Bio::Tools Primer3
SummaryIncluded librariesPackage variablesSynopsisDescriptionGeneral documentationMethods
Bio::Tools::Primer3 - Create input for and work with the output from
the program primer3
Package variables
No package variables defined.
Included modules
Bio::Root::IO Bio::Root::Root
 # parse primer3 output to get some data
# this is also called from Bio::Tools::Run::Primer3
use Bio::Tools::Primer3;
# read a primer3 output file my $p3 = Bio::Tools::Primer3->new(-file=>"data/primer3_output.txt"); # how many results were there? my $num = $p3->number_of_results; print "There were $num results\n"; # get all the results my $all_results = $p3->all_results; print "ALL the results\n"; foreach my $key (keys %{$all_results}) { print "$key\t${$all_results}{$key}\n"; } # get specific results my $result1 = $p3->primer_results(1); print "The first primer is\n"; foreach my $key (keys %{$result1}) { print "$key\t${$result1}{$key}\n"; } # get the results as a Bio::Seq::PrimedSeq stream my $primer = $p3->next_primer; print "The left primer in the stream is ", $primer->get_primer('-left_primer')->seq->seq, "\n";
Bio::Tools::Primer3 creates the input files needed to design primers using
primer3 and provides mechanisms to access data in the primer3 output files.
This module provides a bioperl interface to the program primer3. See for details and to download the software.
This module is based on one written by Chad Matsalla
I have ripped some of his code, and added a lot of my own. I hope he
is not mad at me!
This is probably best run in one of the two following ways:
  i. To parse the output from Bio::Tools::Run::Primer3.
You will most likely just use next_primer to get the results from
ii. To parse the output of primer3 handed to it as a file name.
No description
Methods description
newcode    nextTop
  Title   : new()
Usage : my $primer3 = Bio::Tools::Primer3->new(-file=>$file);
Function: Parse primer3 output
Returns : Does not return anything. If called with a filename will
allow you to retrieve the results
Args : -file (optional) file of primer3 results to parse -verbose
(optional) set verbose output
Notes :
  Title   : number_of_results()
Usage : my $count = $primer3->number_of_results();
Function: Retrieve the number of primers returned from Primer3.
Returns : A scalar
Args : None
Notes : This returns the count of the primers returned by Primer3
(aka how many of them there are).
This is one more than the maximum offset into the zero
based list of primers that is accessed by primer_results().
  Title   : all_results()
Usage : my $results = $primer3->all_results();
my $results = $primer3->all_results('primer3 result name', 'other results');
Function: Retrieve the results returned from Primer3.
Returns : A reference to a hash
Args : Optional array of specific results to retrieve
  Title   : primer_results()
Usage : my $results = $primer3->primer_results(2); # results for third primer
Function: Retrieve the results returned from Primer3 for specific primer pairs.
Returns : A reference to a hash
Args : A number between 0 and the maximum number of primers to retrieve
  Title   : _readfile()
Usage : $self->_readfile();
Function: An internal function that reads a file and sets up the results
Returns : Nothing.
Args : None
Notes :
  Title   : next_primer()
Usage : while (my $primed_seq = $primer3->next_primer()) {
Function: Retrieve the primed sequence and a primer pair, one at a time
Returns : Returns a Bio::Seq::PrimedSeq object, one at a time
Args : None
Notes : Use $primed_seq->annotated_seq to get an annotated sequence
object you can write out.
  Title   : primer_stream()
Usage : while (my $primed_seq = $primer3->primer_stream()) {
Function: Retrieve the primer/sequences one at a time
Returns : Returns a Bio::Seq::PrimedSeq object, one at a time
Args : None
Notes : Deprecated, just a link to next_primer
  Title   : _separate()
Usage : $self->_separate();
Function: An internal function that groups the results by number
(e.g. primer pair 1, etc)
Returns : Nothing.
Args : None
Notes :
  Title   : _set_variable()
Usage : $self->_set_variable('variable name', 'value');
Function: An internal function that sets a variable
Returns : Nothing.
Args : None
Notes : Used to set $self->{results} and $self->seqobject
Methods code
    @PRIMER3_PARAMS = qw(results seqobject);
    foreach my $attr (@PRIMER3_PARAMS) {$OK_FIELD{$attr}++
    my $self = shift;
    my $attr = $AUTOLOAD;
    $attr =~ s/.*:://;
    $self->throw("Unallowed parameter: $attr !") unless $OK_FIELD{$attr};
    $self->{$attr} = shift if @_;
    return $self->{$attr};
sub new {
    my($class,%args) = @_;
    my $self = $class->SUPER::new(%args);
    if ($args{'-file'}) {
    if ($args{'-verbose'}) {
        $self->{'verbose'} = 1;
    return $self;
sub number_of_results {
    my $self = shift;
    return $self->{'maximum_primers_returned'} + 1;
sub all_results {
    my ($self, @results) = @_;
    my %hash;
    if (@results) {
        # we only want a few things
foreach my $result (@results) { $hash{$result} = $self->{'results'}->$result; } } else { foreach my $result (keys %{$self->{'results'}}) { $hash{$result}=$self->{'results'}->{$result}; } } return\% hash;
sub primer_results {
    my ($self, $toget) = @_;
    if ($toget > $self->{'maximum_primers_returned'}) {
        $self->warn("Didn't get any results for $toget");
        return 0;
    } else {
        return\% {$self->{'results_by_number'}->{$toget}};
sub _readfile {
    my ($self, $file) = @_;
    my $line;
    my $id='primer 3 parsed results'; # hopefully we'll get this, but we can set a temp id in case not.
