High-quality DNA extraction method enabling long-read sequencing in challenging native plant species

Abstract

The extraction of high-quality, high-molecular-weight (HMW) DNA from plant tissue is a critical step for successful long-read sequencing, particularly in species with high secondary metabolite content. Myracrodruon urundeuva (“Aroeira”, Anacardiaceae), a native tree species from Brazil, presents significant challenges for DNA extraction due to its rich composition of polyphenols and polysaccharides, which can inhibit enzymatic reactions and compromise sequencing efficiency. In this study, we present an optimized DNA extraction protocol specifically tailored for native forest species with high secondary metabolite content. The protocol integrates a sorbitol pre-wash step to reduce contaminants, an improved CTAB-based extraction method, and a subsequent clean-up process using proteinase K and RNAse A, followed by short-fragment elimination to enrich HMW DNA. Our approach significantly improved DNA purity, achieving 260/280 ratios close to 1.8 and 260/230 ratios above 1.9, with DNA fragment sizes averaging over 60 kbp. The extracted DNA was successfully used in Oxford Nanopore Technologies (ONT) long-read sequencing, yielding high read lengths and sequencing coverage. These findings enhance genomic studies of native species, facilitating conservation efforts, genetic diversity assessments, and sustainable management strategies. The proposed protocol offers an efficient and cost-effective alternative to obtain high-quality DNA in species with challenging biochemical compositions, supporting the advancement of third-generation sequencing technologies in plant genomics.