NanoGBS: A Miniaturized Procedure for GBS Library Preparation

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.

Genome-Wide Association Study of Soybean Germplasm Derived From Canadian × Chinese Crosses to Mine for Novel Alleles to Improve Seed Yield and Seed Quality Traits

“…GBS was conducted following the methods and recommendations outlined by Elshire et al (2011) , Sonah et al (2013) , and Torkamaneh et al (2020a , c) . The GBS library was created with Ape KI restriction enzyme digestion.…”

A bumper crop of SNPs in soybean through high‐density genotyping‐by‐sequencing (HD‐GBS)

“…To validate these simulations experimentally, we constructed GBS libraries for a set of 96 diverse soybean accessions using Bfa I digestion and the standard GBS (Std‐GBS) protocol (Torkamaneh et al ., 2020a). After size selection and PCR amplification, the quality of the GBS library was assessed and the resulting profile (Figure 1b) indicated that the vast majority of size‐selected fragments (including sequencing adapters) ranged between 200 and 600 bp.…”

“…Genotyping cost per sample can be dramatically decreased by miniaturizing library preparation (e.g. NanoGBS) and an increased multiplexing level (Torkamaneh et al ., 2020a). We also developed a plate barcoding strategy (Colston‐Nepali et al ., 2019) that enables the multiplexing of over 4500 GBS samples compatible with the highest throughput Illumina sequencing platforms (such as NovaSeq), thus enabling to tap into the economies of scale in sequencing (Figure 1g; HD‐GBS+).…”

Endosperm variability: from endoreduplication within a seed to higher ploidy across species, and its competence

“…With the availability of computational facilities and robust sequencing technologies, the importance of genomics for the identification of genes and pathways is undisputed. Notably, genome-wide scanning approaches and digital candidate gene identification approaches, such as QTL, LD mapping, GWAS, GBS, and functional annotation, either alone or in combination with traditional candidate gene approaches are now frequently used (Mackay and Powell, 2007;Götz et al, 2008;McCarthy et al, 2008;Chen et al, 2009;Zhang et al, 2010Zhang et al, , 2020Bush and Moore, 2012;Glaubitz et al, 2014;Torkamaneh et al, 2020). The following three subsections provide an overview of the strength of genomics, with special reference towards understanding endosperm variability (both across species and within the seed).…”