the sequence (read-only) samples_length Number of samples in the file (read-only) version SCF version (read-only) code_set Code set used to code bases (read-only) comments Structured comments (read-only) bases Array reference to a list of the base calls index Array reference to a list of the sample position for each of the base calls (e.g. the position of the base calling peak) A An array reference that can be used to determine the probability that the base in position $i is an "A". G An array reference that can be used to determine the probability that the base in position $i is a "G". C An array reference that can be used to determine the probability that the base in position $i is a "C". T An array reference that can be used to determine the probability that the base in position $i is a "T". samples A hash reference with keys "A", "C", "G" and "T". The value of each hash is an array reference to the list of intensity values for each sample. To get the length of the called sequence: $scf{bases_length} To get the value of the called sequence at position 3: $scf{bases}[3] To get the sample position at which base 3 was called: $scf{index}[3] To get the value of the "C" curve under base 3: $scf{samples}{C}[$scf{index}[3]] To get the probability that base 3 is a "C": $scf{C}[3] To print out the chromatogram as a four-column list: my $samples = $scf{samples}; for (my $i = 0; $i<$scf{samples_length}; $i++) { print join "\t",$samples->{C}[$i],$samples->{G}[$i], $samples->{A}[$i],$samples->{T}[$i],"\n"; } =item $scf{bases}[$index] = $new_value The base call probability scores, base call values, base call positions, and sample values are all read/write, so that you can change them: $samples->{C}[500] = 0; =item each %scf Will return keys and values for the tied object. =item delete $scf{$key} =item %scf = () These operations are not supported and will return a run-time error =back =head2 Object Methods =over 4 =item $scf = Bio::SCF->new($scf_file_or_filehandle) Create a new Bio::SCF object. The single argument is the name of a file or a previously-opened filehandle. If successful, new() returns the Bio::SCF object. =item $length = $scf->bases_length Return the length of the called sequence. =item $samples = $scf->samples_length Return the length of the list of chromatogram samples in the file. There are four sample series, one for each base. =item $sample_size = $scf->sample_size Returns the size of each sample (bytes). =item $code_set = $scf->code_set Return the code set used for base calling. =item $base = $scf->base($base_no [,$new_base]) Get the base call at the indicated position. If a new value is provided, will change the base call to the indicated base. =item $index = $scf->index($base_no [,$new_index]) Translates the indicated base position into the sample index for that called base. Here is how to fetch the intensity values at base number 5: my $sample_index = $scf->index(5); my ($g,$a,$t,$c) = map { $scf->sample($_,$sample_index) } qw(G A T C); If you provide a new value for the sample index, it will be updated. =item $base_score = $scf->base_score($base,$base_no [,$new_score]) Get the probability that the indicated base occurs at position $base_no. Here is how to fetch the probabilities for the four bases at base position 5: my ($g,$a,$t,$c) = map { $scf->base_score($_,5) } qw(G A T C); If you provide a new value for the base probability score, it will be updated. =item $score = $scf->score($base_no) Get the quality score for the called base at the indicated position. =item $intensity = $scf->sample($base,$sample_index [,$new_value]) Get the intensity value for the channel corresponding to the indicated base at the indicated sample index. You may update the intensity by providing a new value. =item $scf->write('file_path') Write the updated SCF file to the indicated file path. =item $scf->fwrite($file_handle) Write the updated SCF file to the indicated filehandle. The file must previously have been opened for writing. The filehandle is actually reopened in append mode, so you can call fwrite() multiple times and interperse your own record separators. =back =head1 EXAMPLES Reading information from a preexisting file: tie %scf, 'Bio::SCF', "data.scf"; print "Base calls:\n"; for ( my $i=0; $i<$scf{bases}; $i++ ){ print "$scf{base}[$i] "; } print "\n"; print "Intensity values for the A curve\n"; for ( my $i=0; $i<$scf{samples}; $i++ ){ print "$scf{sample}{A}[$i]; } print "\n"; Another example, where we set all bases to "A", indexes to 10 and write the file back: my $obj = tie %scf,'Bio::SCF','data.scf'; for (0...@{$scf{bases}}-1){ $scf{base}[$_] = "A"; $obj->set('index', $_, 10); } $obj->write('data.scf'); =head1 AUTHOR Dmitri Priimak, priimak@cshl.org (1999) with some cleanups by Lincoln Stein, lstein@cshl.edu (2006) =head1 SEE ALSO perl(1). =cut