Primer Design, Evaluation of Primer Universality, and Estimation of Identification Power of Amplicon Sequences In Silico

Abstract

Primer design for polymerase chain reaction is an essential step for DNA barcoding, metabarcoding, molecular phylogenetics, and population genetic studies. Both forward and reverse primers need to match to annealing positions of template sequences of all subtaxa of target taxa in order to amplify target locus sequences. Although there are many existing such “universal” primer sets, the existing primer sets are not able to amplify target locus sequences of target taxa in some cases. Moreover, recent high-throughput sequencers cannot read long contiguous sequences and require new universal primer sets which can amplify relatively short barcode DNA sequences because most of the existing universal primer sets amplify longer sequences than high-throughput sequencers can read contiguously. Taking accumulation of nucleotide sequences of public DNA databases into consideration, retrieving target locus sequences of target taxa from public DNA databases, and designing primer set based on consensus sequence of retrieved sequences can be the solution for this problem. In this text, we provide methods and procedures to design universal primer pairs and to evaluate primer universality and identification power of amplicons.