=head1 NAME
Bio::Polloc::Typing::bandingPattern::amplification - banding-pattern-based
methods for typing assessment using amplification
=head1 DESCRIPTION
Category 1 of genotyping methods in:
Li, W., Raoult, D., & Fournier, P.-E. (2009).
Bacterial strain typing in the genomic era.
FEMS Microbiology Reviews, 33(5), 892-916.
=head1 IMPLEMENTS OR EXTENDS
=over
=item *
L<Bio::Polloc::Typing::bandingPattern>
=back
=head1 AUTHOR - Luis M. Rodriguez-R
Email lmrodriguezr at gmail dot com
=cut
package Bio::Polloc::Typing::bandingPattern::amplification;
use base qw(Bio::Polloc::Typing::bandingPattern);
use strict;
use Bio::Polloc::Polloc::IO;
use Bio::Polloc::LocusI;
our $VERSION = 1.0503; # [a-version] from Bio::Polloc::Polloc::Version
=head1 APPENDIX
Methods provided by the package
=head2 new
Generic initialization method
=cut
sub new {
my($caller,@args) = @_;
my $self = $caller->SUPER::new(@args);
$self->_initialize(@args);
return $self;
}
=head1 METHODS FROM Bio::Polloc::TypingI
=head2 scan
=head2 cluster
=head2 typing_value
=head2 graph_content
=head1 METHODS FROM Bio::Polloc::Typing::bandingPattern
=head2 fragments
=cut
sub fragments {
my($self, @args) = @_;
my ($locigroup) = $self->_rearrange([qw(LOCIGROUP)], @args);
defined $locigroup or $self->throw('Trying to amplify fragments, but no loci group provided');
my $primers = $self->design_primers(-locigroup=>$locigroup);
return unless defined $primers;
UNIVERSAL::can($primers, 'isa') and $primers->isa('Bio::Polloc::Polloc::IO')
or $self->throw('Wrong primers file', $primers, 'Bio::Polloc::Polloc::UnexpectedException');
defined $primers->file or $self->throw('Impossible to locate primers temporal file', $primers, 'Bio::Polloc::Polloc::UnexpectedException');
my $out = Bio::Polloc::LociGroup->new(-genomes=>$locigroup->genomes);
for my $g (0 .. $#{$locigroup->genomes}){
next unless defined $locigroup->genomes->[$g]->file;
my $run = Bio::Polloc::Polloc::IO->new(-file=>"primersearch '".$locigroup->genomes->[$g]->file."' ".
"'".$primers->file."' '".$self->annealing_errors."' -auto -stdout |");
my $amp = [];
my $k = -1;
while(my $ln = $run->_readline){
chomp $ln;
if($ln =~ m/^Amplimer (\d+)/){
$amp->[$k = $1-1] = Bio::Polloc::LocusI->new(
-type=>'amplicon',
-primersio=>$primers,
-genome=>$locigroup->genomes->[$g]);
}elsif($ln =~ m/^\s*Sequence: ([^\s]*)\s*/){
my $sid=$1;
$amp->[$k]->seq($locigroup->genomes->[$g]->search_sequence($sid));
}elsif($ln =~ m/^\s*\S+ hits forward strand at (\d+) with (\d+) mismatches/){
my($from,$err) = ($1+0, $2+0);
$amp->[$k]->from($from);
$amp->[$k]->errors($err);
}
elsif($ln =~ m/^\s*\S+ hits reverse strand at \[(\d+)\] with (\d+) mismatches/){ $amp->[$k]->errors($2 + $amp->[$k]->errors) }
elsif($ln =~ m/^\s*Amplimer length: (\d+) bp/){ $amp->[$k]->to($1 + $amp->[$k]->from - 1) }
}
$out->add_loci($g, @$amp);
}
return $out;
}
=head2 min_size
=head2 max_size
=head1 SPECIFIC METHODS
=head2 design_primers
Designs the primers to amplify all the loci in the group.
=head3 Arguments
=over
=item -locigroup I<Bio::Polloc::LociGroup>
The loci to be amplified.
=back
=head3 Returns
A <Bio::Polloc::Polloc::IO> object pointing to a file containing the primers
designed in the format required by EMBOSS primerseq:
NAME_1 FWD-SEQ REV-SEQ
...
=cut
sub design_primers {
my($self,@args) = @_;
my($locigroup) = $self->_rearrange([qw(LOCIGROUP)], @args);
defined $locigroup or $self->throw('Trying to design primers, but no loci group provided');
$locigroup->fix_strands;
# Align flanking regions
my $left_aln = $locigroup->align_context(-1, $self->flanking_size, 0) or return;
my $right_aln = $locigroup->align_context(1, $self->flanking_size, 0) or return;
# Consensus
my $left_cons = $left_aln->consensus_string($self->primer_conservation);
my $right_cons = $right_aln->consensus_string($self->primer_conservation);
# Conserved region
my $len = $self->primer_size;
$left_cons =~ s/^.*?([^?]{$len}).*$/$1/;
$right_cons =~ s/^.*?([^?]{$len}).*$/$1/;
return unless length($left_cons)==$len and length($right_cons)==$len;
# Reverse complement
my $uprc = Bio::Seq->new(-seq=>$left_cons )->revcom->seq;
my $downrc = Bio::Seq->new(-seq=>$right_cons)->revcom->seq;
# Output file
my $io = Bio::Polloc::Polloc::IO->new(-createtemp=>1);
$io->_print("Polloc $uprc $downrc\n");
return $io;
}
=head2 primer_conservation
Gets/sets the minimum conservation of a region to design primers. 1 by default.
=cut
sub primer_conservation {
my($self, $value) = @_;
$self->{'_primer_conservation'} = $value+0 if defined $value;
return 1 unless defined $self->{'_primer_conservation'};
return $self->{'_primer_conservation'};
}
=head2 primer_size
Gets/sets the primer size. 20 by default.
=cut
sub primer_size {
my($self, $value) = @_;
$self->{'_primer_size'} = $value+0 if defined $value;
return 20 unless defined $self->{'_primer_size'};
return $self->{'_primer_size'};
}
=head2 flanking_size
Gets/sets the size of the flanking region to take into account for the
primer design. 500 by default.
=cut
sub flanking_size {
my($self, $value) = @_;
$self->{'_flanking_size'} = $value+0 if defined $value;
return 500 unless defined $self->{'_flanking_size'};
return $self->{'_flanking_size'};
}
=head2 annealing_errors
Gets/sets the maximum percentage of errors allowed for a primer to anneal.
0 by default.
=cut
sub annealing_errors {
my($self, $value) = @_;
$self->{'_annealing_errors'} = $value+0 if defined $value;
return $self->{'_annealing_errors'} || 0;
}
=head1 INTERNAL METHODS
Methods intended to be used only within the scope of Bio::Polloc::*
=head2 _initialize
=head2 _initialize_method
=cut
sub _initialize_method {
my($self,@args) = @_;
my($primerConservation, $primerSize, $flankingSize, $annealingErrors) =
$self->_rearrange([qw(PRIMERCONSERVATION PRIMERSIZE FLANKINGSIZE ANNEALINGERRORS)], @args);
$self->type('bandingPattern::amplification');
$self->primer_conservation($primerConservation);
$self->primer_size($primerSize);
$self->flanking_size($flankingSize);
$self->annealing_errors($annealingErrors);
}
1;