3D-GBS: a universal genotyping-by-sequencing approach for genomic selection and other high-throughput low-cost applications in species with small to medium-sized genomes

Abstract

Despite the increased efficiency of sequencing technologies and the development of reduced-representation sequencing (RRS) approaches allowing high-throughput sequencing (HTS) of multiplexed samples, the per-sample genotyping cost remains the most limiting factor in the context of large-scale studies. For example, in the context of genomic selection (GS), breeders need genome-wide markers to predict the breeding value of large cohorts of progenies, requiring the genotyping of thousands candidates. Here, we introduce 3D-GBS, an optimized GBS procedure, to provide an ultra-high-throughput and ultra-low-cost genotyping solution for species with small to medium-sized genome and illustrate its use in soybean. Using a combination of three restriction enzymes (PstI/NsiI/MspI), the portion of the genome that is captured was reduced fourfold (compared to a “standard” ApeKI-based protocol) while reducing the number of markers by only 40%. By better focusing the sequencing effort on limited set of restriction fragments, fourfold more samples can be genotyped at the same minimal depth of coverage. This GBS protocol also resulted in a lower proportion of missing data and provided a more uniform distribution of SNPs across the genome. Moreover, we investigated the optimal number of reads per sample needed to obtain an adequate number of markers for GS and QTL mapping (500–1000 markers per biparental cross). This optimization allows sequencing costs to be decreased by ~ 92% and ~ 86% for GS and QTL mapping studies, respectively, compared to previously published work. Overall, 3D-GBS represents a unique and affordable solution for applications requiring extremely high-throughput genotyping where cost remains the most limiting factor.

Comparative restriction enzyme analysis of methylation (CREAM) reveals methylome variability within a clonal in vitro cannabis population

“…Two sequencing libraries were prepared in parallel for each sample with the extracted DNA using the Comparative Restriction Enzyme Analysis of Methylation (CREAM) approach ( Figure 1 ) at the Plateforme d’Analyses Génomiques 2 at the Institut de Biologie Intégrative et des Systèmes (IBIS) of Université Laval, Quebec, Canada. The CREAM method builds on the 3D-GBS approach ( de Ronne et al., 2023 ). Briefly, in both libraries, DNA molecules were cleaved at one end either by the restriction enzymes Nsi I (New England Biolabs) or Pst I (New England Biolabs), which have distinct restriction sites (5’-ATGCA/T-3’ and 5’-CTGCA/G-3’, respectively) to anchor DNA fragments at specific locations in the genome.…”

Neutral markers reveal complex population structure across the range of a widespread songbird

“…Libraries were prepared using Peterson et al. ( 2013 ) with modifications to include a third enzyme (de Ronne et al., 2023 ). The inclusion of a third restriction enzyme allows for the cutting of adapter dimers which improves adapter ligation and thus improves the yield of amplified reads (Bayona‐Vásquez et al., 2019 ).…”

Tufted Puffins exhibit low levels of genetic differentiation among breeding colonies in North America

“…For 3dRAD sequencing (Graham et al 2015, Bayona Vásquez et al 2019), genomic library preparation was completed at the Université Laval. DNA was digested with 3 restriction enzymes, PstI , NsiI , MspI , and libraries were prepared following the protocol outlined in de Ronne et al (2023). Libraries were then sent to Génome Québec at McGill University and sequenced using NovaSeq6000 S4 100-bp paired-end sequencing.…”