Data from: Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing

High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data.

高通量测序（high-throughput sequencing）已被提出作为微卫星（microsatellites）基因分型的方法，可克服毛细管电泳（capillary electrophoresis）的四大主要技术缺陷：扩增伪影（amplification artifacts）、片段长度定量不准（imprecise sizing）、长度同形现象（length homoplasy）以及有限的多重扩增能力（multiplex capability）。本项目的目标是测试一种用于短串联重复序列（short tandem repeats）片段分析的高通量扩增子测序（amplicon sequencing）方法，并对比传统毛细管电泳，阐明该方法的优缺点。我们对180份麝鼠样本的12个微卫星位点进行了扩增与测序，并针对等位基因分型（allele calling）的准确性、扩增或测序伪影的发生倾向，以及长度同形现象的相关证据，对测序数据展开分析。在我们通过测序检出的共计294个等位基因中，仅有164个可通过毛细管电泳检出，剩余130个等位基因（占比44%）会因长度同形现象而无法被毛细管电泳识别。能够检出更多独特等位基因，意味着可以更清晰地解析群体遗传结构（population genetic structure）。基于测序的片段分析的核心优势在于：可精准定量片段长度、解析长度同形现象、可将大量个体与多个位点整合至单次高通量运行中完成检测，且仅需最低限度的技术校准（technical calibration），即可实现不同项目、不同实验室（当前及未来）间的数据比对。基于测序的片段分析的显著缺陷在于，该方法仅在对包含多个位点的数百份样本批次进行检测时，才具备实用性与成本效益。未来仍需开展相关研究，在优化通量的同时控制成本，并对现有的微卫星等位基因分型与分析程序进行更新，以适配序列感知微卫星数据（sequence-aware microsatellite data）。