#
# BioPerl module for Bio::Assembly::Tools::ContigSpectrum
#
# Copyright by Florent Angly
#
# You may distribute this module under the same terms as Perl itself
#
# POD documentation - main docs before the code
=head1 NAME
Bio::Assembly::Tools::ContigSpectrum - create and manipulate contig spectra
=head1 SYNOPSIS
# Simple contig spectrum creation
my $csp1 = Bio::Assembly::Tools::ContigSpectrum->new(
-id => 'csp1',
-spectrum => { 1 => 10,
2 => 2,
3 => 1 } );
# ...or another way to create a simple contig spectrum
my $csp2 = Bio::Assembly::Tools::ContigSpectrum->new;
$csp2->id('csp2');
$csp2->spectrum({ 1 => 20, 2 => 1, 4 => 1 });
# Get some information
print "This is contig spectrum ".$csp->id."\n";
print "It contains ".$csp->nof_seq." sequences\n";
print "The largest contig has ".$csp->max_size." sequences\n";
print "The spectrum is: ".$csp->to_string($csp->spectrum)."\n";
# Let's add the contig spectra
my $summed_csp = Bio::Assembly::Tools::ContigSpectrum->new;
$summed_csp->add($csp1);
$summed_csp->add($csp2);
print "The summed contig spectrum is ".$summed_csp->to_string."\n";
# Make an average
my $avg_csp = Bio::Assembly::Tools::ContigSpectrum->new;
$avg_csp = $avg_csp->average([$csp1, $csp2]);
print "The average contig spectrum is ".$avg_csp->to_string."\n";
# Get a contig spectrum from an assembly
my $from_assembly = Bio::Assembly::Tools::ContigSpectrum->new(
-assembly => $assembly_object,
-eff_asm_params => 1);
print "The contig spectrum from assembly is ".$from_assembly->to_string."\n";
# Report advanced information (possible because eff_asm_params = 1)
print "Average sequence length: ".$from_assembly->avg_seq_len." bp\n";
print "Minimum overlap length: ".$from_assembly->min_overlap." bp\n";
print "Average overlap length: ".$from_assembly->avg_overlap." bp\n";
print "Minimum overlap match: ".$from_assembly->min_identity." %\n";
print "Average overlap match: ".$from_assembly->avg_identity." %\n";
# Assuming the assembly object contains sequences from several different
# metagenomes, we have a mixed contig spectrum from which a cross contig
# spectrum and dissolved contig spectra can be obtained
my $mixed_csp = $from_assembly;
# Calculate a dissolved contig spectrum
my $meta1_dissolved = Bio::Assembly::Tools::ContigSpectrum->new(
-dissolve => [$mixed_csp, 'metagenome1'] );
my $meta2_dissolved = Bio::Assembly::Tools::ContigSpectrum->new(
-dissolve => [$mixed_csp, 'metagenome2'] );
print "The dissolved contig spectra are:\n".
$meta1_dissolved->to_string."\n".
$meta2_dissolved->to_string."\n";
# Determine a cross contig spectrum
my $cross_csp = Bio::Assembly::Tools::ContigSpectrum->new(
-cross => $mixed_csp );
print "The cross contig spectrum is ".$cross_csp->to_string."\n";
# Score a contig spectrum (the more abundant the contigs and the larger their
# size, the larger the score)
my $csp_score = $csp->score( $csp->nof_seq );
=head1 DESCRIPTION
The Bio::Assembly::Tools::ContigSpectrum Perl module enables to
manually create contig spectra, import them from assemblies,
manipulate them, transform between different types of contig spectra
and output them.
Bio::Assembly::Tools::ContigSpectrum is a module to create, manipulate
and output contig spectra, assembly-derived data used in metagenomics
(community genomics) for diversity estimation.
=head2 Background
A contig spectrum is the count of the number of contigs of different
size in an assembly. For example, the contig spectrum [100 5 1 0 0
...] means that there were 100 singlets (1-contigs), 5 contigs of 2
sequences (2-contigs), 1 contig of 3 sequences (3-contig) and no
larger contigs.
An assembly can be produced from a mixture of sequences from different
metagenomes. The contig obtained from this assembly is a mixed contig
spectrum. The contribution of each metagenome in this mixed contig
spectrum can be obtained by determining a dissolved contig spectrum.
Finally, based on a mixed contig spectrum, a cross contig spectrum can
be determined. In a cross contig spectrum, only contigs containing
sequences from different metagenomes are kept; "pure" contigs are
excluded. Additionally, the total number of singletons (1-contigs)
from each region that assembles with any fragments from other regions
is the number of 1-contigs in the cross contig spectrum.
=head2 Implementation
The simplest representation of a contig spectrum is as a hash
representation where the key is the contig size (number of sequences
making up the contig) and the value the number of contigs of this
size.
In fact, it is useful to have more information associated with the
contig spectrum, hence the Bio::Assembly::Tools::ContigSpectrum module
implements an object containing a contig spectrum hash and additional
information. The get/set methods to access them are:
id contig spectrum ID
nof_rep number of repetitions (assemblies) used
max_size size of (number of sequences in) the largest contig
spectrum hash representation of a contig spectrum
nof_seq number of sequences
avg_seq_len average sequence length
eff_asm_params reports effective assembly parameters
nof_overlaps number of overlaps (needs eff_asm_params)
min_overlap minimum overlap length in a contig (needs eff_asm_params)
min_identity minimum sequence identity percentage (needs eff_asm_params)
avg_overlap average overlap length (needs eff_asm_params)
avg_identity average overlap identity percentage (needs eff_asm_params)
Operations on the contig spectra:
to_string create a string representation of the spectrum
spectrum import a hash contig spectrum
assembly determine a contig spectrum from an assembly, contig or singlet
dissolve calculate a dissolved contig spectrum (depends on assembly)
cross produce a cross contig spectrum (depends on assembly)
add add a contig spectrum to an existing one
average make an average of several contig spectra
score score a contig spectrum: the higher the number of contigs
and the larger their size, the higher the score.
When using operations that rely on knowing "where" (from what
metagenomes) a sequence came from (i.e. when creating a dissolved or
cross contig spectrum), make sure that the sequences used for the
assembly have a name header, e.g. E<gt>metagenome1|seq1,
E<gt>metagenome2|seq1, ...
Note: The following operations require the C<Graph::Undirected> module:
eff_asm_params, cross, dissolve
=head1 FEEDBACK
=head2 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 lists Your participation is much appreciated.
bioperl-l@bioperl.org - General discussion
http://bioperl.org/wiki/Mailing_lists - About the mailing lists
=head2 Support
Please direct usage questions or support issues to the mailing list:
I<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.
=head2 Reporting Bugs
Report bugs to the BioPerl bug tracking system to help us keep track
the bugs and their resolution. Bug reports can be submitted via email
or the web:
bioperl-bugs@bio.perl.org
https://github.com/bioperl/bioperl-live/issues
=head1 AUTHOR - Florent E Angly
Email florent_dot_angly_at_gmail_dot_com
=head1 APPENDIX
The rest of the documentation details each of the object
methods. Internal methods are usually preceded with a "_".
