Bio::Seq::PrimedSeq - A representation of a sequence and two primers flanking a target region for amplification # 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; # we'll just 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); } #(ii) to create a genbank file for a sequence and its cognate primers #For example: 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 $seqin=new Bio::SeqIO(-file=>$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. This module is a slightly glorified capsule containg a primed sequence. It was created to address the fact that a primer is more the 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 instatiated first. The simplest way to initiate a PrimedSeq object is as follows: my $primedseq=Bio::Seq::PrimedSeq->new( -seq=>Bio::Seq object, -left_primer=>Bio::SeqFeature::Primer object, -right_primer=>Bio::SeqFeature::Primer object, ); From the PrimedSeq object you should be able to retrieve information about Tm's and what not of 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. 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://bio.perl.org/MailList.html - About the mailing lists

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 http://bugzilla.bioperl.org/ Rob Edwards, redwards@utmem.edu Based on a module written by Chad Matsalla, bioinformatics1@dieselwurks.com The rest of the documentation details each of the object methods. Internal methods are usually preceded with a _

new
Title : new() Usage : $primed_sequence = new Bio::SeqFeature::Primer( -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 : -target_sequence => 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 some of these parameters (most...) 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.

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