Isolating Linum usitatissimum L. Nuclear DNA Enabled Assembling High-Quality Genome

Abstract

High-quality genome sequences help to elucidate the genetic basis of numerous biological processes and track species evolution. For flax (Linum usitatissimum L.)—a multifunctional crop, high-quality assemblies from Oxford Nanopore Technologies (ONT) data were unavailable, largely due to the difficulty of isolating pure high-molecular-weight DNA. This article proposes a scheme for gaining a contiguous L. usitatissimum assembly using Nanopore data. We developed a protocol for flax nuclei isolation with subsequent DNA extraction, which allows obtaining about 5 μg of pure high-molecular-weight DNA from 0.5 g of leaves. Such an amount of material can be collected even from a single plant and yields more than 30 Gb of ONT data in two MinION runs. We performed a comparative analysis of different genome assemblers and polishers on the gained data and obtained the final 447.1-Mb assembly of L. usitatissimum line 3896 genome using the Canu—Racon (two iterations)—Medaka combination. The genome comprised 1695 contigs and had an N50 of 6.2 Mb and a completeness of 93.8% of BUSCOs from eudicots_odb10. Our study highlights the impact of the chosen genome construction strategy on the resulting assembly parameters and its eligibility for future genomic studies.