The rhAmpSeq Design Tool aims to maximize PCR target specificity at intended loci and minimize any amplification of unintended loci that might consume valuable sequencing reads. Therefore, the design output can sometimes include not only the main panel, but also a secondary pool and possibly individual (singleton) primer sets requiring independent amplification.
Several factors can lead to more than one panel being designed:
(1) A primer overlaps common single nucleotide polymorphisms (SNPs) leading to potential inefficient amplification.
(2) The GC content of a primer or amplicon significantly differs from the panel’s average GC content.
(3) The Tm or secondary structure of the primers is not ideal.
(4) The amplicons contain homopolymers.
(5) The locus and design constraints do not allow the design of a primer pair that is expected to uniformly amplify with the assays in the primary pool.