Targeted Genotyping of a Whole-Gene Repertoire by an Ultrahigh-Multiplex and Flexible HD-Marker Approach

Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important. However, for non-model organisms, large-scale target genotyping in a cost-effective manner remains a major challenge. To address this issue, we present an ultrahigh-multiplex, in-solution probe array-based high-throughput diverse marker genotyping (HD-Marker) approach that is capable of targeted genotyping of up to 86 000 loci, with coverage of the whole gene repertoire, in what is a 27-fold and six-fold multiplex increase in comparison with the conventional Illumina GoldenGate and original HD-Marker assays, respectively. We perform extensive analyses of various ultrahigh-multiplex levels of HD-Marker (30 k-plex, 56 k-plex, and 86 k-plex) and show the power and excellent performance of the proposed method with an extremely high capture rate (about 96%) and genotyping accuracy (about 96%). With great advantages in terms of cost (as low as 0.0006 USD per genotype) and high technical flexibility, HD-Marker is a highly efficient and powerful tool with broad application potential for genetic, ecological, and evolutionary studies of non-model organisms.