=cut
package Bio::Assembly::Tools::ContigSpectrum;
use strict;
use Bio::Root::Root;
use Bio::Assembly::Scaffold;
use Bio::SimpleAlign;
use Bio::LocatableSeq;
use base 'Bio::Root::Root';
=head2 new
Title : new
Usage : my $csp = Bio::Assembly::Tools::ContigSpectrum->new();
or
my $csp = Bio::Assembly::Tools::ContigSpectrum->new(
-id => 'some_name',
-spectrum => { 1 => 90 , 2 => 3 , 4 => 1 },
);
or
my $csp = Bio::Assembly::Tools::ContigSpectrum->new(
-assembly => $assembly_obj
);
Function: create a new contig spectrum object
Returns : reference to a contig spectrum object
Args : none
=cut
sub new {
my ($class, @args) = @_;
my $self = $class->SUPER::new(@args);
my ( $id, $nof_seq, $nof_rep, $max_size, $nof_overlaps, $min_overlap,
$min_identity, $avg_overlap, $avg_identity, $avg_seq_len, $spectrum,
$assembly, $eff_asm_params, $dissolve, $cross) = $self->_rearrange(
[qw(ID NOF_SEQ NOF_REP MAX_SIZE NOF_OVERLAPS MIN_OVERLAP MIN_IDENTITY
AVG_OVERLAP AVG_IDENTITY AVG_SEQ_LEN SPECTRUM ASSEMBLY EFF_ASM_PARAMS
DISSOLVE CROSS)], @args );
# First set up some defauts
$self->{'_id'} = 'NoName';
$self->{'_nof_seq'} = 0;
$self->{'_nof_rep'} = 0;
$self->{'_max_size'} = 0;
$self->{'_nof_overlaps'} = 0;
$self->{'_min_overlap'} = undef;
$self->{'_min_identity'} = undef;
$self->{'_avg_overlap'} = 0;
$self->{'_avg_identity'} = 0;
$self->{'_avg_seq_len'} = 0;
$self->{'_eff_asm_params'} = 0;
$self->{'_spectrum'} = {1 => 0}; # contig spectrum hash representation
$self->{'_assembly'} = []; # list of assembly, contigs and singlet objects
# Then, according to user desires, override defaults
$self->{'_id'} = $id if (defined $id);
$self->{'_nof_seq'} = $nof_seq if (defined $nof_seq);
$self->{'_nof_rep'} = $nof_rep if (defined $nof_rep);
$self->{'_max_size'} = $max_size if (defined $max_size);
$self->{'_nof_overlaps'} = $nof_overlaps if (defined $nof_overlaps);
$self->{'_min_overlap'} = $min_overlap if (defined $min_overlap);
$self->{'_avg_overlap'} = $avg_overlap if (defined $avg_overlap);
$self->{'_min_identity'} = $min_identity if (defined $min_identity);
$self->{'_avg_identity'} = $avg_identity if (defined $avg_identity);
$self->{'_avg_seq_len'} = $avg_seq_len if (defined $avg_seq_len);
$self->{'_eff_asm_params'} = $eff_asm_params if (defined $eff_asm_params);
# Finally get stuff that can be obtained in an automated way
$self->_import_spectrum($spectrum) if defined($spectrum);
$self->_import_assembly($assembly) if defined($assembly);
$self->_import_cross_csp($cross) if defined($cross);
if (defined($dissolve)) {
my ($mixed_csp, $header) = (@$dissolve[0], @$dissolve[1]);
$self->_import_dissolved_csp($mixed_csp, $header);
}
return $self;
}
=head2 id
Title : id
Usage : $csp->id
Function: get/set contig spectrum id
Returns : string
Args : string [optional]
=cut
sub id {
my ($self, $id) = @_;
if (defined $id) {
$self->{'_id'} = $id;
}
$id = $self->{'_id'};
return $id;
}
=head2 nof_seq
Title : nof_seq
Usage : $csp->nof_seq
Function: get/set the number of sequences making up the contig spectrum
Returns : integer
Args : integer [optional]
=cut
sub nof_seq {
my ($self, $nof_seq) = @_;
if (defined $nof_seq) {
$self->throw("The number of sequences must be strictly positive. Got ".
"'$nof_seq'") if $nof_seq < 1;
$self->{'_nof_seq'} = $nof_seq;
}
$nof_seq = $self->{'_nof_seq'};
return $nof_seq;
}
=head2 nof_rep
Title : nof_rep
Usage : $csp->nof_rep
Function: Get/Set the number of repetitions (assemblies) used to create the
contig spectrum
Returns : integer
Args : integer [optional]
=cut
sub nof_rep {
my ($self, $nof_rep) = @_;
if (defined $nof_rep) {
$self->throw("The number of repetitions must be strictly positive. Got ".
"'$nof_rep'") if $nof_rep < 1;
$self->{'_nof_rep'} = $nof_rep;
}
$nof_rep = $self->{'_nof_rep'};
return $nof_rep;
}
=head2 max_size
Title : max_size
Usage : $csp->max_size
Function: get/set the size of (number of sequences in) the largest contig
Returns : integer
Args : integer [optional]
=cut
sub max_size {
my ($self, $max_size) = @_;
if (defined $max_size) {
$self->throw("The contig maximum size must be strictly positive. Got ".
"'$max_size'") if $max_size < 1;
$self->{'_max_size'} = $max_size;
}
$max_size = $self->{'_max_size'};
return $max_size;
}
=head2 nof_overlaps
Title : nof_overlaps
Usage : $csp->nof_overlaps
Function: Get/Set the number of overlaps in the assembly.
Returns : integer
Args : integer [optional]
=cut
sub nof_overlaps {
my ($self, $nof_overlaps) = @_;
if (defined $nof_overlaps) {
$self->throw("The number of overlaps must be strictly positive. Got ".
"'$nof_overlaps'") if $nof_overlaps < 1;
$self->{'_nof_overlaps'} = $nof_overlaps;
}
$nof_overlaps = $self->{'_nof_overlaps'};
return $nof_overlaps;
}
=head2 min_overlap
Title : min_overlap
Usage : $csp->min_overlap
Function: get/set the assembly minimum overlap length
Returns : integer
Args : integer [optional]
=cut
sub min_overlap {
my ($self, $min_overlap) = @_;
if (defined $min_overlap) {
$self->throw("The minimum of overlap length must be strictly positive. Got".
" '$min_overlap'") if $min_overlap < 1;
$self->{'_min_overlap'} = $min_overlap;
}
$min_overlap = $self->{'_min_overlap'};
return $min_overlap;
}
=head2 avg_overlap
Title : avg_overlap
Usage : $csp->avg_overlap
Function: get/set the assembly average overlap length
Returns : decimal
Args : decimal [optional]
=cut
sub avg_overlap {
my ($self, $avg_overlap) = @_;
if (defined $avg_overlap) {
$self->throw("The average overlap length must be strictly positive. Got ".
"'$avg_overlap'") if $avg_overlap < 1;
$self->{'_avg_overlap'} = $avg_overlap;
}
$avg_overlap = $self->{'_avg_overlap'};
return $avg_overlap;
}
=head2 min_identity
Title : min_identity
Usage : $csp->min_identity
Function: get/set the assembly minimum overlap identity percent
Returns : 0 < decimal < 100
Args : 0 < decimal < 100 [optional]
=cut
sub min_identity {
my ($self, $min_identity) = @_;
if (defined $min_identity) {
$self->throw("The minimum overlap percent identity must be strictly ".
"positive. Got '$min_identity'") if $min_identity < 1;
$self->{'_min_identity'} = $min_identity;
}
$min_identity = $self->{'_min_identity'};
return $min_identity;
}
=head2 avg_identity
Title : avg_identity
Usage : $csp->avg_identity
Function: get/set the assembly average overlap identity percent
Returns : 0 < decimal < 100
Args : 0 < decimal < 100 [optional]
=cut
sub avg_identity {
my ($self, $avg_identity) = @_;
if (defined $avg_identity) {
$self->throw("The average overlap percent identity must be strictly ".
"positive. Got '$avg_identity'") if $avg_identity < 1;
$self->{'_avg_identity'} = $avg_identity;
}
$avg_identity = $self->{'_avg_identity'};
return $avg_identity;
}
=head2 avg_seq_len
Title : avg_seq_len
Usage : $csp->avg_seq_len
Function: get/set the assembly average sequence length
Returns : avg_seq_len
Args : real [optional]
=cut
sub avg_seq_len {
my ($self, $avg_seq_len) = @_;
if (defined $avg_seq_len) {
$self->throw("The average sequence length must be strictly positive. Got ".
"'$avg_seq_len'") if $avg_seq_len < 1;
$self->{'_avg_seq_len'} = $avg_seq_len;
}
$avg_seq_len = $self->{'_avg_seq_len'};
return $avg_seq_len;
}
=head2 eff_asm_params
Title : eff_asm_params
Usage : $csp->eff_asm_params(1)
Function: Get/set the effective assembly parameters option. It defines if the
effective assembly parameters should be determined when a contig
spectrum based or derived from an assembly is calculated. The
effective assembly parameters include avg_seq_length, nof_overlaps,
min_overlap, avg_overlap, min_identity and avg_identity.
1 = get them, 0 = don't.
Returns : integer
Args : integer [optional]
=cut
sub eff_asm_params {
my ($self, $eff_asm_params) = @_;
if (defined $eff_asm_params) {
$self->throw("eff_asm_params can only take values 0 or 1. Input value was ".
