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#!/usr/local/bin/perl
package main;
our $SEE;
package CDNA::PASA_alignment_assembler;
=head1 NAME
CDNA::PASA_alignment_assembler
=cut
=head1 DESCRIPTION
This module is used to assemble compatible cDNA alignments. The algorithm is as follows:
must describe this here.
=cut
use strict;
use CDNA::CDNA_alignment;
use Data::Dumper;
use Carp;
## File scoped globals:
my $DELIMETER = "$;,";
our $FUZZLENGTH = 20;
=item new()
=over 4
B<Description:> instantiates a new cDNA assembler obj.
B<Parameters:> none
B<Returns:> $obj_href
$obj_href is the object reference newly instantiated by this new method.
=back
=cut
sub new {
my $package_name = shift;
my $self = {};
bless ($self, $package_name);
$self->_init(@_);
return ($self);
}
sub _init {
my $self = shift;
$self->{incoming_alignments} = []; #these are the alignments to be assembled.
$self->{assemblies} = []; #contains list of all singletons and assemblies.
$self->{fuzzlength} = $FUZZLENGTH; #default setting.
my $pasa_bin = `which pasa`;
$pasa_bin =~ s/\s//g;
unless (-x $pasa_bin) {
confess "Error, pasa binary [$pasa_bin] isn't executable or couldn't be found.";
}
$self->{pasa_bin} = $pasa_bin;
}
=item assemble_alignments()
=over 4
B<DESCRIPTION:> assembles a series of cDNA aligmnments into one or more cDNA assemblies using a directed acyclic graph.
B<Parameters:> @alignments
@alignments is an array of CDNA::CDNA_alignment objects
B<Returns:> none.
=back
=cut
sub assemble_alignments {
my $self = shift;
my @alignments = @_;
@alignments = sort {$a->{lend}<=>$b->{lend}} @alignments; #keep in order of lend across genomic sequence to provide a layout.
$self->{incoming_alignments} = [@alignments];
my %accs;
my %spliced_orientations; # track so can set later in each assembly based on content.
my %aligned_orientations;
my %FL_accs;
foreach my $alignment (@alignments) {
my $acc = $alignment->get_acc();
$accs{$acc} = 0;
my $spliced_orient = $alignment->get_spliced_orientation();
$spliced_orientations{$acc} = $spliced_orient;
my $aligned_orient = $alignment->get_orientation();
$aligned_orientations{$acc} = $aligned_orient;
$FL_accs{$acc} = $alignment->is_fli();
}
$self->{accs_in_assemblies} = \%accs;
my $num_alignments = $#alignments + 1;
$self->force_flexorient('+');
my @assemblies = $self->pasa_cpp_assemblies('+');
$self->force_flexorient('-');
push (@assemblies, $self->pasa_cpp_assemblies('-'));
# sort in order of decreasing score
@assemblies = reverse sort {$a->{num_contained_aligns}<=>$b->{num_contained_aligns}} @assemblies;
if ($SEE) {
print "\n\nScore summary for all assemblies (nr set unchosen):\n\n";
foreach my $assembly (@assemblies) {
print "score: " . $assembly->{num_contained_aligns} . ", " . $assembly->toToken . "\n";
}
print "\n\n";
}
my @report_assemblies;
my $still_missing = 1;
foreach my $assembly (@assemblies) {
print "\nAnalyzing assembly.\n" if $SEE;
my $contained_aligns_aref = $assembly->{contained_aligns};
my $have_unseen = 0;
my $spliced_orient = '?';
my $is_fli = 0;
my %aligned_orient_counts;
foreach my $acc (@$contained_aligns_aref) {
print "got: $acc\n" if $SEE;
# check to see if we've encountered this one yet.
unless ($accs{$acc}) {
$have_unseen = 1;
}
$accs{$acc} = 1;
my $curr_spliced_orient = $spliced_orientations{$acc};
print "curr_spliced_orient: $curr_spliced_orient\n" if $SEE;
if ($curr_spliced_orient ne '?') {
if ($spliced_orient ne '?' && $spliced_orient ne $curr_spliced_orient) {
## cannot have conflicting spliced orientations in the assembly: corruption.
die "Fatal: conflicting spliced orientations in current PASA assembly results (spliced_orient: $spliced_orient, $acc has $curr_spliced_orient).\n";
}
$spliced_orient = $curr_spliced_orient; ## retain original spliced orientation.
