Genotyping-in-Thousands by sequencing (GT-seq) panel development and application to minimally-invasive DNA samples to support studies in molecular ecology

Minimally-invasive sampling (MIS) is widespread in wildlife studies; however, its utility for massively parallel DNA sequencing (MPS) is limited. Poor sample quality and contamination by exogenous DNA can make MIS challenging to use with modern genotyping-by-sequencing approaches, which have been traditionally developed for high-quality DNA sources. Given that MIS is often more appropriate in many contexts, there is a need to make such samples practical for harnessing MPS. Here, we test the ability for Genotyping-in-Thousands by sequencing (GT-seq), a multiplex amplicon sequencing approach, to effectively genotype minimally-invasive cloacal DNA samples collected from the Western Rattlesnake (Crotalus oreganus), a threatened species in British Columbia, Canada. As there was no previous genetic information for this species, an optimized panel of 362 SNPs was selected for use with GT-seq from a de novo restriction-site associated DNA sequencing (RADseq) assembly. Comparisons of genotypes g...