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.