}
if ( (!$is_fli) && $FL_accs{$acc}) {
$is_fli = 1;
}
## track aligned orientation
$aligned_orient_counts{ $aligned_orientations{$acc} } ++;
}
## set assembly orientation
$assembly->set_spliced_orientation($spliced_orient);
if ($spliced_orient eq '?') {
## set aligned orientation based on a majority vote
my @orients = reverse sort { $aligned_orient_counts{$a} <=> $aligned_orient_counts{$b} } keys %aligned_orient_counts;
my $winning_aligned_orient = shift @orients;
$assembly->set_orientation($winning_aligned_orient);
}
else {
# got spliced orientation, use it for aligned orientation too.
$assembly->set_orientation($spliced_orient);
}
$assembly->set_fli_status($is_fli);
if ($have_unseen) {
print $assembly->toToken . "\n" if $SEE;
push (@report_assemblies, $assembly);
}
$still_missing = 0;
foreach my $key (keys %accs) {
if (! $accs{$key}) {
$still_missing = 1;
print "still missing: $key\n" if $SEE;
}
}
if (! $still_missing) {
last; #got them all.
}
}
$self->{assemblies} = \@report_assemblies;
if ($still_missing) {
die "Didn't obtain assemblies describing all maximal assemblies.\n";
}
}
sub pasa_cpp_assemblies {
my $self = shift;
my $forced_orient = shift;
my $prev_input_sep = $/;
$/ = "\n";
my $sequence_ref;
my $incoming_alignments_aref = $self->{incoming_alignments};
# create input file for pasa-cpp implementation:
my $pasa_input = "pasa.$$.$forced_orient.in";
my $pasa_output = "pasa.$$.$forced_orient.out";
my @assemblies;
open (TMPIN, ">$pasa_input") or die "Can't open file $pasa_input";
foreach my $alignment (@$incoming_alignments_aref) {
my $acc = $alignment->get_acc();
## commas not allowed in acc name:
if ($acc =~ /,/) {
die "ERROR, $acc accession contains comma(s). This is not allowed.\n";
}
my $orient = $alignment->{fixed_orient};
my $alignText = "$acc,$orient";
unless (ref $sequence_ref) {
$sequence_ref = $alignment->get_genomic_seq_ref();
}
foreach my $seg ($alignment->get_alignment_segments()) {
my ($lend, $rend) = $seg->get_coords();
$alignText .= ",$lend-$rend";
}
print TMPIN $alignText . "\n";
}
close TMPIN;
if ($SEE) {
print "PASA_INPUT ($forced_orient):\n====\n";
system "cat $pasa_input";
print "====\n";
}
my $cmd = $self->{pasa_bin} . " $pasa_input > $pasa_output";
my $ret = system $cmd;
if ($ret) {
system "mv $pasa_input pasa_killer.input";
print STDERR "PASA died on input file. See pasa_killer.input";
die;
} else {
# process the output.
open (TMPOUT, $pasa_output) or die "Can't open $pasa_output";
while (<TMPOUT>) {
if (/assembly:\s\(\d+\)\scontains\salignments:\s\[([^\]]+)\]\swith\sstructure\s\[([^\]]+)\]/) {
print "Extracting assembly output: $_" if $SEE;
my $acclist = $1;
my $aligndescript = $2;
my @x = split (/,/, $aligndescript);
shift @x;
my $orient = shift @x;
my @alignSegs;
my $length = 0;
foreach my $coordset (@x) {
my ($lend, $rend) = sort {$a<=>$b} split (/-/, $coordset);
my $seg = new CDNA::Alignment_segment($lend, $rend);
$length += ($rend - $lend) + 1;
push (@alignSegs, $seg);
}
my $assembly = new CDNA::CDNA_alignment($length, \@alignSegs, $sequence_ref);
my @accs = split (/,/, $acclist);
my $num_accs = $#accs + 1;
$assembly->{contained_aligns} = [@accs];
$assembly->{num_contained_aligns} = $num_accs;
$acclist =~ s/,/\//g; #convert list of accessions into a new accession representing a single entry (unity)