"'$eff_asm_params'") unless $eff_asm_params == 0 || $eff_asm_params == 1;
$self->{'_eff_asm_params'} = $eff_asm_params;
}
$eff_asm_params = $self->{'_eff_asm_params'};
return $eff_asm_params;
}
=head2 spectrum
Title : spectrum
Usage : my $spectrum = $csp->spectrum({1=>10, 2=>2, 3=>1});
Function: Get the current contig spectrum represented as a hash / Update a
contig spectrum object based on a contig spectrum represented as a
hash
The hash representation of a contig spectrum is as following:
key -> contig size (in number of sequences)
value -> number of contigs of this size
Returns : contig spectrum as a hash reference
Args : contig spectrum as a hash reference [optional]
=cut
sub spectrum {
my ($self, $spectrum) = @_;
if (defined $spectrum) {
$self->_import_spectrum($spectrum);
}
$spectrum = $self->{'_spectrum'};
return $spectrum;
}
=head2 assembly
Title : assembly
Usage : my @obj_list = $csp->assembly();
Function: get/set the contig spectrum object by adding an assembly, contig or
singlet object to it, or get the list of objects associated with it
Returns : arrayref of assembly, contig and singlet objects used in the contig
spectrum object (Bio::Assembly::Scaffold, Bio::Assembly::Contig and
Bio::Assembly::Singlet objects)
Args : Bio::Assembly::Scaffold, Contig or Singlet object
=cut
sub assembly {
my ($self, $assembly) = @_;
if (defined $assembly) {
$self->_import_assembly($assembly);
}
my @obj_list = @{$self->{'_assembly'}} if defined $self->{'_assembly'};
return @obj_list;
}
=head2 drop_assembly
Title : drop_assembly
Usage : $csp->drop_assembly();
Function: Remove all assembly objects associated with a contig spectrum.
Assembly objects can take a lot of memory, which can be freed by
calling this method. Don't call this method if you need the assembly
object later on, for example for creating a dissolved or cross
contig spectrum.
Returns : 1 for success
Args : none
=cut
sub drop_assembly {
my ($self) = @_;
$self->{'_assembly'} = [];
return 1;
}
=head2 dissolve
Title : dissolve
Usage : $dissolved_csp->dissolve($mixed_csp, $seq_header);
Function: Dissolve a mixed contig spectrum for the set of sequences that
contain the specified header, i.e. determine the contribution of
these sequences to the mixed contig spectrum. The mixed contig
spectrum object must have one or several assembly object(s). In
addition, min_overlap, min_identity and eff_asm_params are taken
from the mixed contig spectrum, unless they are specified manually
for the dissolved contig spectrum. The dissolved contigs underlying
the contig spectrum can be obtained by calling the assembly() method.
Returns : 1 for success
Args : Bio::Assembly::Tools::ContigSpectrum reference
sequence header string
=cut
sub dissolve {
my ($self, $mixed_csp, $seq_header) = @_;
$self->_import_dissolved_csp($mixed_csp, $seq_header);
return 1;
}
=head2 cross
Title : cross
Usage : $cross_csp->cross($mixed_csp);
Function: Calculate a cross contig_spectrum based on a mixed contig_spectrum.
The underlying cross-contigs themselves can be obtained by calling
the assembly() method.
Returns : 1 for success
Args : Bio::Assembly::Tools::ContigSpectrum reference
=cut
sub cross {
my ($self, $mixed_csp) = @_;
$self->_import_cross_csp($mixed_csp);
return 1;
}
=head2 to_string
Title : to_string
Usage : my $csp_string = $csp->to_string;
Function: Convert the contig spectrum into a string (easy to print!!).
Returns : string
Args : element separator (integer) [optional]
1 -> space-separated
2 -> tab-separated
3 -> newline-separated
=cut
sub to_string {
my ($self, $element_separator) = @_;
return 0 if $self->{'_max_size'} == 0;
$element_separator ||= 1;
if ($element_separator == 1) {
$element_separator = ' ';
} elsif ($element_separator == 2) {
$element_separator = "\t";
} elsif ($element_separator == 3) {
$element_separator = "\n";
} else {
$self->throw("Unknown separator type '$element_separator'\n");
}
my $str = '';
for (my $q = 1 ; $q <= $self->{'_max_size'} ; $q++) {
my $val = 0;
if (exists $self->{'_spectrum'}{$q}) {
$val = $self->{'_spectrum'}{$q};
}
$str .= $val.$element_separator;
}
$str =~ s/\s$//;
return $str;
}
=head2 add
Title : add
Usage : $csp->add($additional_csp);
Function: Add a contig spectrum to an existing one: sums the spectra, update
the number of sequences, number of repetitions, ...
Returns : 1 for success
Args : Bio::Assembly::Tools::ContigSpectrum object
=cut
sub add {
my ($self, $csp) = @_;
# Sanity check
if( !ref $csp || ! $csp->isa('Bio::Assembly::Tools::ContigSpectrum') ) {
$self->throw("Unable to process non Bio::Assembly::Tools::ContigSpectrum ".
"object [".ref($csp)."]");
}
# Update overlap statistics
if ( $self->{'_eff_asm_params'} > 0 ) {
# Warnings
if ( $csp->{'_eff_asm_params'} == 0 ) {
$self->warn("The parent contig spectrum needs effective assembly ".
"parameters (eff_asm_params = ".$self->{'_eff_asm_params'}.") but the ".
"child contig spectrum doesn't have them (eff_asm_params = ".
$csp->{'_eff_asm_params'}."). Skipping them...");
} elsif ( $csp->{'_eff_asm_params'} != $self->{'_eff_asm_params'} ) {
$self->warn("The parent contig spectrum needs a different method for ".
"detecting the effective assembly parameters (eff_asm_params = ".
$self->{'_eff_asm_params'}.") than the one specified for the child ".
"contig spectrum (eff_asm_params = ".$csp->{'_eff_asm_params'}."). ".
"Ignoring the differences...");
}
# Update existing stats
( $self->{'_avg_overlap'} , $self->{'_avg_identity'}, $self->{'_min_overlap'},
$self->{'_min_identity'}, $self->{'_nof_overlaps'} ) = $self->_update_overlap_stats(
$self->{'_avg_overlap'} , $self->{'_avg_identity'}, $self->{'_min_overlap'},
$self->{'_min_identity'}, $self->{'_nof_overlaps'},
$csp->{'_avg_overlap'} , $csp->{'_avg_identity'} , $csp->{'_min_overlap'},
$csp->{'_min_identity'} , $csp->{'_nof_overlaps'} );
}
# Update sequence average length (not number of sequences)
( $self->{'_avg_seq_len'} ) = $self->_update_seq_stats(
$self->{'_avg_seq_len'}, $self->{'_nof_seq'}, $csp->{'_avg_seq_len'},
$csp->{'_nof_seq'} );
# Update spectrum (and nof_seq, max_size, and increment nof_rep by 1)
$self->_import_spectrum($csp->{'_spectrum'});
# Update nof_rep
$self->{'_nof_rep'}--;
$self->{'_nof_rep'} += $csp->{'_nof_rep'};
# Update list of assembly objects used
push @{$self->{'_assembly'}}, @{$csp->{'_assembly'}}
if defined $csp->{'_assembly'};
return 1;
}
=head2 average
Title : average
Usage : my $avg_csp = $csp->average([$csp1, $csp2, $csp3]);
Function: Average one contig spectrum or the sum of several contig spectra by
the number of repetitions
Returns : Bio::Assembly::Tools::ContigSpectrum
Args : Bio::Assembly::Tools::ContigSpectrum array reference
eff_asm_params
=cut
sub average {
my ($self, $list) = @_;
# Sanity check
if ( ! ref $list || ! ref $list eq 'ARRAY') {
$self->throw("Average takes an array reference but got [".ref($list)."]");
}
# New average contig spectrum object
my $avg = Bio::Assembly::Tools::ContigSpectrum->new;
$avg->{'_eff_asm_params'} = $self->{'_eff_asm_params'};
# Cycle through contig spectra
my $tot_nof_rep = 0;
for my $csp (@$list) {
# Sanity check
if (not $csp->isa('Bio::Assembly::Tools::ContigSpectrum')) {
$csp->throw("Unable to process non Bio::Assembly::Tools::ContigSpectrum ".
