Feasibility of using Oxford Nanopore Technology for population genomics in Fagus sylvatica

We conducted a pilot study to evaluate the feasibility of using Oxford Nanopore Technology (ONT) to analyze genetic variation among cohorts of European beech (Fagus sylvatica L.) individuals in the context of a long-term monitoring program. Oxford Nanopore guarantees support of reference chemistry for long-term studies, and ONT offers potential to recover structural and epigenetic variants as well as single nucleotide polymorphisms (SNPs). However, population genomics requires scalable approaches. We extracted DNA from dormant leaf buds of 18 adult trees and sequenced these either with individual barcodes, or as a single pool (ca. equimolar pooling) with the aim of saving costs. The comparison of individually barcoded versus pooled samples was conducted in duplicate (two replicate fl ow cells) using a PromethION. Results were generally reproducible across fl ow cells, but numbers of reads across individually barcoded samples varied, likely due to differences in DNA intactness or small differences in size distribution that may have been further biased during library preparation. Furthermore, a disproportionately low number of sequencing reads was obtained for the pooled sample. Although seemingly a cost-saving measure, we do not recommend sample pooling, as ensuring equal representation requires detailed, often cost-intensive characterization of samples prior to pooling, making low-coverage sequencing of individual samples often the more feasible option. The low-coverage data we obtained yielded interpretable SNP variation (which, however, remains more accurate using short-read sequencing techniques), but failed to yield reproducible data on epigenetic and structural variants. In conclusion, scaling ONT to population genomics would benefi t from the development of more cost-effective methods to ensure even representation across samples and to obtain more reproducible information on variants, especially structural and epigenetic variants, with lower sequencing coverage.