# if we keep the commas, use of this assembly in future PASA runs will break the assembler
# because of the input file requirements.
$assembly->set_acc($acclist);
push (@assemblies, $assembly);
}
}
close TMPOUT;
if ($SEE) {
print "PASA_OUTPUT ($forced_orient):\n####\n";
system "cat $pasa_output";
print "####\n";
}
unlink ($pasa_input, $pasa_output) unless $SEE;
}
$/ = $prev_input_sep; ## restore
return (@assemblies);
}
sub force_flexorient {
my $self = shift;
my $orient = shift;
my $alignments_aref = $self->{incoming_alignments};
my $num_alignments = $#{$alignments_aref} + 1;
## Fix orientations of fli and multi-segment alignments:
for (my $i = 0; $i < $num_alignments; $i++) {
my $alignment = $alignments_aref->[$i];
my $num_segments = $alignment->get_num_segments();
my $spliced_orientation = $alignment->get_spliced_orientation();
if ($spliced_orientation =~ /^[+-]$/) { # set specifically
$alignment->{fixed_orient} = $spliced_orientation; ## adding tag, using only in this module.
} else {
print "Setting $i to flex orient: $orient.\n" if $SEE;
$alignment->{fixed_orient} = $orient;
}
}
}
sub unique_entries {
my @x = @_;
my %z;
foreach my $y (@x) {
$z{$y}=1;
}
return (keys %z);
}
=item get_assemblies()
=over 4
B<Description:> returns all the alignment assemblies resulting from the assembly procedure.
B<Parameters:> none.
B<Returns:> @assemblies
@assemblies is an array of CDNA::CDNA_alignment objects.
use the get_acc() method of the alignment object to retrieve all the accessions of the cDNAs that were merged into the assembly.
=back
=cut
sub get_assemblies {
my $self = shift;
return (@{$self->{assemblies}});
}
=item toAlignIllustration()
=over 4
B<Description:> illustrates the individual cDNAs to be assembled along with the final products.
B<Parameters:> $max_line_chars(optional)
$max_line_chars is an integer representing the maximum number of characters in a single line of output to the terminal. The default is 100.
B<Returns:> $alignment_illustration_text
$alignment_illustration_text is a string containing a paragraph of text which illustrates the alignments and assemblies. An example is below:
---> <--> <-----> <---> <---------------- (+)gi|1199466
---> <--> <-----> <---> <------------ (+)gi|1209702
----> <--> <---- (+)AV827070
----> <--> <--- (+)AV828861
----> <--> <--- (+)AV830936
---> <--> <- (+)H36350
ASSEMBLIES: (1)
----> <--> <-----> <---> <---------------- (+) gi|1199466, gi|1209702, AV827070, AV828861, AV830936, H36350
=back
=cut
;
sub toAlignIllustration () {
my $self = shift;
my $max_line_chars = shift;
$max_line_chars = ($max_line_chars) ? $max_line_chars : 100; #if not specified, 100 chars / line is default.
## Get minimum coord for relative positioning.
my @coords;
my @alignments = @{$self->{incoming_alignments}};
foreach my $alignment (@alignments) {
my @c = $alignment->get_coords();
push (@coords, @c);
}
@coords = sort {$a<=>$b} @coords;
print "coords: @coords\n" if $::SEE;
my $min_coord = shift @coords;
my $max_coord = pop @coords;
my $rel_max = $max_coord - $min_coord;
my $alignment_text = "";
## print each alignment followed by assemblies:
my $num_alignments = $#alignments + 1;
$alignment_text .= "Individual Alignments: ($num_alignments)\n";
my $i = 0;
foreach my $alignment (@alignments) {
$alignment_text .= (sprintf ("%3d ", $i)) . $alignment->toAlignIllustration($min_coord, $rel_max, $max_line_chars) . "\n";
$i++;
}
my @assemblies = @{$self->{assemblies}};
my $num_assemblies = $#assemblies + 1;
$alignment_text .= "\n\nASSEMBLIES: ($num_assemblies)\n";
foreach my $assembly (@assemblies) {
$alignment_text .= " " . $assembly->toAlignIllustration($min_coord, $rel_max, $max_line_chars) . "\n";
}
return ($alignment_text);
}
1;
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