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The effects of read length, quality and quantity on microsatellite discovery and primer development: from I llumina to P ac B io
Author(s) -
Wei Na,
Bemmels Jordan B.,
Dick Christopher W.
Publication year - 2014
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12245
Subject(s) - microsatellite , biology , computational biology , dna sequencing , workflow , nanopore sequencing , genetics , computer science , database , dna , gene , allele
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, I llumina, and I on T orrent platforms, but other methods including single‐molecule real‐time DNA sequencing ( P acific B iosciences or P ac B io) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using P ac B io circular consensus sequencing ( CCS ). We then evaluate the performance of P ac B io CCS in comparison with other NGS platforms for microsatellite isolation, through simulations that focus on variations in read length, read quantity and sequencing error rate. 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 each species. The simulations quantitatively validate the advantages of long reads and emphasize the detrimental effects of sequencing errors on NGS ‐enabled microsatellite development. In view of the continuing improvement in read length on NGS platforms, sequence quality and the corresponding strategies of quality control will become the primary factors to consider for effective microsatellite isolation. Among current options, P ac B io CCS may be optimal for rapid, small‐scale microsatellite development due to its flexibility in scaling sequencing effort, while platforms such as I llumina M i S eq will provide cost‐efficient solutions for multispecies microsatellite projects.