Overview

The prymer Python library is intended to be used for three main purposes:

1. Clustering targets into larger amplicons prior to designing primers.
2. Designing primers (single and paired) and internal hybridization probes using Primer3 for each target from (1).
3. Build and Picking a set of primer pairs from the design candidates produced in (2).

Clustering Targets

Optionally, input targets may be clustered into larger amplicons prior to designing primers. These amplicons wholly contain the input targets, and are used as input for primer design. The cluster_intervals() method in the prymer.api.clustering module is used to cluster targets into larger amplicons prior to primer design.

Designing Primers

Designing primers (left or right) or primer pairs using Primer3 is primarily performed using the Primer3 class, which wraps the primer3 command line tool. The design() method facilitates the design of primers (single and paired) and internal hybridization probes for a single target. The Primer3 instance is intended to be re-used to design primers across multiple targets, or re-design (after changing parameters) for the same target, or both!

Common input parameters for designing primers are specified in PrimerAndAmpliconParameters() and PrimerAndAmpliconWeights(), while the task type (left primer, right primer, or primer pair design) is specified with the corresponding Primer3Task. Design specifications for designing probes are stored in ProbeParameters(). Penalty weights for designing internal probes are specified in ProbeWeights().

The result of a primer design is encapsulated in the Primer3Result class. It provides the primers, probes, or primer pairs that were designed, as well as a list of reasons some primers were not returned, for example exceeding the melting temperature threshold, too high GC content, and so on. These failures are encapsulated in the Primer3Failures class.

The Primer3Result returned by the primer design contains either a list of Oligos or PrimerPairs, depending on the Primer3Task specified in the input parameters. These can be subsequently filtered or examined.

Build and Picking Primer Pairs

It is recommended to design left and right primers individually, then to screen each primer for off-target mappings using the OffTargetDetector class, which wraps bwa aln to identify off-target primer mappings.

The remaining primers may then be used with the build_primer_pairs() method to build primer pairs from all combinations of the left and right primers that are within acceptable amplicon size and tm ranges. Optionally, if a max_heterodimer_tm is provided, primer pairs will be screened to ensure that the left and right primers do not dimerize with a Tm greater than the one provided. The produced primer pairs are scored in a manner similar to Primer3 using the score() method.