HD-Marker: a highly multiplexed and flexible approach for targeted genotyping of more than 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 and ), 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.