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Bridging the Gap Between Legacy PCR-based Microsatellite Data with High-Throughput Sequencing Data in Conservation

Dalya Salih1,2, Ellie E Armstrong3,4, Charles T Robbins5

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Whole-genome sequencing accurately genotypes microsatellites in brown bears, aligning historical data with modern methods. Careful variant interpretation is key for reliable genetic monitoring of endangered wildlife.

Keywords:
Genotypingbrown bearconcordanceconservationcoverageshort tandem repeat

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Area of Science:

  • Wildlife genetics
  • Conservation genomics
  • Population genetics

Background:

  • Microsatellites are vital genetic markers for wildlife population studies due to high polymorphism.
  • Polymerase chain reaction (PCR) fragment analysis is the traditional method for microsatellite genotyping.
  • High-throughput sequencing offers enhanced resolution for genetic variation analysis.

Purpose of the Study:

  • To evaluate the concordance between whole-genome sequencing (WGS) and PCR-based genotyping for microsatellites.
  • To assess the impact of sequencing depth on microsatellite genotype accuracy in brown bears (Ursus arctos).
  • To determine the feasibility of integrating WGS data with historical PCR-derived microsatellite datasets for conservation.

Main Methods:

  • Genotyping of 15 microsatellite loci in 11 North American brown bears using both WGS and PCR.
  • Comparison of genotype concordance between WGS and PCR data.
  • Downsampling WGS data from 30x to 2x coverage to evaluate the effect of sequencing depth.

Main Results:

  • Achieved a 94.5% microsatellite genotype concordance rate between WGS and PCR.
  • Discrepancies were primarily observed at complex loci with multiple insertions/deletions (indels) or linked single nucleotide polymorphisms (SNPs).
  • High concordance was maintained at 20-30x coverage; accuracy dropped significantly below 10x, with 2x and 5x showing insufficient data or discordant genotypes.

Conclusions:

  • Short-read WGS can accurately recover microsatellite genotypes when coupled with careful variant interpretation, especially for complex loci.
  • Sufficient sequencing depth and read coverage across repeat regions are critical for accurate microsatellite genotyping.
  • Integrating WGS with historical PCR data enhances long-term genetic monitoring of at-risk populations like the endangered brown bear.