/ 
BioPerl-1.6.901
River stage two • 60 direct dependents • 65 total dependents
/ Bio::Seq::PrimedSeq

NAME

Bio::Seq::PrimedSeq - A representation of a sequence and two primers flanking a target region

SYNOPSIS

# The easiest way to use this is probably either, (i), get the
# output from Bio::Tools::Run::Primer3, Bio::Tools::Primer3, or 
# Bio::Tools::PCRSimulation:

    # For example, start with a fasta file
    use Bio::SeqIO;
    use Bio::Tools::Run::Primer3;

    my $file = shift || die "need a file to read";
    my $seqin = Bio::SeqIO->new(-file => $file);
    my $seq = $seqin->next_seq;

    # use primer3 to design some primers
    my $primer3run = Bio::Tools::Run::Primer3->new(-seq => $seq);
    $primer3run -> run; # run it with the default parameters

    # create a file to write the results to
    my $seqout = Bio::SeqIO->new(-file => ">primed_sequence.gbk", 
                                 -format => 'genbank');

    # now just get all the results and write them out.
    while (my $results = $primer3run->next_primer) {
       $seqout->write_seq($results->annotated_seq);
    }

# Or, (ii), to create a genbank file for a sequence and its cognate
# primers:

   use Bio::SeqIO;
   use Bio::Seq::PrimedSeq;

   # have a sequence file ($file) with the template, and two primers
   # that match it, in fasta format

   my $file = shift || die "$0 <file>";
   my $seqin = Bio::SeqIO->new(-file => $file);

   # read three sequences
   my ($template, $leftprimer, $rightprimer) =
         ($seqin->next_seq, $seqin->next_seq, $seqin->next_seq);
   # set up the primed sequence object
   my $primedseq = Bio::Seq::PrimedSeq->new(-seq => $template, 
                                            -left_primer => $leftprimer,
                                            -right_primer => $rightprimer);
   # open a file for output
   my $seqout = Bio::SeqIO->new(-file => ">primed_sequence.gbk",
                                -format => 'genbank');
   # print the sequence out
   $seqout->write_seq($primedseq->annotated_sequence);

# This should output a genbank file with the two primers labeled.

DESCRIPTION

This module is a slightly glorified capsule containing a primed sequence. It was created to address the fact that a primer is more than a seqfeature and there need to be ways to represent the primer-sequence complex and the behaviors and attributes that are associated with the complex.

The primers are represented as Bio::SeqFeature::Primer objects, and should be instantiated first.

A simple way to create a PrimedSeq object is as follows:

my $primedseq = Bio::Seq::PrimedSeq->new(
        -seq          => $seq,  # Bio::Seq object,
        -left_primer  => $left, # Bio::SeqFeature::Primer object,
        -right_primer => $right # Bio::SeqFeature::Primer object,
);

From the PrimedSeq object you should be able to retrieve information about melting temperatures and what not on each of the primers and the amplicon.

This is based on the PrimedSeq.pm module started by Chad Matsalla, with additions/improvements by Rob Edwards.

FEEDBACK

User feedback is an integral part of the evolution of this and other Bioperl modules. Send your comments and suggestions preferably to one of 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

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 the bugs and their resolution. Bug reports can be submitted via the web:

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

AUTHOR

Rob Edwards, redwards@utmem.edu

Based on a module written by Chad Matsalla, bioinformatics1@dieselwurks.com

APPENDIX

The rest of the documentation details each of the object methods. Internal methods are usually preceded with a _

new

Title   : new()
Usage   : $primed_sequence = Bio::SeqFeature::Primer->new( 
                                    -seq => $sequence,
                                    -left_primer => $left_primer,
                                    -right_primer => $right_primer);
Function: A constructor for an object representing a primed sequence 
Returns : A Bio::Seq::PrimedSeq object
Args    :  -seq => a Bio::Seq object (required)
           -left_primer => a Bio::SeqFeature::Primer object (required)
           -right_primer => a Bio::SeqFeature::Primer object (required)

          Many other parameters can be included including all of the output
          parameters from the primer3 program. At the moment most of these
          parameters will not do anything.

get_primer

 Title   : get_primer();
 Usage   : $primer = $primedseq->get_primer(l, left, left_primer, 
           -left_primer) to return the left primer or 
	        $primer = $primedseq->get_primer(r, right, right_primer, 
           -right_primer) to return the right primer or
	        $primer = $primedseq->get_primer(b, both, both_primers, 
           -both_primers)
           to return the left primer, right primer array
 Function: A getter for the left primer in thie PrimedSeq object.
 Returns : A Bio::SeqFeature::Primer object
 Args    : Either of (l, left, left_primer, -left_primer) to get left 
           primer.
           Either of (r, right, right_primer, -right_primer) to get 
           right primer
	        Either of (b, both, both_primers, -both_primers) to get 
           both primers. 
           Note that this is plural. [default]

annotated_sequence

Title   : annotated_sequence
Usage   : $annotated_sequence_object = $primedseq->annotated_sequence()
Function: Get an annotated sequence object containg the left and right 
          primers
Returns : An annotated sequence object or 0 if not defined.
Args    : 
Note    : Use this method to return a sequence object that you can write
          out (e.g. in GenBank format). See the example above.

amplicon

Title   : amplicon
Usage   : my $amplicon = $primedseq->amplicon()
Function: Retrieve the amplicon as a sequence object
Returns : A seq object. To get the sequence use $amplicon->seq
Args    : None
Note    :

seq

Title   : seq
Usage   : my $seqobj = $primedseq->seq()
Function: Retrieve the target sequence as a sequence object
Returns : A seq object. To get the sequence use $seqobj->seq
Args    : None
Note    :

_place_seqs

Title   : _place_seqs
Usage   : $self->_place_seqs()
Function: An internal method to place the primers on the sequence and 
          set up the ranges of the sequences
Returns : Nothing
Args    : None
Note    : Internal use only

_set_seqfeature

Title   : _set_seqfeature
Usage   : $self->_set_seqfeature()
Function: An internal method to create Bio::SeqFeature::Generic objects
          for the primed seq
Returns : Nothing
Args    : None
Note    : Internal use only. Should only call this once left and right 
          primers have been placed on the sequence. This will then set 
          them as sequence features so hopefully we can get a nice output 
          with write_seq.