The effects of read length, quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio

The advent of next-generation sequencing (NGS) technologies has transformed the way microsatellites are isolated for ecological and evolutionary investigations. Recent attempts to employ NGS for microsatellite discovery have used the 454, Illumina, and Ion Torrent platforms, but other methods including single-molecule real-time DNA sequencing (Pacific Biosciences, or PacBio) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using PacBio circular consensus sequencing (CCS). We then evaluate the performance of PacBio CCS in comparison to other NGS platforms for microsatellite isolation, through simulations that focus on variations in read lengths, read quantity, and sequencing error rates. Although quality control of CCS reads reduced microsatellite yield by around 50%, hundreds of microsatellite loci that are expected to have improved conversion efficiency to functional markers were retrieved for eac...