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

Abstract

Targeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals, but studies describing detailed workflows of GTseq panel development are rare. Here, we develop a dual-purpose GT-seq panel for walleye (Sander vitreus) and discuss trade-offs associated with different development and genotyping approaches. Our GT-seq panel was developed using restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We then conducted simulations to test the utility of loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We conducted three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. Optimization focused on reducing variation in amplification rate among loci and minimizing the proportion of off-target sequence, both of which are important considerations for developing large GT-seq panels. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (1) inexpensive Chelex extractions performed well for genotyping, (2) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was likely unnecessary, and (3) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings should aid in their construction in myriad taxa.

The ghosts of propagation past: haplotype information clarifies the relative influence of stocking history and phylogeographic processes on contemporary population structure of walleye (Sander vitreus)

“…Restriction site‐associated DNA sequencing data were available from Bootsma et al (2020) for 954 walleye from 23 populations, with an average of 42 individuals per population. Sequencing yielded 1,463,269 retained reads per individual, on average (range = 22,506–8,156,976).…”

“…In the current study, we analyzed genotypes from 954 walleye sampled from 23 populations across Wisconsin and Minnesota. All samples were previously genotyped using restriction site‐associated DNA (RAD) sequencing by Bootsma et al (2020), and represent each of the major drainages across the study range. The highest sampling effort was weighted toward the upper Chippewa and upper Wisconsin drainages in Wisconsin, two watersheds that border each other and were challenging to differentiate using microsatellites (Hammen & Sloss, 2019).…”

“…We conducted de novo construction of loci rather than aligning to a related reference genome as suggested by Paris et al, (2017). A raw vcf file containing genotypes for all individuals, available from Bootsma et al (2020), was used to select SNPs for downstream analyses; this vcf was produced in the populations module of STACKS v2.2 with two loose filtering parameters: retain only SNPs present in >5% of individuals and a minimum minor allele frequency >0.005.…”

“…Red lines intersecting connections show boundaries between neighbors, with line thickness representing the order in which the boundary was identified (i.e., strength. Thickest line is strongest) DNA (RAD) sequencing by Bootsma et al (2020), and represent each of the major drainages across the study range. The highest sampling effort was weighted toward the upper Chippewa and upper Wisconsin drainages in Wisconsin, two watersheds that border each other and were challenging to differentiate using microsatellites (Hammen & Sloss, 2019).…”

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

“…Moving forward, GT-seq and other targeted amplicon sequencing approaches hold great promise for informing conservation and management of biodiversity [66]. Originally demonstrated in steelhead trout [24], GT-seq has been subsequently applied to the study of other fish species, including redband trout [66], brook trout [67], Pacific lamprey [68], and walleye [69]. More recently, the efficacy of using GT-seq for genotyping minimally-invasive samples has been demonstrated [46], and successfully applied to further understanding of the molecular ecology and conservation status of the at-risk Western rattlesnake [70].…”