Targeted genome-wide SNP genotyping in feral horses using non-invasive fecal swabs

Abstract

The development of high-throughput sequencing has prompted a transition in wildlife genetics from using microsatellites toward sets of single nucleotide polymorphisms (SNPs). However, genotyping large numbers of targeted SNPs using non-invasive samples remains challenging due to relatively large DNA input requirements. Recently, target enrichment has emerged as a promising approach requiring little template DNA. We assessed the efficacy of Tecan Genomics’ Allegro Targeted Genotyping (ATG) for generating genome-wide SNP data in feral horses using DNA isolated from fecal swabs. Total and host-specific DNA were quantified for 989 samples collected as part of a long-term individual-based study of feral horses on Sable Island, Nova Scotia, Canada, using dsDNA fluorescence and a host-specific qPCR assay, respectively. Forty-eight samples representing 44 individuals containing at least 10 ng of host DNA (ATG’s recommended minimum input) were genotyped using a custom multiplex panel targeting 279 SNPs. Genotyping accuracy and consistency were assessed by contrasting ATG genotypes with those obtained from the same individuals with SNP microarrays, and from multiple samples from the same horse, respectively. 62% of swabs yielded the minimum recommended amount of host DNA for ATG. Ignoring samples that failed to amplify, ATG recovered an average of 88.8% targeted sites per sample, while genotype concordance between ATG and SNP microarrays was 98.5%. The repeatability of genotypes from the same individual approached unity with an average of 99.9%. This study demonstrates the suitability of ATG for genome-wide, non-invasive targeted SNP genotyping, and will facilitate further ecological and conservation genetics research in equids and related species.

Development of a high-density sub-species-specific targeted SNP assay for Rocky Mountain bighorn sheep (Ovis canadensis canadensis)

“…The 10 and 50k assays only retained ~79% and ~43% of faecal samples, respectively, and had accuracies of 93% and 92%, respectively. These values are low relative to the overall genotyping success of 85% and accuracy of 99.9% reported by Gavriliuc et al (2022) for ATG genotyping faecal samples from domestic horses ( Equus caballus ). However, there are methodological differences between the studies.…”

“…This form of SPET uses a simplified single primer design and functions by sequencing flanking regions around a probe to sequence target regions of interest ( Barchi et al, 2019 ). Despite being a relatively new technology, it has proven applications in humans ( Nairismägi et al, 2016 ; Saber et al, 2017 ; Scolnick et al, 2015 ), non-human mammals ( Andrews et al, 2021 ; Gavriliuc et al, 2022 ), plants ( Barchi et al, 2019 ; Gramazio et al, 2020 ; Scaglione et al, 2019 ), arthropods ( Chang et al, 2020 ; Vu et al, 2023 ), and bacteria ( Benjamino et al, 2021 ; Homeier-Bachmann et al, 2022 ).…”

“…Thus, all the resources required to develop a targeted-GBS assay are available. Additionally, Tecan’s ATG requires modest quantities of genomic DNA which make it well suited for field studies relying on non-invasively collected sample types such as faeces ( Albaugh et al, 1992 ; Gavriliuc et al, 2022 ).…”

Pitfalls and recommendations for large-scale SNP genotyping in a non-model endangered species: the southern damselflies (Coenagrion mercuriale) as a case study

“…Additionally, for about a third of the targeted SNPs, only one of the probes of the pair was found (Table S4). This drives variability in the sequencing depth between sites covered by two probes or by just a single probe (Scaglione et al ., 2019; Gavriliuc et al ., 2022).…”

“…Based on a single primer enrichment technology (SPET), ATG relies on the design of ∼40 bp long probes based on reference genomes or transcriptomes, enabling to target with high reproducibility specific genomic regions carrying genetic variants of interest. The method has so far mainly been used in human health (Scolnick et al ., 2015; Nairismägi et al ., 2016; Saber et al ., 2017) or plants of agronomic interest (Barchi et al ., 2019; Scaglione et al ., 2019; Baccichet et al ., 2022; Tripodi et al ., 2023) and its use on wildlife species for conservation purposes is only starting to develop (Gramazio et al ., 2020; Gavriliuc et al ., 2022).…”