Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

Abstract

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.

Next Gen Pop Gen: implementing a high-throughput approach to population genetics in boarfish (Capros aper)

“…Additionally, as the combinatorial barcoding method uses universal tails for barcode annealing rather than platform-specific adapters and indexes the method is not limited to a single sequencing platform. This means the method is easily transferable between platforms, with adequate read length, as new technology become available, which is in contrast to other recently reported microsatellite GBS methods [23,65], which used Illumina specific dual indexing primers.…”

“…In summary, the current study implements a novel and high-throughput approach to undertaking a complete population genetics study that can be faster and cheaper than current approaches while offering better and more data [22,23]. The method was demonstrated in a ‘real’ scenario, on a species with no existing population genetic information, which required urgent and robust answers.…”

“…Furthermore, it is important to remember that many questions in molecular ecology can be efficiently addressed with a limited number of highly polymorphic markers, such as microsatellites [5]. Many of the issues associated with microsatellite-based population studies could be mitigated using an NGS-based microsatellite GBS approach leading to faster and cheaper genotyping in large-scale population genetics studies while offering better and more data than conventional methods [22,23]. …”

“…[22,24] and later Darby et al . [23] demonstrated the potential of NGS for microsatellite GBS using de novo and existing capillary/gel electrophoresis multiplex marker panels and validated the GBS approach against microsatellite data generated by traditional capillary electrophoresis. Vartia et al .…”

“…[22] and Darby et al . [23] also demonstrated that the GBS approach detected a greater number of unique alleles than capillary electrophoresis and resulted in the ability to resolve greater population genetic structure. In addition, Vartia et al .…”