"object [".ref($csp)."]");
}
# Import contig spectrum
$avg->add($csp);
}
# Average sum of contig spectra by number of repetitions
for (my $q = 1 ; $q <= $avg->{'_max_size'} ; $q++) {
$avg->{'_spectrum'}{$q} /= $avg->{'_nof_rep'}
if (defined $avg->{'_spectrum'}{$q});
}
# Average number of sequences
$avg->{'_nof_seq'} /= $avg->{'_nof_rep'};
# Average number of overlaps
$avg->{'_nof_overlaps'} /= $avg->{'_nof_rep'};
return $avg;
}
=head2 score
Title : score
Usage : my $score = $csp->score();
Function: Score a contig spectrum (or cross-contig spectrum) such that the
higher the number of contigs (or cross-contigs) and the larger their
size, the higher the score.
Let n : total number of sequences
c_q : number of contigs of size q
q : number of sequence in a contig
We define: score = n/(n-1) * (X - 1/n)
where X = sum ( c_q * q^2 ) / n**2
The score ranges from 0 (singlets only) to 1 (a single large contig)
It is possible to specify a value for the number of sequences to
assume in the contig spectrum.
Returns : contig score, or undef if there were no sequences in the contig spectrum
Args : number of total sequences to assume [optional]
=cut
sub score {
my ($self, $nof_seqs) = @_;
# Sanity check
my $n = $self->nof_seq;
return undef if ($n <= 0);
# Calculate X
my $score = 0;
my $q_max = $self->max_size;
my $spec = $self->spectrum;
for my $q ( 1 .. $q_max ) {
my $c_q = $spec->{$q};
if ( $q == 1 && $nof_seqs ) {
$c_q += $nof_seqs - $n;
$n = $nof_seqs;
}
next if not $c_q;
$score += $c_q * $q ** 2;
}
$score /= $n ** 2;
# Rescale X to obtain the score
$score = $n/($n-1) * ($score - 1/$n);
return $score;
}
=head2 _naive_assembler
Title : _naive_assembler
Usage :
Function: Reassemble the specified sequences only based on their position in
the contig. This naive assembly only verifies that the minimum
overlap length and percentage identity are respected. No actual
alignment is done
Returns : arrayref of contigs and singlets
Args : Bio::Assembly::Contig
array reference of sequence IDs to use [optional]
minimum overlap length (integer) [optional]
minimum percentage identity (integer) [optional]
=cut
sub _naive_assembler {
my ($self, $contig, $seqlist, $min_overlap, $min_identity) = @_;
# Use all reads if none was specified:
if (not defined $seqlist) {
for my $seq ($contig->each_seq) {
push @$seqlist, $seq->id;
}
}
# Sanity checks
if ( ! ref $seqlist || ! ref($seqlist) eq 'ARRAY') {
$self->throw('Expecting an array reference. Got ['.ref($seqlist)."] \n");
}
my $max = scalar @$seqlist;
$self->throw("Expecting at least 2 sequences as input for _naive_assembler")
if ($max < 2);
# Build contig graph
my %seq_hash = map { $_ => undef } (@$seqlist) if (scalar @$seqlist > 0);
my ($g, $overlaps) = $self->_contig_graph($contig, \%seq_hash, $min_overlap, $min_identity);
# Construct sub-contigs
my @contig_objs;
my $num = 1;
if (defined $g) {
for my $connected_reads ($g->connected_components) { # reads that belong in contigs
my $sub_id = $contig->id.'_'.$num;
my $sub_contig = $self->_create_subcontig($contig, $connected_reads, $sub_id);
push @contig_objs, $sub_contig;
$num++;
for my $read_id ( @$connected_reads ) {
delete $seq_hash{$read_id};
}
}
}
# Construct sub-singlets
my @singlet_objs;
for my $read_id ( keys %seq_hash ) {
my $read = $contig->get_seq_by_name($read_id);
my $sub_singlet = Bio::Assembly::Singlet->new(
-id => $contig->id.'_'.$num,
-seqref => $self->_obj_copy($read)
);
$num++;
push @singlet_objs, $sub_singlet;
}
return [@contig_objs, @singlet_objs];
}
=head2 _create_subcontig
Title : _create_subcontig
Usage :
Function: Create a subcontig from another contig
Returns : Bio::Assembly::Contig object
Args : Bio::Assembly::Contig
arrayref of the IDs of the reads to includes in the subcontig
ID to give to the subcontig
=cut
sub _create_subcontig {
my ($self, $contig, $read_ids, $sub_contig_id) = @_;
my $sub_contig = Bio::Assembly::Contig->new( -id => $sub_contig_id );
# Get min and max read coordinates
my ($min, $max) = (undef, undef);
for my $read_id ( @$read_ids ) {
my ($aln_coord) = $contig->get_features_collection->get_features_by_type("_aligned_coord:$read_id");
my $seq_start = $aln_coord->location->start;
my $seq_end = $aln_coord->location->end;
$min = $seq_start if (not defined $min) || ((defined $min) && ($seq_start < $min));
$max = $seq_end if (not defined $max) || ((defined $max) && ($seq_end > $max));
}
# Add reads to subcontig
for my $read_id (@$read_ids) {
my $read = $self->_obj_copy($contig->get_seq_by_name($read_id));
my $coord = $self->_obj_copy($contig->get_seq_coord($read));
if ($min > 1) {
# adjust read coordinates
$coord->start( $coord->start - $min + 1 );
$coord->end( $coord->end - $min + 1 );
}
$sub_contig->set_seq_coord($coord, $read);
}
# Truncate copy of original consensus to new boundaries
my $cons_seq = $contig->get_consensus_sequence;
$sub_contig->set_consensus_sequence( $self->_obj_copy($cons_seq, $min, $max) );
my $cons_qual = $contig->get_consensus_quality;
if ($cons_qual) {
$sub_contig->set_consensus_quality( $self->_obj_copy($cons_qual, $min, $max) );
}
return $sub_contig;
}
=head2 _obj_copy
Title : _obj_copy
Usage :
Function: Copy (most of) an object, and optionally truncate it
Returns : another a Bio::LocatableSeq, Bio::Seq::PrimaryQual, or
Bio::SeqFeature::Generic object
Args : a Bio::LocatableSeq, Bio::Seq::PrimaryQual, or
Bio::SeqFeature::Generic object
a start position
an end position
=cut
sub _obj_copy {
my ($self, $obj, $start, $end) = @_;
my $new;
if ($obj->isa('Bio::Seq::PrimaryQual')) {
my $qual = [@{$obj->qual}]; # copy of the quality scores
if (defined $start && defined $end && $start !=1 && $end != scalar(@$qual)) {
# Truncate the quality scores
$qual = [ splice @$qual, $start - 1, $end - $start + 1 ];
}
$new = Bio::Seq::PrimaryQual->new(
-qual => $qual,
-id => $obj->id,
);
} elsif ($obj->isa('Bio::LocatableSeq')) {
my $seq = $obj->seq;
if (defined $start && defined $end && $start != 1 && $end != length($seq)) {
# Truncate the aligned sequence
$seq = substr $seq, $start - 1, $end - $start + 1;
}
$new = Bio::LocatableSeq->new(
-seq => $seq,
-id => $obj->id,
-start => $obj->start,
-strand => $obj->strand,
-alphabet => $obj->alphabet,
);
} elsif ($obj->isa('Bio::SeqFeature::Generic')) {
$new = Bio::SeqFeature::Generic->new(
-start => $obj->start,
-end => $obj->end
);
}
return $new;
}
=head2 _new_from_assembly
Title : _new_from_assembly
Usage :
Function: Creates a new contig spectrum object based solely on the result of
an assembly, contig or singlet
Returns : Bio::Assembly::Tools::ContigSpectrum object
Args : Bio::Assembly::Scaffold, Contig or Singlet object
=cut
sub _new_from_assembly {
# Create new contig spectrum object based purely on what we can get from the
# assembly object
my ($self, $assemblyobj) = @_;
my $csp = Bio::Assembly::Tools::ContigSpectrum->new();
# 1: Set id
$csp->{'_id'} = $assemblyobj->id;
# 2: Set overlap statistics: nof_overlaps, min_overlap, avg_overlap,
# min_identity and avg_identity
$csp->{'_eff_asm_params'} = $self->{'_eff_asm_params'};
$csp->{'_min_overlap'} = $self->{'_min_overlap'};
$csp->{'_min_identity'} = $self->{'_min_identity'};
if ( $csp->{'_eff_asm_params'} > 0 ) {
( $csp->{'_avg_overlap'}, $csp->{'_avg_identity'}, $csp->{'_min_overlap'},
$csp->{'_min_identity'}, $csp->{'_nof_overlaps'} )
= $csp->_get_assembly_overlap_stats($assemblyobj);
}
# 3: Set sequence statistics: nof_seq and avg_seq_len
($csp->{'_avg_seq_len'}, $csp->{'_nof_seq'}) = $self->_get_assembly_seq_stats($assemblyobj);
