Bio::Tools::SiRNA::Ruleset::tuschl - Perl object implementing the

BioPerl documentation Contained in the BioPerl distribution.

Index

Code Index:

NAME

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Bio::Tools::SiRNA::Ruleset::tuschl - Perl object implementing the tuschl group's rules for designing small inhibitory RNAs

SYNOPSIS

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Do not use this module directly. Instead, use Bio::Tools::SiRNA and specify the tuschl ruleset:

  use Bio::Tools::SiRNA;

  my $sirna_designer = Bio::Tools::SiRNA->new( -target => $bio_seq,
                                               -rules  => 'tuschl'
    );
  my @pairs = $sirna_designer->design;

  foreach $pair (@pairs) {
      my $sense_oligo_sequence = $pair->sense->seq;
      my $antisense_oligo_sequence = $pair->antisense->seq;

      # print out results
      print join ("\t", $pair->start, $pair->end, $pair->rank,
                  $sense_oligo_sequence, $antisense_oligo_sequence), "\n";
  }

DESCRIPTION

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This package implements the rules for designing siRNA reagents developed by Tuschl and colleagues (see http://www.rockefeller.edu/labheads/tuschl/sirna.html). It looks for oligos that match the following patterns in the target sequence:

  1. AA(N19)TT (rank 1)
  2. AA(N21) (rank 2)
  3. NA(N21) (rank 3)

The package also supports selection of siRNA seqences that can be transcribed by pol3:

    A[A,G]N17[C,T]

SEE ALSO

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Bio::Tools::SiRNA, Bio::SeqFeature::SiRNA::Pair, Bio::SeqFeature::SiRNA::Oligo.

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:

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

AUTHOR

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Donald Jackson (donald.jackson@bms.com)

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	: Do not call directly - use Bio::Tools::SiRNA->new instead.
  Returns : Bio::Tools::SiRNA::Ruleset::saigo object
  Args	: none
BioPerl documentation Contained in the BioPerl distribution. 
#
#
# BioPerl module for Bio::Tools::SiRNA::Ruleset::tuschl
#
# Please direct questions and support issues to <bioperl-l@bioperl.org> 
#
# Cared for by Donald Jackson, donald.jackson@bms.com
#
# Copyright Bristol-Myers Squibb
#
# You may distribute this module under the same terms as perl itself

# POD documentation - main docs before the code


package Bio::Tools::SiRNA::Ruleset::tuschl;

use strict;
use warnings;

use base qw(Bio::Tools::SiRNA);

our %PATTERNS = ( 1 	=> '(AA.{19}TT)',
		  2 	=> '(AA.{19}[ACG][ACG])',
		  3 	=> '([CGT]A.{21})',
		  Pol3	=> '(.A[AG].{17}[CT]..)'
		  );

our $DEFAULT_CUTOFF = 2;


sub new {
    my ($proto, %args) = @_;
    my $class = ref($proto) || $proto;
    
    $args{'RULES'} = 'tuschl';

    return $class->SUPER::new(%args);
}

sub _regex {
    my ($self, $rank) = @_;
    return $PATTERNS{$rank};
}

sub cutoff {
    my ($self, $cutoff) = @_;
    if ($cutoff) {
	$self->{'cutoff'} = $cutoff;
    }
    elsif (!$self->{'cutoff'}) {
	$self->{'cutoff'} = $DEFAULT_CUTOFF;
    }
    return $self->{'cutoff'};
}


sub _get_oligos {
    #use regular expressions to pull out oligos
    my ($self) = @_;

    my @ranks;
    if ($self->cutoff eq 'pol3') {
	@ranks = ('pol3');
    }
    else {
	@ranks = (1 .. $self->cutoff);
    }
    
    foreach my $rank (@ranks) {
	my $regex = $self->_regex($rank);
	#my @exclude;


# 	my ($targregion) = grep { $_->primary_tag eq 'Target' } $self->target->top_SeqFeatures;
# 	my $seq = $targregion->seq->seq;
# 	# but this way I loose start info
# 	my $targstart = $targregion->start;
	my ($seq, $targstart) = $self->_get_targetregion();

	while ( $seq =~ /(.*?)$regex/gi ) {
	    my $target = $2;

	    # check for too many Gs (or Cs on the other strand)
	    next if ( $target =~ /G{ $self->gstring,}/io );
	    next if ( $target =~ /C{ $self->gstring,}/io );
# 	skip Ns (for filtering)
	    next if ( $target =~ /N/i);

	    my $start = length($1) + $targstart;
	    my $stop = $start + length($target) -1;

	    my @gc = ( $target =~ /G|C/gi);
	    my $fxGC = sprintf("%2.2f", (scalar(@gc) / length($target)));
	    next if ($fxGC < $self->min_gc);
	    next if ($fxGC > $self->max_gc);
	    
	    $self->add_oligos($target, $start, $rank);
	}
    }
}

	 
sub _get_sense {
    my ($self, $target) = @_;
    # trim off 1st 2 nt to get overhang
    $target =~ s/^..//;
    # convert T's to U's (transcribe)
    $target =~ s/T/U/gi;
    # force last 2 nt to be T's
    $target =~ s/..$/TT/;

    return $target;
}

sub _get_anti {
    my ($self, $target) = @_;
    my @target = split(//, $target);
    my ($nt,@antitarget);

    while ($nt = pop @target) {
	push(@antitarget, $self->_comp($nt));
    }
    my $anti = join('', @antitarget);
    # trim off 1st 2 nt to get overhang
    $anti =~ s/^..//;
    # convert T's to U's
    $anti =~ s/T/U/gi;
    # convert last 2 NT's to T
    $anti =~ s/..$/TT/;

    return $anti;
}


1;