Journal
ECOLOGY AND EVOLUTION
Volume 6, Issue 13, Pages 4502-4512Publisher
WILEY
DOI: 10.1002/ece3.2221
Keywords
Genotyping by sequencing; length homoplasy; marker; microsatellite; sequencing; short tandem repeat
Categories
Funding
- North Dakota Game and Fish from the Wildlife and Sport Fish Restoration Program (CFDA) [15.611]
- National Science Foundation EPSCoR Advanced Undergraduate Research Award (NSF) [IAA-1355466]
- University of North Dakota College of Arts and Sciences Undergraduate Research Fund
- Office Of The Director
- Office of Integrative Activities [1355466] Funding Source: National Science Foundation
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available