### any use in using _naive_assembler here to re-assemble with specific minmum criteria?
# 4: Set the spectrum: spectrum and max_size
for my $contigobj ( $self->_get_contig_like($assemblyobj) ) {
my $size = $contigobj->num_sequences;
if (defined $csp->{'_spectrum'}{$size}) {
$csp->{'_spectrum'}{$size}++;
} else {
$csp->{'_spectrum'}{$size} = 1;
}
$csp->{'_max_size'} = $size if $size > $csp->{'_max_size'};
}
# 5: Set list of assembly objects used
push @{$csp->{'_assembly'}}, $assemblyobj;
# 6: Set number of repetitions
$csp->{'_nof_rep'} = 1;
return $csp;
}
=head2 _new_dissolved_csp
Title : _new_dissolved_csp
Usage :
Function: create a dissolved contig spectrum object
Returns : dissolved contig spectrum
Args : mixed contig spectrum
header of sequences to keep in this contig spectrum
=cut
sub _new_dissolved_csp {
my ($self, $mixed_csp, $seq_header) = @_;
# Sanity checks on the mixed contig spectrum
# min_overlap and min_identity must be specified if there are some overlaps
# in the mixed contig
if ($mixed_csp->{'_nof_overlaps'} > 0) {
unless ( defined $self->{'_min_overlap'} ||
defined $mixed_csp->{'_min_overlap'} ) {
$self->throw("min_overlap must be defined in the dissolved contig spectrum".
" or mixed contig spectrum to dissolve a contig");
}
unless ( defined $self->{'_min_identity'} ||
defined $mixed_csp->{'_min_identity'} ) {
$self->throw("min_identity must be defined in the dissolved contig spectrum".
" or mixed contig spectrum");
}
}
# there must be at least one assembly in mixed contig spectrum
if (!defined $mixed_csp->{'_assembly'} ||
scalar @{$mixed_csp->{'_assembly'}} < 1) {
$self->throw("The mixed contig spectrum must be based on at least one
assembly");
}
# New dissolved contig spectrum object
my $dissolved = Bio::Assembly::Tools::ContigSpectrum->new();
# Take attributes of the parent contig spectrum if they exist, or those of the
# mixed contig spectrum otherwise
my ($eff_asm_params, $min_overlap, $min_identity);
if ($self->{'_eff_asm_params'}) {
$eff_asm_params = $self->{'_eff_asm_params'};
} else {
$eff_asm_params = $mixed_csp->{'_eff_asm_params'};
}
if ($self->{'_min_overlap'}) {
$min_overlap = $self->{'_min_overlap'};
} else {
$min_overlap = $mixed_csp->{'_min_overlap'};
}
if ($self->{'_min_identity'}) {
$min_identity = $self->{'_min_identity'};
} else {
$min_identity = $mixed_csp->{'_min_identity'};
}
($dissolved->{'_eff_asm_params'}, $dissolved->{'_min_overlap'},
$dissolved->{'_min_identity'}) = ($eff_asm_params, $min_overlap,
$min_identity);
# Dissolve each assembly
for my $obj (@{$mixed_csp->{'_assembly'}}) {
for my $contig ( $self->_get_contig_like($obj) ) {
# Dissolve this assembly/contig/singlet for the given sequences
my $dissolved_objs = $self->_dissolve_contig( $contig, $seq_header,
$min_overlap, $min_identity );
# Add dissolved contigs to contig spectrum
for my $dissolved_obj (@$dissolved_objs) {
$dissolved->assembly($dissolved_obj);
$dissolved->{'_nof_rep'}--;
}
}
}
# Update nof_rep
$dissolved->{'_nof_rep'} += $mixed_csp->{'_nof_rep'};
return $dissolved;
}
=head2 _dissolve_contig
Title : _dissolve_contig
Usage :
Function: dissolve a contig
Returns : arrayref of contigs and singlets
Args : mixed contig spectrum
header of sequences to keep in this contig spectrum
minimum overlap
minimum identity
=cut
sub _dissolve_contig {
my ($self, $contig, $wanted_origin, $min_overlap, $min_identity) = @_;
# List of reads
my @seqs;
if ($contig->isa('Bio::Assembly::Singlet')) {
@seqs = $contig->seqref;
} elsif ($contig->isa('Bio::Assembly::Contig')) {
@seqs = $contig->each_seq;
}
# Get sequences from the desired metagenome
my @contig_seqs;
for my $seq (@seqs) {
my $seq_id = $seq->id;
# get sequence origin
next unless $self->_seq_origin($seq_id) eq $wanted_origin;
# add it to hash
push @contig_seqs, $seq_id;
}
# Update spectrum
my $size = scalar @contig_seqs;
my $objs;
if ($size == 1) {
# create a singlet and add it to list of objects
my $id = $contig_seqs[0];
my $seq = $contig->get_seq_by_name($id);
push @$objs, Bio::Assembly::Singlet->new(-id => $contig->id, -seqref => $self->_obj_copy($seq) );
} elsif ($size > 1) {
# Reassemble good sequences
my $contig_objs = $self->_naive_assembler( $contig, \@contig_seqs,
$min_overlap, $min_identity );
push @$objs, @$contig_objs;
}
return $objs;
}
=head2 _new_cross_csp
Title : _new_cross_csp
Usage :
Function: create a cross contig spectrum object
Returns : cross-contig spectrum
Args : mixed contig spectrum
=cut
sub _new_cross_csp {
my ($self, $mixed_csp) = @_;
# Sanity check on the mixed contig spectrum
# There must be at least one assembly
if (!defined $mixed_csp->{'_assembly'} ||
scalar @{$mixed_csp->{'_assembly'}} < 1) {
$self->throw("The mixed contig spectrum must be based on at least one ".
"assembly.");
}
# New dissolved contig spectrum object
my $cross = Bio::Assembly::Tools::ContigSpectrum->new();
# Take attributes from parent or from mixed contig spectrums
my ($eff_asm_params, $min_overlap, $min_identity);
if ($self->{'_eff_asm_params'}) {
$eff_asm_params = $self->{'_eff_asm_params'};
} else {
$eff_asm_params = $mixed_csp->{'_eff_asm_params'};
}
if ($self->{'_min_overlap'}) {
$min_overlap = $self->{'_min_overlap'};
} else {
$min_overlap = $mixed_csp->{'_min_overlap'};
}
if ($self->{'_min_identity'}) {
$min_identity = $self->{'_min_identity'};
} else {
$min_identity = $mixed_csp->{'_min_identity'};
}
($cross->{'_eff_asm_params'},$cross->{'_min_overlap'},$cross->{'_min_identity'})
= ($eff_asm_params, $min_overlap, $min_identity);
# Get cross contig spectrum for each assembly
for my $obj ( @{$mixed_csp->{'_assembly'}} ) {
for my $contig ( $self->_get_contig_like($obj) ) {
# Go through contigs and skip the pure ones
my ($cross_contigs, $nof_cross_singlets) = $self->_cross_contig($contig,
$min_overlap, $min_identity);
# Add cross-contig
for my $cross_contig ( @$cross_contigs ) {
$cross->assembly($cross_contig);
$cross->{'_nof_rep'}--;
}
# Add cross-singlets
$cross->{'_spectrum'}->{'1'} += $nof_cross_singlets;
}
}
# Update nof_rep
$cross->{'_nof_rep'} += $mixed_csp->{'_nof_rep'};
return $cross;
}
=head2 _cross_contig
Title : _cross_contig
Usage :
Function: calculate cross contigs
Returns : arrayref of cross-contigs
number of cross-singlets
Args : contig
minimum overlap
minimum identity
=cut
sub _cross_contig {
my ($self, $contig, $min_overlap, $min_identity) = @_;
my $nof_cross_singlets = 0;
my @cross_contigs;
# Weed out pure contigs
my %all_origins;
for my $seq ($contig->each_seq) {
my $seq_id = $seq->id;
my $seq_origin = $self->_seq_origin($seq_id);
if (not defined $seq_origin) {
$self->warn("Sequence $seq_id doesn't have any header. Skipping it...");
next;
}
if ( scalar keys %all_origins > 1 ) {
# a cross-contig spectrum
last;
}
$all_origins{$seq_origin} = undef;
}
if ( scalar keys %all_origins <= 1 ) {
# a pure contig
return \@cross_contigs, $nof_cross_singlets;
}
%all_origins = ();
# Break the cross-contigs using the specified stringency
my $test_contigs = $self->_naive_assembler($contig, undef, $min_overlap, $min_identity);
# Find cross contigs and singlets
for my $test_contig ( @$test_contigs ) {
# Find cross-contigs
my %origins;
for my $seq ($test_contig->each_seq) {
my $seq_id = $seq->id;
my $seq_origin = $self->_seq_origin($seq_id);
next if not defined $seq_origin;
push @{$origins{$seq_origin}}, $seq_id;
}
if (scalar keys %origins > 1) {
# Found a cross-contig
push @cross_contigs, $test_contig;
} else {
next;
}
# Find cross-singlets
for my $origin (keys %origins) {
my @ori_ids = @{$origins{$origin}};
if (scalar @ori_ids == 1) {
$nof_cross_singlets++;
