Data from: 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 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.

下一代测序（next-generation sequencing, NGS）技术的问世，彻底变革了用于生态学与进化研究的微卫星（microsatellite）分离流程。此前利用NGS开展微卫星发掘的研究多采用454、Illumina及Ion Torrent测序平台，但包括单分子实时DNA测序（Pacific Biosciences，即PacBio）在内的其他方法，仍是可行的替代方案。本文详述了一套基于PacBio环形共识测序（circular consensus sequencing, CCS）技术的研究流程，涵盖从测序质量控制到三种植物物种的微卫星标记（microsatellite marker）验证环节。随后，我们通过聚焦读长、读取数量与测序错误率差异的模拟实验，对比评估了PacBio CCS与其他NGS平台在微卫星分离工作中的性能表现。尽管对CCS读取序列的质量控制环节使微卫星位点产出量降低了约50%，但每个物种仍可获得数百个有望在转化为功能性标记时具备更高效率的微卫星位点。该模拟实验从量化角度验证了长读长的技术优势，并强调了测序错误对基于NGS的微卫星开发工作的不利影响。鉴于NGS平台的读长在持续优化，序列质量及其对应的质量控制策略，将成为实现高效微卫星分离的首要考量因素。在当前可选的技术方案中，PacBio CCS因其可灵活调整测序投入规模，或可成为快速开展小规模微卫星开发项目的最优选择；而Illumina MiSeq等平台则可为多物种微卫星研究项目提供高性价比的解决方案。