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

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.