} elsif (scalar @ori_ids > 1) {
# Dissolve contig for the given origin
### consider using the minimum overlap and identity here again?
my $ori_contigs = $self->_naive_assembler($test_contig, \@ori_ids, undef, undef);
for my $ori_contig (@$ori_contigs) {
$nof_cross_singlets++ if $ori_contig->num_sequences == 1;
}
}
}
}
return \@cross_contigs, $nof_cross_singlets;
}
=head2 _seq_origin
Title : _seq_origin
Usage :
Function: determines where a sequence comes from using its header. For example
the origin of the sequence 'metagenome1|gi|9626988|ref|NC_001508.1|'
is 'metagenome1'
Returns : origin
Args : sequence ID
=cut
sub _seq_origin {
# Current sequence origin. Example: sequence with ID
# 'metagenome1|gi|9626988|ref|NC_001508.1|' has header 'metagenome1'
my ($self, $seq_id) = @_;
my $origin;
if ( $seq_id =~ m/^(.+?)\|/ ) {
$origin = $1;
}
return $origin;
}
=head2 _import_assembly
Title : _import_assembly
Usage : $csp->_import_assembly($assemblyobj);
Function: Update a contig spectrum object based on an assembly, contig or
singlet object
Returns : 1 for success
Args : Bio::Assembly::Scaffold, Contig or Singlet object
=cut
sub _import_assembly {
my ($self, $assemblyobj) = @_;
# Sanity check
if ( ! ref $assemblyobj ||
( ! $assemblyobj->isa('Bio::Assembly::ScaffoldI') &&
! $assemblyobj->isa('Bio::Assembly::Contig') )) {
$self->throw("Unable to process non Bio::Assembly::ScaffoldI, Contig or ".
"Singlet object [".ref($assemblyobj)."]");
}
# Create new object from assembly
my $csp = $self->_new_from_assembly($assemblyobj);
# Update current contig spectrum object with new one
$self->add($csp);
return 1;
}
=head2 _import_spectrum
Title : _import_spectrum
Usage : $csp->_import_spectrum({ 1 => 90 , 2 => 3 , 4 => 1 })
Function: update a contig spectrum object based on a contig spectrum
represented as a hash (key: contig size, value: number of contigs of
this size)
Returns : 1 for success
Args : contig spectrum as a hash reference
=cut
sub _import_spectrum {
my ($self, $spectrum) = @_;
# Sanity check
if( ! ref $spectrum || ! ref $spectrum eq 'HASH') {
$self->throw("Spectrum should be a hash reference, but it is [".
ref($spectrum)."]");
}
# Update the spectrum (+ nof_rep, max_size and nof_seq)
for my $size (keys %$spectrum) {
# Get the number of contigs of different size
if (defined $self->{'_spectrum'}{$size}) {
$self->{'_spectrum'}{$size} += $$spectrum{$size};
} else {
$self->{'_spectrum'}{$size} = $$spectrum{$size};
}
# Update nof_seq
$self->{'_nof_seq'} += $size * $$spectrum{$size};
# Update max_size
$self->{'_max_size'} = $size if $size > $self->{'_max_size'};
}
# If the contig spectrum has only zero 1-contigs, max_size is zero
$self->{'_max_size'} = 0 if scalar keys %{$self->{'_spectrum'}} == 1 &&
defined $self->{'_spectrum'}{'1'} && $self->{'_spectrum'}{'1'} == 0;
# Update nof_rep
$self->{'_nof_rep'}++;
return 1;
}
=head2 _import_dissolved_csp
Title : _import_dissolved_csp
Usage : $csp->_import_dissolved_csp($mixed_csp, $seq_header);
Function: Update a contig spectrum object by dissolving a mixed contig
spectrum based on the header of the sequences
Returns : 1 for success
Args : Bio::Assembly::Tools::ContigSpectrum
sequence header string
=cut
sub _import_dissolved_csp {
my ($self, $mixed_csp, $seq_header) = @_;
# Sanity check
if (not defined $mixed_csp || not defined $seq_header) {
$self->throw("Expecting a contig spectrum reference and sequence header as".
" arguments");
}
# Create new object from assembly
my $dissolved_csp = $self->_new_dissolved_csp($mixed_csp, $seq_header);
# Update current contig spectrum object with new one
$self->add($dissolved_csp);
return 1;
}
=head2 _import_cross_csp
Title : _import_cross_csp
Usage : $csp->_import_cross_csp($mixed_csp);
Function: Update a contig spectrum object by calculating the cross contig
spectrum based on a mixed contig spectrum
Returns : 1 for success
Args : Bio::Assembly::Tools::ContigSpectrum
=cut
sub _import_cross_csp {
my ($self, $mixed_csp) = @_;
# Sanity check
if (not defined $mixed_csp) {
$self->throw("Expecting a contig spectrum reference as argument");
}
# Create new object from assembly
my $cross_csp = $self->_new_cross_csp($mixed_csp);
my $nof_1_cross_contigs = $cross_csp->spectrum->{1};
# Update current contig spectrum object with new one
$self->add($cross_csp);
# Remove 1-contigs
$self->{'_nof_seq'} -= $nof_1_cross_contigs;
return 1;
}
=head2 _get_contig_like
Title : _get_contig_like
Usage : my @contig_like_objs = $csp->_get_contig_like($assembly_obj);
Function: Get contigs and singlets from an assembly, contig or singlet
Returns : array of Bio::Assembly::Contig and Singlet objects
Args : a Bio::Assembly::Scaffold, Contig or singlet object
=cut
sub _get_contig_like {
my ($self, $assembly_obj) = @_;
my @contig_objs;
if ($assembly_obj->isa('Bio::Assembly::ScaffoldI')) {
# all contigs and singlets in the scaffold
push @contig_objs, ($assembly_obj->all_contigs, $assembly_obj->all_singlets);
} else {
# a contig or singlet
@contig_objs = $assembly_obj;
}
return @contig_objs;
}
=head2 _get_assembly_seq_stats
Title : _get_assembly_seq_stats
Usage : my $seqlength = $csp->_get_assembly_seq_stats($assemblyobj);
Function: Get sequence statistics from an assembly:
average sequence length, number of sequences
Returns : average sequence length (decimal)
number of sequences (integer)
Args : Bio::Assembly::Scaffold, Contig or singlet object
hash reference with the IDs of the sequences to consider [optional]
=cut
sub _get_assembly_seq_stats {
my ($self, $assemblyobj, $seq_hash) = @_;
# Sanity checks
if ( !defined $assemblyobj ||
( !$assemblyobj->isa('Bio::Assembly::ScaffoldI') &&
!$assemblyobj->isa('Bio::Assembly::Contig') ) ) {
$self->throw("Must provide a Bio::Assembly::Scaffold, Contig or Singlet object");
}
$self->throw("Expecting a hash reference. Got [".ref($seq_hash)."]")