while (defined($line = $self->_readline()) ) { chomp $line; next unless ($line); my ($return, $value) = split /=/, $line; if (uc($return) eq "SEQUENCE") { $self->{seqobject} = Bio::Seq->new(-seq=>$value, $id=>$id); next; } if (uc($return) eq "PRIMER_SEQUENCE_ID") { if ($self->{seqobject}) {$self->{seqobject}->id($value)} else {$id=$value} } $self->{'results'}->{$return} = $value; } # convert the results to individual results
sub next_primer {
    my $self = shift;
    # here we are going to convert the primers to Bio::SeqFeature::Primer objects
# and the primer/sequence to Bio::Seq::PrimedSeq objects
# the problem at the moment is that PrimedSeq can only take one sequence/primer pair, and
# yet for each sequence we can have lots of primer pairs. We need a way to overcome this.
# at the moment we can do this as a stream, I guess.
$self->warn("No primers were found for: ".$self->{'seqobject'}->{'primary_id'}) if (! $self->number_of_results); $self->{'next_to_return'} = 0 unless ($self->{'next_to_return'}); return if ($self->{'next_to_return'} >= $self->number_of_results); my $results = $self->primer_results($self->{'next_to_return'}); $self->throw("No left primer sequence") unless (${$results}{'PRIMER_LEFT_SEQUENCE'}); $self->throw("No right primer sequence") unless (${$results}{'PRIMER_RIGHT_SEQUENCE'}); $self->throw("No target sequence") unless ($self->{'seqobject'}); my $left_seq = Bio::SeqFeature::Primer->new( -id => 'left_primer', -seq => ${$results}{'PRIMER_LEFT_SEQUENCE'}, -display_id => ($self->{'next_to_return'} + 1), ); my $right_seq = Bio::SeqFeature::Primer->new( -id => "right_primer", -seq => ${$results}{'PRIMER_RIGHT_SEQUENCE'}, -display_id => ($self->{'next_to_return'} + 1) ); # add data to the Primer objects
for my $key (%$results) { # skip the primer sequence data, already added above
next if ($key =~ /PRIMER_(LEFT|RIGHT)_SEQUENCE/i ); if ($key =~ /PRIMER_LEFT/i) { $left_seq->add_tag_value($key, $$results{$key}); } elsif ($key =~ /PRIMER_RIGHT/i) { $right_seq->add_tag_value($key, $$results{$key}); } } my $primed_seq = Bio::Seq::PrimedSeq->new( -target_sequence => $self->{'seqobject'}->clone, -left_primer => $left_seq, -right_primer => $right_seq, ); # add data to the the PrimedSeq object that's not specific to the Primers
for my $key (%$results) { next if ($key =~ /PRIMER_(LEFT|RIGHT)/i ); $primed_seq->add_tag_value($key, $$results{$key}); } $self->{'next_to_return'}++; return $primed_seq;
sub primer_stream {
    my $self = shift;
    my $primedseq = $self->next_primer;
    return $primedseq;
sub _separate {
    my $self = shift;
    my %results; # the results that we find
my $maxlocation = -1; # the maximum number of primers returned
foreach my $key (keys %{$self->{'results'}}) { next if (${$self->{'input_options'}}{$key}); # don't process it if it is an input key
my $location; # the number of the primer pair
# names will have values like
# PRIMER_PRODUCT_SIZE_3 hence we need to find and remove the number
my $tempkey = $key; if ($tempkey =~ s/_(\d+)//) { $location = $1; if ($location > $maxlocation) {$maxlocation = $location} } elsif ( $tempkey =~ /PRIMER_(RIGHT|LEFT)_SEQUENCE/ ) { # first primers reported without a number, therefore set $location to 0
$location = 0; if ($location > $maxlocation) {$maxlocation = $location} } else { $location = 0; } # we will hash the results by number, and then by name
${$results{$location}}{$tempkey}=${$self->{'results'}}{$key}; } $self->{'results_by_number'} =\% results; $self->{'maximum_primers_returned'} = $maxlocation;
sub _set_variable {
    my ($self, $name, $value) = @_;
    next unless ($name);
    $self->{$name} = $value;


General documentation
Mailing ListsTop
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.                  - General discussion - About the mailing lists
Support Top
Please direct usage questions or support issues to the mailing list:
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 BugsTop
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:
  Rob Edwards
Based heavily on work of Chad Matsalla
  Brian Osborne bosborne at
The rest of the documentation details each of the object methods.
Internal methods are usually preceded with a _