A GT‐seq panel for walleye (Sander vitreus) provides important insights 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. Development of GT-seq panels is nontrivial, but studies describing trade-offs associated with different steps of GT-seq panel development are rare. Here, we construct a dual-purpose GT-seq panel for walleye (Sander vitreus), discuss trade-offs associated with different development and genotyping approaches, and provide suggestions for researchers constructing their own GT-seq panels. Our GT-seq panel was developed using an ascertainment set consisting of restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We conducted simulations to test the utility of all loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We then performed three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. 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: (i) inexpensive Chelex extractions performed well for genotyping; (ii) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was probably unnecessary; and (iii) 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 and suggestions should aid in their construction in myriad taxa.

Mixed-stock analysis in the age of genomics: Rapture genotyping enables evaluation of stock-specific exploitation in a freshwater fish population with weak genetic structure

“…Genotyping-by-sequencing methods, like Rapture, are a feasible way to obtain this level of diagnostic power in a non-model species and can provide reliable mixture estimates. Once stock differences are identified, markers from high-density marker panels can be filtered to identify smaller subsets of highly-informative markers that can be incorporated into even higher throughput genotyping-in-thousands (GT-seq) panels for long-term monitoring and annual mixed-stock assessments (Bootsma et al 2020). For this reason, we are optimistic that our approach will offer a new means for agencies managing fisheries with weak population structure to discern local spawning stocks such that their relative contributions can be quantified.…”

eDNA metabarcoding outperforms traditional fisheries sampling and reveals fine‐scale heterogeneity in a temperate freshwater lake

“…We then produced the final metabarcoding libraries using latter steps in the genotyping‐in‐thousands by sequencing (GT‐seq) protocol (Campbell et al, 2015) following Bootsma et al (2020). Briefly, each individual and plate was barcoded in a second indexing PCR, and barcoded products were normalized using SequalPrep DNA Normalization plates (Invitrogen).…”

“…We then produced the final metabarcoding libraries using latter steps in the genotyping-in-thousands by sequencing (GT-seq) protocol (Campbell et al, 2015) following Bootsma et al (2020). Briefly,…”

It’s complicated: Heterogeneous patterns of genetic structure in five fish species from a fragmented river suggest multiple processes can drive differentiation

“…STACKS parameters for all species were as follows: process_radtags (‐e SbfI, ‐c, ‐q, ‐filter_illumina, ‐r, ‐‐bestrad, ‐t 140), ustacks (‐‐disable‐gapped, ‐‐model_type bounded, ‐‐bound_high 0.05, ‐M 3, ‐max_locus_stacks 4, ‐m 3, ‐H, ‐p 32), cstacks (‐n 3, ‐p 6 ‐‐disable_gapped). These STACKs parameters were derived based on review papers by Mastretta‐Yanes et al ( 2015 ) and Paris et al ( 2017 ) and have been shown to work well for similar fish species with nonduplicated genomes (e.g., Bootsma et al, 2020 ). SNPs genotyped in >30% of individuals (parameter flag: ‐r 0.3) were exported with the subprogram populations in variant call format (vcf) files.…”