Abstract
Non-invasive DNA sampling to identify and enumerate species is critical to population monitoring and for developing effective management strategies. However, individual DNA identification is often limited by degraded and low template DNA (LT-DNA) that routinely yields partial profiles prone to technical artifacts, thus limiting their utility/reliability. Massively parallel, genotyping-by-sequencing (GBS) assays present an opportunity to amplify not only a large suite of molecular markers simultaneously, providing higher resolution to identify individuals, but also higher levels of sequence redundancy to enable quality metric evaluations of profiles from LT-DNA. Taxidea taxus jacksoni is an endangered badger subspecies in Canada, with low levels of genetic diversity, complicating individual identifications from closely related DNA sequences. Challenges arise from the small number of hairs collected from snag traps set in badger burrows that rarely provide full profiles. We designed a GBS assay to obtain microsatellite profiles compatible with pre-existing databases generated with conventional capillary electrophoresis (CE) genotyping. We assessed the assay’s reproducibility via a dilution series to mimic LT-DNA and tested if the assay produced similar CE-generated results. While GBS offers the potential to genotype large numbers of individuals and markers at the same time, we found low concordance between GBS- and CE-based genotypes from DNA templates < 250 pg. We recommend existing wildlife genetic databases focus on tetra-nucleotide microsatellite or SNP markers to reduce or eliminate sequencing artifacts (i.e., stutter) that present challenges for GBS genotypes from degraded and LT-DNA, and the use of sample replicates to form consensus genotypes.
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Data availability
All sequence data is available through the NCBI Sequence Read Archive (accession number SRP233447).
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Acknowledgements
This research was funded by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada to CJK. Funding for the Ontario Badger Project was provided in part by the Ontario Ministry of Natural Resource and Forestry Species-at-Risk Stewardship Fund and Species-at-Risk Research Fund, Environment and Climate Change Canada’s Habitat Stewardship Program for Species-at-Risk, Earth Rangers, World Wildlife Fund, and Norfolk Field Naturalists. This research was enabled in part by support provided by Compute Canada (www.computecanada.ca; RRG gme-665-ab). We thank: the Ontario Badger Recovery Team, Ontario Ministry of Natural Resources and Forestry, and the Ontario Badger Project for their collection of badger specimens; the Natural Heritage Information Centre for providing archival records; Matt Harnden, Sarah Dolynskyj, Nguyen Thi-Xuan Nguyen, and Brad Seyler from the Natural Resources DNA Profiling and Forensics Centre at Trent University for technical assistance; and Jeffrey Gross from the Advanced Analysis Centre at the University of Guelph for performing the Illumina sequencing.
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Conceived and designed the experiments: MED CJK. Sample collection: JS. Performed the experiments: KJ. Analyzed the data: KJ YR MED. Wrote the manuscript: MED KJ CJK. Revised the manuscript: MED KJ YR JS DME CJK.
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Donaldson, M.E., Jackson, K., Rico, Y. et al. Development of a massively parallel, genotyping-by-sequencing assay in American badger (Taxidea taxus) highlights the need for careful validation when working with low template DNA. Conservation Genet Resour 12, 601–610 (2020). https://doi.org/10.1007/s12686-020-01146-8
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DOI: https://doi.org/10.1007/s12686-020-01146-8