Bio::Tools::AmpliconSearch - Find amplicons in a template using degenerate PCR primers


   use Bio::PrimarySeq;
   use Bio::Tools::AmpliconSearch;

   my $template = Bio::PrimarySeq->new(
      -seq => 'aaaaaCCCCaaaaaaaaaaTTTTTTaaaaaCCACaaaaaTTTTTTaaaaaaaaaa',
   my $fwd_primer = Bio::PrimarySeq->new(
      -seq => 'CCNC',
   my $rev_primer = Bio::PrimarySeq->new(
      -seq => 'AAAAA',

   my $search = Bio::Tools::AmpliconSearch->new(
      -template   => $template,
      -fwd_primer => $fwd_primer,
      -rev_primer => $rev_primer,
   while (my $amplicon = $search->next_amplicon) {
      print "Found amplicon at position ".$amplicon->start.'..'.$amplicon->end.":\n";
      print $amplicon->seq->seq."\n\n";

   # Now change the template (but you could change the primers instead) and look
   # for amplicons again

   $template = Bio::PrimarySeq->new(
      -seq => 'aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa',

   while (my $amplicon = $search->next_amplicon) {
      print "Found amplicon at position ".$amplicon->start.'..'.$amplicon->end.":\n";
      print $amplicon->seq->seq."\n\n";


Perform an in silico PCR reaction, i.e. search for amplicons in a given template sequence using the specified degenerate primer.

The template sequence is a sequence object, e.g. Bio::Seq, and the primers can be a sequence or a Bio::SeqFeature::Primer object and contain ambiguous residues as defined in the IUPAC conventions. The primer sequences are converted into regular expressions using Bio::Tools::IUPAC and the matching regions of the template sequence, i.e. the amplicons, are returned as Bio::Seq::PrimedSeq objects.

AmpliconSearch will look for amplicons on both strands (forward and reverse- complement) of the specified template sequence. If the reverse primer is not provided, an amplicon will be returned and span a match of the forward primer to the end of the template. Similarly, when no forward primer is given, match from the beginning of the template sequence. When several amplicons overlap, only the shortest one to more accurately represent the biases of PCR. Future improvements may include modelling the effects of the number of PCR cycles or temperature on the PCR products.


Future improvements may include:

  • Allowing a small number of primer mismatches

  • Reporting all amplicons, including overlapping ones

  • Putting a limit on the length of amplicons, in accordance with the processivity of the polymerase used


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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 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:


Florent Angly <>


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


 Title    : new
 Usage    : my $search = Bio::Tools::AmpliconSearch->new( );
 Function : Initialize an amplicon search
 Args     : -template       Sequence object for the template sequence. This object
                            will be converted to Bio::Seq if needed in since features
                            (amplicons and primers) will be added to this object.
            -fwd_primer     A sequence object representing the forward primer
            -rev_primer     A sequence object representing the reverse primer
            -primer_file    Read primers from a sequence file. It replaces
                            -fwd_primer and -rev_primer (optional)
            -attach_primers Whether or not to attach primers to Amplicon objects. Default: 0 (off)
 Returns  : A Bio::Tools::AmpliconSearch object


 Title    : template
 Usage    : my $template = $search->template;
 Function : Get/set the template sequence. Setting a new template resets any
            search in progress.
 Args     : Optional Bio::Seq object
 Returns  : A Bio::Seq object


 Title    : fwd_primer
 Usage    : my $primer = $search->fwd_primer;
 Function : Get/set the forward primer. Setting a new forward primer resets any
            search in progress.
 Args     : Optional sequence object or primer object or '' to match beginning
            of sequence.            
 Returns  : A sequence object or primer object or undef


 Title    : rev_primer
 Usage    : my $primer = $search->rev_primer;
 Function : Get/set the reverse primer. Setting a new reverse primer resets any
            search in progress.
 Args     : Optional sequence object or primer object or '' to match end of
 Returns  : A sequence object or primer object or undef


 Title    : primer_file
 Usage    : my ($fwd, $rev) = $search->primer_file;
 Function : Get/set a sequence file to read the primer from. The first sequence
            must be the forward primer, and the second is the optional reverse
            primer. After reading the file, the primers are set using fwd_primer()
            and rev_primer() and returned.
 Args     : Sequence file
 Returns  : Array containing forward and reverse primers as sequence objects.


 Title    : attach_primers
 Usage    : my $attached = $search->attach_primers;
 Function : Get/set whether or not to attach primer objects to the amplicon
 Args     : Optional integer (1 for yes, 0 for no)
 Returns  : Integer (1 for yes, 0 for no)


 Title    : next_amplicon
 Usage    : my $amplicon = $search->next_amplicon;
 Function : Get the next amplicon
 Args     : None
 Returns  : A Bio::SeqFeature::Amplicon object


 Title    : annotate_template
 Usage    : my $template = $search->annotate_template;
 Function : Search for all amplicons and attach them to the template.
            This is equivalent to running:
               while (my $amplicon = $self->next_amplicon) {
                  # do something
               my $annotated = $self->template;
 Args     : None
 Returns  : A Bio::Seq object with attached Bio::SeqFeature::Amplicons (and
            Bio::SeqFeature::Primers if you set -attach_primers to 1).