Development of a massively parallel, genotyping-by-sequencing assay in American badger ( Taxidea taxus ) highlights the
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METHODS AND RESOURCES ARTICLE
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 Michael E. Donaldson1 · Katelyn Jackson2 · Yessica Rico3,4 · Josh B. Sayers5 · Danielle M. Ethier6 · Christopher J. Kyle1,7 Received: 30 August 2019 / Accepted: 30 March 2020 © Springer Nature B.V. 2020
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 4% and filtered out individuals with 4% per individual is converted from the MPAF value to either present (1) or absent (0). To determine the presence of novel DRB exon 2 alleles, we compared the resulting DRB exon 2 sequences from alleles with an MPAF > 4% to the 26 T. taxus MHC class II antigen DRB alleles in GenBank (accession numbers KU059084.1–KU059109.1; Rico et al. 2016). Finally, to determine the presence of an X or Y amplification (and infer sex of the sample), we calculated the total number of ZFX or SRY sequences detected in each sample and used a threshold of > 50 to indicate successful amplification of those regions.
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Conservation Genetics Resources
Results and discussion Individual identification via non-invasive sampling methods has the potential to provide critical insight into the life history of elusive and endangered species that are difficult to observe directly (e.g., Manlick et al. 2017), and guide conservation and management decisions. Herein, we used DNA isolated from T. t. jacksoni hair and tissue samples to amplify a set of 28 dinucleotide repeats, two sex-determining markers, along with a highly polymorphic immunoge
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