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

Abstract

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 generated within and among RADseq and GT‐seq for the same individuals found low rates of genotyping error (GT‐seq: 0.50%; RADseq: 0.80%) and discordance (2.57%), the latter likely due to the different genotype calling models employed. GT‐seq mean genotype discordance between blood and cloacal swab samples collected from the same individuals was also minimal (1.37%). Estimates of population diversity parameters were similar across GT‐seq and RADseq data sets, as were inferred patterns of population structure. Overall, GT‐seq can be effectively applied to low‐quality DNA samples, minimizing the inefficiencies presented by exogenous DNA typically found in minimally invasive samples and continuing the expansion of molecular ecology and conservation genetics in the genomics era.

RapidRat: Development, validation and application of a genotyping-by-sequencing panel for rapid biosecurity and invasive species management

“…Of these, four individuals were replicated to explicitly assess genotyping error rate (see below), resulting in a total 96 samples in our initial library. GT-seq library preparation followed the original protocol [24] with some modifications [46]. DNA extractions for all individuals were normalized to 20 ng/uL to ensure even amplification across samples and diluted the PCR1 products 1:10 before use in PCR2.…”

“…While GT-seq does offer many advantages over other approaches (e.g., reduced representation sequencing, including ddRAD), panel development does require existing genomic data for locus identification and primer design [24,46]. For this study, we had previously genotyped brown and black rats across their distributions in Haida Gwaii, as well as characterized population structure among island populations [25].…”

A GT-seq panel for walleye (Sander vitreus) provides a generalized workflow for efficient development and implementation of amplicon panels in non-model organisms

“…GT-seq and other amplicon sequencing methods have tremendous potential for facilitating high-throughput genotyping in non-model organisms (Meek & Larson, 2019). The general steps for GT-seq panel development: SNP ascertainment, SNP selection, primer design, and panel optimization have been previously detailed (Baetscher et al, 2018; McKinney et al, 2019; Schmidt et al, 2019); however, the process of GT-seq panel development is not static. Here, we leverage our experiences developing a GT-seq panel for walleye with testing various aspects of the GT-seq methodological process to provide additional guidelines usable by other researchers to simplify panel construction and validation, particularly in non-model species.…”

“…Pedigree analysis in wild populations is highly dependent upon the ability to genotype large sample sizes to increase the likelihood of detecting kin relationships, toward which GT-seq is ideally suited. Moreover, GT-seq has proven capable of generating high-quality genotypes from low-quality DNA samples (Natesh et al, 2019; Schmidt et al, 2019), making it a viable approach for monitoring endangered or elusive species.…”

“…Moreover, developing GT-seq panels is a relatively involved task and, to this point, there are limited resources providing standardized workflows and guidelines for efficient panel construction (but see Campbell et al, 2015; McKinney et al, 2019). At a basic level, panel construction involves SNP discovery, SNPs selection, primer design, and panel optimization (see Baetscher et al, 2018; McKinney et al, 2019; Schmidt et al, 2019); however, within this general framework there are many decision points in panel development related to primer selection, multiplexing approaches, laboratory protocols, and analysis parameters that have yet to be addressed. We used walleye ( Sander vitreus ) from Minnesota and Wisconsin, USA, as a test case to investigate various tradeoffs associated with GT-seq panel development and optimization and leveraged our collective experience to provide guidelines for researchers developing GT-seq panels.…”