if (defined $seq_hash && ! ref($seq_hash) eq 'HASH');
# Update sequence stats
my @asm_stats = (0,0);
# asm_stats = (avg_seq_len, nof_seq)
for my $contigobj ( $self->_get_contig_like($assemblyobj) ) {
@asm_stats = $self->_update_seq_stats( @asm_stats,
$self->_get_contig_seq_stats($contigobj, $seq_hash) );
}
return @asm_stats;
}
=head2 _get_contig_seq_stats
Title : _get_contig_seq_stats
Usage : my $seqlength = $csp->_get_contig_seq_stats($contigobj);
Function: Get sequence statistics from a contig:
average sequence length, number of sequences
Returns : average sequence length (decimal)
number of sequences (integer)
Args : contig object reference
hash reference with the IDs of the sequences to consider [optional]
=cut
sub _get_contig_seq_stats {
my ($self, $contigobj, $seq_hash) = @_;
my @contig_stats = (0, 0);
# contig_stats = (avg_length, nof_seq)
for my $seqobj ($contigobj->each_seq) {
next if defined $seq_hash && !defined $$seq_hash{$seqobj->id};
my $seq_string;
if ($contigobj->isa('Bio::Assembly::Singlet')) { # a singlet
$seq_string = $contigobj->seqref->seq;
} else { # a contig
$seq_string = $seqobj->seq;
}
# Number of non-gap characters in the sequence
my $seq_len = $seqobj->_ungapped_len;
my @seq_stats = ($seq_len);
@contig_stats = $self->_update_seq_stats(@contig_stats, @seq_stats);
}
return @contig_stats;
}
=head2 _update_seq_stats
Title : _update_seq_stats
Usage :
Function: Update the number of sequences and their average length 1
average identity 1
minimum length 1
minimum identity 1
number of overlaps 1 average sequence length
Returns : average sequence length
number of sequences
Args : average sequence length 1
number of sequences 1
average sequence length 2
number of sequences 2
=cut
sub _update_seq_stats {
my ($self, $p_avg_length, $p_nof_seq, $n_avg_length, $n_nof_seq) = @_;
# Defaults
if (not defined $n_nof_seq) {
$n_nof_seq = 1;
}
# Update overlap statistics
my $avg_length = 0;
my $nof_seq = $p_nof_seq + $n_nof_seq;
if ($nof_seq != 0) {
$avg_length = ($p_avg_length * $p_nof_seq + $n_avg_length * $n_nof_seq) / $nof_seq;
}
return $avg_length, $nof_seq;
}
=head2 _get_assembly_overlap_stats
Title : _get_assembly_overlap_stats
Usage : my ($avglength, $avgidentity, $minlength, $min_identity, $nof_overlaps)
= $csp->_get_assembly_overlap_stats($assemblyobj);
Function: Get statistics about pairwise overlaps in contigs of an assembly
Returns : average overlap length
average identity percent
minimum overlap length
minimum identity percent
number of overlaps
Args : Bio::Assembly::Scaffold, Contig or Singlet object
hash reference with the IDs of the sequences to consider [optional]
=cut
sub _get_assembly_overlap_stats {
my ($self, $assembly_obj, $seq_hash) = @_;
# Sanity check
if ( !defined $assembly_obj ||
( !$assembly_obj->isa('Bio::Assembly::ScaffoldI') &&
!$assembly_obj->isa('Bio::Assembly::Contig') ) ) {
$self->throw("Must provide a Bio::Assembly::ScaffoldI, Contig or Singlet object");
}
$self->throw("Expecting a hash reference. Got [".ref($seq_hash)."]")
if (defined $seq_hash && ! ref($seq_hash) eq 'HASH');
# Look at all the contigs (no singlets!)
my @asm_stats = (0, 0, undef, undef, 0);
# asm_stats = (avg_length, avg_identity, min_length, min_identity, nof_overlaps)
for my $contig_obj ( $self->_get_contig_like($assembly_obj) ) {
@asm_stats = $self->_update_overlap_stats( @asm_stats,
$self->_get_contig_overlap_stats($contig_obj, $seq_hash) );
}
return @asm_stats;
}
=head2 _get_contig_overlap_stats
Title : _get_contig_overlap_stats
Usage : my ($avglength, $avgidentity, $minlength, $min_identity, $nof_overlaps)
= $csp->_get_contig_overlap_stats($contigobj);
Function: Get statistics about pairwise overlaps in a contig or singlet. The
statistics are obtained using graph theory: each read is a node
and the edges between 2 reads are weighted by minus the number of
conserved residues in the alignment between the 2 reads. The
minimum spanning tree of this graph represents the overlaps that
form the contig. Overlaps that do not satisfy the minimum overlap
length and similarity get a malus on their score.
Note: This function requires the optional BioPerl dependency
module called 'Graph'
Returns : average overlap length
average identity percent
minimum overlap length
minimum identity percent
number of overlaps
Args : Bio::Assembly::Contig or Singlet object
hash reference with the IDs of the sequences to consider [optional]
=cut
sub _get_contig_overlap_stats {
my ($self, $contig_obj, $seq_hash) = @_;
# Sanity check
$self->throw("Must provide a Bio::Assembly::Contig object")
if (!defined $contig_obj || !$contig_obj->isa("Bio::Assembly::Contig"));
$self->throw("Expecting a hash reference. Got [".ref($seq_hash)."]")
if (defined $seq_hash && ! ref($seq_hash) eq 'HASH');
my @contig_stats = (0, 0, undef, undef, 0);
# contig_stats = (avg_length, avg_identity, min_length, min_identity, nof_overlaps)
# Build contig graph
### consider providing the minima to _contig_graph here too?
my ($g, $overlaps) = $self->_contig_graph($contig_obj, $seq_hash);
if ( defined $g ) {
# Graph minimum spanning tree (tree that goes through strongest overlaps)
$g = $g->MST_Kruskal();
# Calculate minimum overlap length and identity for this contig
for my $edge ( $g->edges ) {
# Retrieve overlap information
my ($id1, $id2) = @$edge;
if (not exists $$overlaps{$id1}{$id2}) {
($id2, $id1) = @$edge;
}
my ($score, $length, $identity) = @{$$overlaps{$id1}{$id2}};
# Update contig stats
my @overlap_stats = ($length, $identity);
@contig_stats = $self->_update_overlap_stats(@contig_stats, @overlap_stats);
}
}
return @contig_stats;
}
=head2 _update_overlap_stats
Title : _update_overlap_stats
Usage :
Function: update the number of overlaps and their minimum and average length
and identity
Returns :
Args : average length 1
average identity 1
minimum length 1
minimum identity 1
number of overlaps 1
average length 2
average identity 2
minimum length 2
minimum identity 2
number of overlaps 2
=cut
sub _update_overlap_stats {
my ($self,
$p_avg_length, $p_avg_identity, $p_min_length, $p_min_identity, $p_nof_overlaps,
$n_avg_length, $n_avg_identity, $n_min_length, $n_min_identity, $n_nof_overlaps)
= @_;
# Defaults
if (not defined $n_nof_overlaps) { $n_nof_overlaps = 1 };
if ((not defined $n_min_length) && ($n_avg_length != 0)) { $n_min_length = $n_avg_length };
if ((not defined $n_min_identity) && ($n_avg_identity != 0)) { $n_min_identity = $n_avg_identity };
# Update overlap statistics
my ($avg_length, $avg_identity, $min_length, $min_identity, $nof_overlaps)
= (0, 0, undef, undef, 0);
$nof_overlaps = $p_nof_overlaps + $n_nof_overlaps;
if ($nof_overlaps > 0) {
$avg_length = ($p_avg_length * $p_nof_overlaps + $n_avg_length * $n_nof_overlaps) / $nof_overlaps;
$avg_identity = ($p_avg_identity * $p_nof_overlaps + $n_avg_identity * $n_nof_overlaps) / $nof_overlaps;
}
if ( not defined $p_min_length ) {
$min_length = $n_min_length;
} elsif ( not defined $n_min_length ) {
$min_length = $p_min_length;
} else { # both values are defined
if ($n_min_length < $p_min_length) {
$min_length = $n_min_length;
} else {
$min_length = $p_min_length;
}
}
if ( not defined $p_min_identity ) {
$min_identity = $n_min_identity;
} elsif ( not defined $n_min_identity ) {
$min_identity = $p_min_identity;
} else { # both values are defined
if ($n_min_identity < $p_min_identity) {
$min_identity = $n_min_identity;
} else {
$min_identity = $p_min_identity;
}
}
return $avg_length, $avg_identity, $min_length, $min_identity, $nof_overlaps;
}
=head2 _overlap_alignment
Title : _overlap_alignment
Usage :
Function: Produce an alignment of the overlapping section of two sequences of
a contig. Minimum overlap length and percentage identity can be
specified. Return undef if the sequences do not overlap or do not
meet the minimum overlap criteria.
