HD-Marker: a highly multiplexed and flexible approach for targeted genotyping of over 10,000 genes in a single-tube assay.

Targeted genotyping of transcriptome-scale genetic markers is highly attractive for genetic, ecological, and evolutionary studies, but achieving this goal in a cost-effective manner remains a major challenge, especially for laboratories working on nonmodel organisms. Here, we develop a high-throughput, sequencing-based GoldenGate approach (called HD-Marker), which addresses the array-related issues of original GoldenGate methodology and allows for highly multiplexed and flexible targeted genotyping of more than 12,000 loci in a single-tube assay (in contrast to fewer than 3100 in the original GoldenGate assay). We perform extensive analyses to demonstrate the power and performance of HD-Marker on various multiplex levels (296, 795, 1293, and 12,472 genic SNPs) across two sequencing platforms in two nonmodel species (the scallops Chlamys farreri and Patinopecten yessoensis), with extremely high capture rate (98%-99%) and genotyping accuracy (97%-99%). We also demonstrate the potential of HD-Marker for high-throughput targeted genotyping of alternative marker types (e.g., microsatellites and indels). With its remarkable cost-effectiveness (as low as $0.002 per genotype) and high flexibility in choice of multiplex levels and marker types, HD-Marker provides a highly attractive tool over array-based platforms for fulfilling genome/transcriptome-wide targeted genotyping applications, especially in nonmodel organisms.