Genotyping-in-thousands by sequencing (GT-seq) of non-invasive fecal and degraded samples: a new panel to enable ongoing monitoring of Canadian polar bear populations

Abstract

Genetic monitoring using non-invasive samples provides a complement or alternative to traditional population monitoring methods. However, Next Generation Sequencing approaches to monitoring typically require high quality DNA and the use of non-invasive samples (e.g. scat) is often challenged by poor DNA quality and contamination by non-target species. One promising solution is a highly multiplexed sequencing approach called Genotyping-in-thousands by sequencing (GT-seq), which can enable cost-efficient genomics-based monitoring for populations based on non-invasively collected samples. Here, we develop and validate a GT-seq panel of 324 single nucleotide polymorphisms (SNPs) optimized for genotyping of polar bears based on DNA from non-invasively collected fecal samples. We demonstrate 1) successful GT-seq genotyping of DNA from a range of sample sources, including successful genotyping of 85.7% of non-invasively collected fecal samples determined to contain polar bear DNA, and 2) that we can reliably differentiate individuals, ascertain sex, assess relatedness, and resolve population structure of Canadian polar bear subpopulations based on a GT-seq panel of 324 SNPs. Our GT-seq data reveal similar spatial-genetic patterns as previous polar bear studies but at lesser cost per sample and using non-invasively collected samples, indicating the potential of this approach for population monitoring. This GT-seq panel provides the foundation for a non-invasive toolkit for polar bear monitoring and contribute to community-based programs – a framework which may serve as a model for wildlife management and contribute to conservation and policy for species worldwide.

A genotyping‐in‐thousands by sequencing panel to inform invasive deer management using noninvasive fecal and hair samples

“…Here, we optimized a GT‐seq panel for utility with noninvasive fecal and hair samples; by adjusting the PCR1 conditions to account for low‐quantity and degraded DNA, we demonstrated high sample retention (97.6%), genotyping success (90.3%), and genotyping concordance (99.5%) relative to those produced from high‐quality tissue sample at the same panel of 244 SNPs. Importantly, the adjustments we made did not require additional laboratory equipment or expertise, and maintained the rapidity and cost‐effectiveness that has elevated GT‐seq as an MTAS approach of choice in molecular ecology (Eriksson et al, 2020; Hayward et al, 2022; Schmidt, Campbell, et al, 2020).…”

“…However, molecular studies involving elusive or at‐risk species often rely on noninvasive samples (e.g., feces, hair, or feathers) that can pose challenges for massively parallel DNA sequencing because of poor DNA quality, low DNA quantity, and exogenous DNA contamination (Andrews et al, 2018; Russello et al, 2015). Such impediments are now starting to be overcome, driven in part by multiplexed targeted amplicon sequencing (MTAS) approaches (Eriksson et al, 2020; Hayward et al, 2022; Natesh et al, 2019; Schmidt et al, 2020). MTAS can target hundreds of single nucleotide polymorphisms (SNP) within a single PCR to genotype thousands of individuals in parallel (Campbell et al, 2015), yet genotyping success and sample retention has varied widely when applying these approaches to noninvasive samples (Eriksson et al, 2020; Hayward et al, 2022; Natesh et al, 2019).…”

“…Such impediments are now starting to be overcome, driven in part by multiplexed targeted amplicon sequencing (MTAS) approaches (Eriksson et al, 2020; Hayward et al, 2022; Natesh et al, 2019; Schmidt et al, 2020). MTAS can target hundreds of single nucleotide polymorphisms (SNP) within a single PCR to genotype thousands of individuals in parallel (Campbell et al, 2015), yet genotyping success and sample retention has varied widely when applying these approaches to noninvasive samples (Eriksson et al, 2020; Hayward et al, 2022; Natesh et al, 2019).…”

“…For example, Eriksson et al (2020) demonstrated high levels of genotyping success and sample retention using coyote feces, but only multiplexed a small number of loci ( n = 26). In contrast, Hayward et al (2022) successfully genotyped several hundred loci using polar bear feces, but experienced only ~60% sample retention even following initial sample screening using species‐specific qPCR. Importantly, such studies employing MTAS have been largely limited to fecal samples despite the fact that hair remains a valuable DNA source for many mammalian systems dating back to the first studies employing noninvasive genetic sampling (Andrews et al, 2018; Morin & Woodruff, 1992; Taberlet & Bouvet, 1992; Waits & Paetkau, 2005).…”

“…Genotyping‐in‐Thousands by sequencing (GT‐seq; Campbell et al, 2015) is a MTAS approach currently available that has seen widespread utility across studies in ecology, evolution, and conservation (Chang et al, 2021, 2022; Hayward et al, 2022; Jo et al, 2021; Natesh et al, 2019; Schmidt et al, 2020; Setzke et al, 2021; Sjodin et al, 2020). Here, we employed a scalable workflow to develop a GT‐seq panel for invasive Sitka black‐tailed deer in Gwaii Haanas.…”