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 non-model 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 over 12,000 loci in a single-tube assay (in contrast to fewer than 3,100 in the original GoldenGate assay). We perform extensive analyses to demonstrate the power and performance of HD-Marker on various multiplex levels (296, 793, 1,293 and 12,472 genic SNPs) across two sequencing platforms in two non-model 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 non-model organisms.