DOI: 10.5061/dryad.0vt4b8h1n
title: Data from: Targeted genome-wide SNP genotyping in feral horses using non-invasive fecal swabs

description: 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 10ng 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 86.7%
 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.
description: <em><strong>Targeted SNP sequencing panel
 design </strong></em> To design the panel
 for ATG, 300 SNPs present on the Illumina Equine GGP65 Plus array and
 shown to be polymorphic in the Sable Island population were selected. SNPs
 with minor allele frequency (MAF) &gt; 0.30 and exhibiting limited
 linkage disequilibrium as determined by the PLINK
 <em>--indep-pairwise</em> command with a window size of 50, a
 step size of 5 and a variance inflation factor of 0.5 (Purcell et al.,
 2007) were selected. An assay was then developed by Tecan Genomics
 (Redwood City, United States) covering 279 of the 300 originally submitted
 SNPs (Appendix 1). In this assay, 237 targets were covered by 2 probes
 while 42 were covered by a single probe.
 <em><strong>Fecal DNA samples
 collection</strong></em> Horse DNA was
 collected by swabbing the mucus layer surrounding freshly deposited feces
 using a polyester swab attached to a 5mL vial (SIMPORT T307-5A). Vials
 were preloaded with 400µl of Aquastool<sup>TM</sup> solution
 (MultiTarget Pharmaceuticals) and kept in insulated bags containing
 icepacks after collection in the field and transferred to -20°C when
 returning to the laboratory on the same day. Samples were transported by
 air to the mainland (frozen) at the end of each field season and archived
 at -80°C until DNA extraction.
 <em><strong>Fecal DNA
 extraction</strong></em> DNA was isolated
 using a modified version of the Aquastool<sup>TM</sup>
 Solution recommended protocol (MultiTarget Pharmaceuticals). First, thawed
 swab vials were vortexed at full speed for 1 minute, and 200µl of
 homogenized solution was transferred to a 1.5ml microfuge tube. Samples
 were then incubated at room temperature for 15 minutes, vortexed for 60
 seconds, and centrifuged at full speed on a microcentrifuge (14,000 rpm)
 for 5 minutes to pellet debris. The clear supernatant (~200µl) was
 transferred to a 1.5ml tube pre-loaded with 160µl of isopropanol and
 vortexed for 10 seconds. Tubes where then centrifuged at full speed for 5
 minutes, and the supernatant removed and discarded. DNA pellets were then
 rinsed twice with 70% ethanol before being air dried and resuspended in
 60µl of molecular grade water. Once DNA pellets dissolved, samples were
 centrifuged at full speed for 5 minutes to pellet contaminants. Clear
 supernatant containing DNA were transferred to new cryotubes, and archived
 at -80°C. <em><strong>Fecal DNA
 quantification</strong></em> We quantified
 total (host + exogenous) DNA concentration in samples using 2µl of
 template DNA and a Qubit 4 or BioTek Synergy LX Multi-Mode Microplate
 Reader with a Qubit or Quant-It dsDNA High-Sensitivity or Broad Range
 Assay Kit (Thermo Fisher Scientific) according to manufacturer protocols.
 To assess how much of the total DNA was attributable to host, we applied a
 qPCR approach targeting the single copy nuclear F2 gene using
 equine-specific primers known to be effective across horse breeds
 (Forward: 5’-GCCAGCAGGCTGAGAACG-3’, Reverse:
 5’-TGGTGCAGTTGATTCTGGAATAGGAAATTT-3’; Floren et al., 2015) and horse DNA
 extracted from muscle tissue as a standard (10x dilution series: 20ng/μl -
 0.0002ng/μl). Samples, standards, and negative controls were run in
 duplicate using a Bio-Rad CFX96 qPCR System, with each reaction containing
 2μl of template, 10.0μl of 2xSEnsiFAST SYBR MIX, 0.8μl of 10μM forward
 primer, 0.8μl of 10μM reverse primer, and 6.4μl molecular grade
 water. Thermocycling conditions consisted of 95°C for 3 minutes (for
 polymerase activation) followed by 40 amplification cycles (95°C for 5
 seconds, 60°C for 10 seconds, and 72°C for 10 seconds).
description: The presented data are used in our pilot study of Allegro
 Targeted Genotyping and consists of paired, raw sequence reads in .fastq
 format. Code to call variants is available at https://github.com/sgavril/Nugen-Pilot-Analysis