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

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 amplicon sequencing panels is non-trivial, but studies describing detailed workflows of GTseq panel development are rare. Here, we develop a dual-purpose GT-seq panel for walleye (Sander vitreus), outline a generalized workflow for panel development, and discuss trade-offs associated with different development and genotyping approaches. 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 then performed 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 conducted three rounds of primer optimization to remove loci that overamplified, yielding a final panel 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: (1) inexpensive Chelex extractions performed well for genotyping, (2) the exonuclease I and shrimp alkaline phosphatase (Exo-SAP) 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 workflow and findings should aid in their construction in myriad taxa.