A Practical Framework for <scp>GT</scp> ‐Seq Panel Optimization

Genotyping-in-thousands by sequencing (GT-seq) panels are powerful tools in ecological, evolutionary and conservation genomics, yet the optimization process critical for robust and reproducible genotyping remains poorly formalized. Here, we present an iterative workflow for GT-seq panel optimization that emphasizes systematic refinement, quality control and structured decision-making to improve panel performance across diverse populations and study contexts. We illustrate this framework through the development and optimization of a GT-seq panel for white-tailed deer, a widely distributed and ecologically important North American species. From an initial set of 1200 candidate SNPs selected from a commercial microarray (OVSNP60, containing 72,728 SNPs) and prioritized for high heterozygosity, primers were designed for 646 loci. The final optimized panel contains 508 high-performing markers retained after iterative removal of overamplifying primer pairs, adjustment of primer concentrations, PCR conditions and bioinformatic filtering. The overall proportion of SNPs with more than 70% genotype rate increased from 25.5% in the first optimization round to 87.8% in the final round. Consequently, the overall genotype rate increased from 39.4% to 84%. We also identify key quality-control checkpoints and practical criteria to guide panel refinement and ensure consistent performance. By prioritizing optimization as an integral component of GT-seq panel development, this work provides a reproducible framework for generating robust, high-throughput genotyping tools in non-model species and underscores the importance of iterative refinement to maximize data quality and utility.