DOI: 10.5061/dryad.r20t0
title: Data from: The effects of read length, quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio

description: 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 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 lengths 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, PacBio CCS may be optimal for rapid, small-scale
 microsatellite development due to its flexibility in scaling sequencing
 effort, while platforms such as Illumina MiSeq will provide cost-efficient
 solutions for multi-species microsatellite projects.
description: Read length simulations
 without sequencing errorsSequences of uniform
 length at each of 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800,
 1000 and 1200 bp were drawn at random from Populus trichocarpa chromosomes
 1–19 (v3.0, DOE-JGI, http://www.phytozome.net/poplar) using 0.1x coverage.
 Simulations were conducted using Grinder v0.5.3 without the inclusion of
 sequencing errors.ReadLengthSim_noErrors.zipRead length simulations with sequencing errorsSequences of uniform length at each of 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 1000 and 1200 bp were drawn at random from Populus trichocarpa chromosomes 1–19 (v3.0, DOE-JGI, http://www.phytozome.net/poplar) using 0.1x coverage. Simulations were conducted using PBSIM v1.0.3 with built-in PacBio CCS error profiles (substitutions/insertions/deletions ratio of 6:21:73; read accuracy of 98 ± 2%).ReadLengthSim_withErrors.zipSequencing error simulationsSequencing errors of substitutions and indels (insertions and deletions) were introduced to reads of uniformly 350 bp simulated from Populus trichocarpa chromosomes 1–19 (v3.0, DOE-JGI, http://www.phytozome.net/poplar) at 0.1x coverage using Grinder v0.5.3. In terms of sequencing error type, simulations included substitution-biased (substitutions/indels ratio of 90:10) and indel-biased (substitutions/indels ratio of 10:90) sequencing errors. In terms of sequencing error rate distribution, we assumed that sequencing errors occurred either uniformly or linearly from the 5′ end to 3′ end of each read. With uniformly distributed sequencing errors, reads were simulated with an error rate of 0, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, and 5%. With linearly distributed errors, the error rate doubled from the 5′ end to 3′ end: 0, 0.01–0.02%, 0.1–0.2%, 0.2–0.4%, 0.5–1%, 1–2%, and 2–4%. For the file name of each simulation, the first three letters reflect the error rate distribution (e.g. Lin for linear error rate distribution, Uni for uniform distribution); the following 4 digits represent the sequencing error type (e.g. 9010 for substitutions/indels ratio of 90:10); the remaining digits indicate the error rate (e.g. 0.1%). When error rate was 0, the simulation was designated as ErrorRate_0.SeqErrorSim.zipError trimming simulationSequences of uniform length of 350 bp were drawn at random from Populus trichocarpa chromosomes 1–19 (v3.0, DOE-JGI, http://www.phytozome.net/poplar) at 0.1x coverage using PBSIM v1.0.3, following the observed read length distribution and quality profiles of Triplaris cumingiana CCS reads in this study.ErrorTrimSim.fastq