PrimerMiner: an r package for development and in silico validation of DNA metabarcoding primers

Abstract

Summary DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus, the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that can serve as a backbone for primer design is currently available. We developed the r package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI data bases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic than the strategy employed in another available software for that purpose, ecoPCR. We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both data bases, we were able to increase the amount of reference data 249‐fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner. With PrimerMiner, we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU‐based reference alignments generated with PrimerMiner can be used for manual primer design or processed with bioinformatic tools for primer development.