Abstract
High-throughput reduced-representation sequencing (RRS)-based genotyping methods, such as genotyping-by-sequencing (GBS), have provided attractive genotyping solutions in numerous species. Here, we present NanoGBS, a miniaturized and eco-friendly method for GBS library construction. Using acoustic droplet ejection (ADE) technology, NanoGBS libraries were constructed in tenfold smaller volumes compared to standard methods (StdGBS) and leading to a reduced use of plastics of up to 90%. A high-quality DNA library and SNP catalogue were obtained with extensive overlap (96%) in SNP loci and 100% agreement in genotype calls compared to the StdGBS dataset with a high level of accuracy (98.5%). A highly multiplexed pool of GBS libraries (768-plex) was sequenced on a single Ion Proton PI chip and yielded enough SNPs (~4K SNPs; 1.5 SNP per cM, on average) for many high-volume applications. Combining NanoGBS library preparation and increased multiplexing can dramatically reduce (72%) genotyping cost per sample. We believe that this approach will greatly facilitate the adoption of marker applications where extremely high throughputs are required and cost is still currently limiting.