Return : Bio::SimpleAlign object reference
alignment overlap length
alignment overlap identity
Args : Bio::Assembly::Contig object reference
Bio::LocatableSeq contig sequence 1
Bio::LocatableSeq contig sequence 2
minimum overlap length [optional]
minimum overlap identity percentage[optional]
=cut
sub _overlap_alignment {
my ($self, $contig, $qseq, $tseq, $min_overlap, $min_identity) = @_;
# get query and target sequence position
my $qpos = $contig->get_seq_coord($qseq);
my $tpos = $contig->get_seq_coord($tseq);
# check that there is an overlap
my $qend = $qpos->end;
my $tstart = $tpos->start;
return if $qend < $tstart;
my $qstart = $qpos->start;
my $tend = $tpos->end;
return if $qstart > $tend;
# get overlap boundaries and check overlap length
my $left;
if ($qstart >= $tstart) {
$left = $qstart
} else {
$left = $tstart;
}
my $right;
if ($qend > $tend) {
$right = $tend;
} else {
$right = $qend;
}
my $overlap = $right - $left + 1;
return if defined $min_overlap && $overlap < $min_overlap;
# slice query and target sequence to overlap boundaries
my $qleft =
$contig->change_coord('gapped consensus', "aligned ".$qseq->id, $left);
my $qstring = substr($qseq->seq, $qleft - 1, $overlap);
my $tleft =
$contig->change_coord('gapped consensus', "aligned ".$tseq->id, $left);
my $tstring = substr($tseq->seq, $tleft - 1, $overlap);
# remove gaps present in both sequences at the same position
for (my $pos = 0 ; $pos < $overlap ; $pos++) {
my $qnt = substr($qstring, $pos, 1);
if ($qnt eq '-') {
my $tnt = substr($tstring, $pos, 1);
if ($tnt eq '-') {
substr($qstring, $pos, 1, '');
substr($tstring, $pos, 1, '');
$pos--;
$overlap--;
}
}
}
return if defined $min_overlap && $overlap < $min_overlap;
# count the number of gaps remaining in each sequence
my $qgaps = ($qstring =~ tr/-//);
my $tgaps = ($tstring =~ tr/-//);
# make an alignment object with the query and target sequences
my $aln = Bio::SimpleAlign->new;
my $alseq = Bio::LocatableSeq->new(
-id => 1,
-seq => $qstring,
-start => 1,
-end => $overlap - $qgaps,
-alphabet => 'dna',
);
$aln->add_seq($alseq);
$alseq = Bio::LocatableSeq->new(
-id => 2,
-seq => $tstring,
-start => 1,
-end => $overlap - $tgaps,
-alphabet => 'dna',
);
$aln->add_seq($alseq);
# check overlap percentage identity
my $identity = $aln->overall_percentage_identity;
return if defined $min_identity && $identity < $min_identity;
# all checks passed, return alignment
return $aln, $overlap, $identity;
}
=head2 _contig_graph
Title : _contig_graph
Usage :
Function: Creates a graph data structure of the contig.The graph is undirected.
The vertices are the reads of the contig and edges are the overlap
between the reads. The edges are weighted by the opposite of the
overlap, so it is negative and the better the overlap, the lower the
weight.
Return : Graph object or undef
hashref of overlaps (score, length, identity) for each read pair
Args : Bio::Assembly::Contig object reference
hash reference with the IDs of the sequences to consider [optional]
minimum overlap length (integer) [optional]
minimum percentage identity (integer) [optional]
=cut
sub _contig_graph {
my ($self, $contig_obj, $seq_hash, $min_overlap, $min_identity) = @_;
# Sanity checks
if( !ref $contig_obj || ! $contig_obj->isa('Bio::Assembly::Contig') ) {
$self->throw("Unable to process non Bio::Assembly::Contig ".
"object [".ref($contig_obj)."]");
}
if (not eval { require Graph::Undirected }) {
$self->throw("Error: the module 'Graph' is needed by the method ".
"_contig_graph but could not be found\n$@");
}
# Skip contigs of 1 sequence (they have no overlap)
my @seq_objs = $contig_obj->each_seq;
my $nof_seqs = scalar @seq_objs;
return if ($nof_seqs <= 1);
# Calculate alignment between all pairs of reads
my %overlaps;
for my $i (0 .. $nof_seqs-1) {
my $seq_obj = $seq_objs[$i];
my $seq_id = $seq_obj->id;
# Skip this read if not in list of wanted sequences
next if defined $seq_hash && !exists $$seq_hash{$seq_id};
# What is the best sequence to align to?
my ($best_score, $best_length, $best_identity);
for my $j ($i+1 .. $nof_seqs-1) {
# Skip this sequence if not in list of wanted sequences
my $target_obj = $seq_objs[$j];
my $target_id = $target_obj->id;
next if defined $seq_hash && !exists $$seq_hash{$target_id};
# How much overlap with this sequence?
my ($aln_obj, $length, $identity)
= $self->_overlap_alignment($contig_obj, $seq_obj, $target_obj, $min_overlap, $min_identity);
next if ! defined $aln_obj; # there was no sequence overlap or overlap not good enough
# Score the overlap as the number of conserved residues. In practice, it
# seems to work better than giving +1 for match and -3 for errors
# (mismatch or indels)
my $score = $length * $identity / 100;
# Apply a malus (square root) for scores that do not satisfy the minimum
# overlap length similarity. It is necessary for overlaps that get a high
# score without satisfying both the minimum values.
if ( ( $min_overlap && ($length < $min_overlap ) ) ||
( $min_identity && ($identity < $min_identity) ) ) {
$score = sqrt($score);
}
$overlaps{$seq_id}{$target_id} = [$score, $length, $identity];
}
}
# Process overlaps
my $g; # the Graph object
if (scalar keys %overlaps >= 1) {
# At least 1 overlap. Create a weighted undirected graph
$g = Graph::Undirected->new();
for my $seq_id (keys %overlaps) {
for my $target_id (keys %{$overlaps{$seq_id}}) {
my $score = @{$overlaps{$seq_id}{$target_id}}[0];
my $weight = -$score;
$g->add_weighted_edge($seq_id, $target_id, $weight);
}
}
}
return $g, \%overlaps;
}
=head2 _draw_graph
Title : _draw_graph
Usage :
Function: Generates a PNG picture of the contig graph. It is mostly for
debugging purposes.
Return : 1 for success
Args : a Graph object
hashref of overlaps (score, length, identity) for each read pair
name of output file
overlap info to display: 'score' (default), 'length' or 'identity'
=cut
sub _draw_graph {
my ($self, $g, $overlaps, $outfile, $edge_type) = @_;
$self->throw("Error: need to provide a graph as input\n") if not defined $g;
if (not eval { require GraphViz }) {
$self->throw("Error: the module 'GraphViz' is needed by the method ".
"_draw_graph but could not be found\n$@");
}
$edge_type ||= 'score';
my $viz = GraphViz->new( directed => 0 );
for my $edge ( $g->edges ) {
# Retrieve overlap information
my ($id1, $id2) = @$edge;
if (not exists $$overlaps{$id1}{$id2}) {
($id2, $id1) = @$edge;
}
my ($score, $length, $identity) = @{$$overlaps{$id1}{$id2}};
my $edge_val;
if ($edge_type eq 'score') {
$edge_val = $score;
} elsif ($edge_type eq 'length') {
$edge_val = $length;
} elsif ($edge_type eq 'identity') {
$edge_val = $identity;
} else {
$self->throw("Error: invalid edge type to display, '$edge_val'");
}
$viz->add_edge($id1 => $id2, label => $edge_val);
}
open my $fh, '>', $outfile or $self->throw("Error: Could not write file '$outfile': $!");
print $fh $viz->as_png;
close $fh;
return 1;
}
1;
__END__