More affordable and effective noninvasive single nucleotide polymorphism genotyping using high-throughput amplicon sequencing

Noninvasive genotyping methods have become key elements of wildlife research over the last two decades, but their widespread adoption is limited by high costs, low success rates and high error rates. The information lost when genotyping success is low may lead to decreased precision in animal population densities, which could misguide conservation and management actions. Single nucleotide polymorphisms (SNPs) provide a promising alternative to traditionally used microsatellites as SNPs allow amplification of shorter DNA fragments, are less prone to genotyping errors and produce results that are easily shared among laboratories. Here, we outline a detailed protocol for cost-effective and accurate noninvasive SNP genotyping using multiplexed amplicon sequencing optimized for degraded DNA. We validated this method for individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (Canis latrans) using 26 SNPs. Our genotyping success rate for scat samples was 93%, and 100% for hair and tissue, representing a substantial increase compared to previous microsatellite-based studies while remaining at a low cost of under $5 per PCR replicate (excluding labour). The accuracy of the genotypes was further corroborated in that genotypes from scats matching known, GPS-collared coyotes were always located within the territory of the known individual. We also show that different levels of multiplexing produced similar results, but that PCR product cleanup strategies can have substantial effects on genotyping success. By making noninvasive genotyping more affordable, accurate and efficient, this research may allow for a substantial increase in the use of noninvasive methods to monitor and conserve free-ranging wildlife populations.