Development of a Noninvasive Genotyping-In-Thousands (GTseq) Panel for Long-Term Conservation of Western Great Lakes Gray Wolves ().

The application of noninvasive genetic methods toward the field of conservation has increased our understanding of many wildlife populations that are difficult to sample, allowing for better management. In molecular ecology, the use of noninvasive sampling became widely feasible with the advent of microsatellites, a highly polymorphic, short-length marker that could be genotyped from low-quality DNA sources. Despite decades of use, many microsatellite panels continue to suffer from high genotyping error rates, allelic dropout, and limited reproducibility across laboratories. To address these issues, single nucleotide polymorphisms (SNPs) offer advantages such as lower genotyping error rates, avoidance of allelic dropout due to consistent allele length, and automated calling through bioinformatic pipelines, reducing human subjectivity and error. Given the advantages SNPs provide relative to microsatellites as a molecular marker, the use of SNP panels and specifically, the method of genotyping-in-thousands by sequencing (GTseq) has gained popularity. Here, we developed a GTseq panel for western Great Lakes canids comprised of 196 loci, capable of species identification, accurately inferring sex (97.2%), identifying unique individuals (probability of identity = 6.71e), assigning relationships (false positive rate = 9.34e), and assigning genotypes with low error (0.39%). In an attempt to improve genotyping success with low-quality samples, we found that while increasing the number of PCR cycles yielded a higher percentage of genotyped loci, it also increased on-target reads in negative PCR controls. We suggest approaching this manipulation with caution and emphasize the importance of including and reporting negative PCR controls. Further, quantitative PCR was a powerful method to estimate host-specific DNA concentrations, enabling conservative sample selection for library preparation with respect to GTseq affordability.