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.
Highlights
Genome-wide genotyping of large sets of samples, an essential component in a wide range of genetic studies, is greatly facilitated by genotyping polymorphic loci, called genetic variants or markers, using next-generation sequencing (NGS)–based methods (Rasheed et al, 2017)
GBS libraries were constructed for a set of 96 soybean samples using StdGBS and NanoGBS methods
By miniaturizing GBS library preparation, the NanoGBS method saved 90% in reagent usage and reduced handling time by 75% (Table 1) compared to StdGBS
Summary
Genome-wide genotyping of large sets of samples, an essential component in a wide range of genetic studies, is greatly facilitated by genotyping polymorphic loci, called genetic variants or markers, using next-generation sequencing (NGS)–based methods (Rasheed et al, 2017). GBS methods have been widely applied for genome-wide genotyping of large multiplexed samples of both model [e.g., human (Luca et al, 2011); Arabidopsis (Begali, 2018)] and nonmodel species [e.g., cattle (De Donato et al, 2013); pigs (Chen et al, 2013); maize (Elshire et al, 2011); cucumber (Zhu et al, 2016); fungi (Leboldus et al, 2015); insects (Dupuis et al, 2017); nematodes (Mimee et al, 2015)] where alternative genotyping tools (e.g., SNP arrays) are typically unavailable (Fonseca et al, 2016). The strengths and limitations of GBS methods have been comprehensively discussed and reviewed in plants (He et al, 2014), livestock (Gurgul et al, 2019), fisheries and aquaculture (Li and Wang, 2017), and ecological and conservation genomics (Narum et al, 2013)
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