High-molecular-weight DNA extraction for long-read sequencing of plant genomes: An optimization of standard methods.

Premise: Developing an effective and easy-to-use high-molecular-weight (HMW) DNA extraction method is essential for genomic research, especially in the era of third-generation sequencing. To efficiently use technologies capable of generating long-read sequences, it is important to maximize both the length and purity of the extracted DNA; however, this is frequently difficult to achieve with plant samples. Methods and Results: We present a HMW DNA extraction method that combines (1) a nuclei extraction method followed by (2) a traditional cetyltrimethylammonium bromide (CTAB) DNA extraction method for plants with optimized extraction conditions that influence HMW DNA recovery. Our protocol produced DNA fragments (percentage of fragments >20 kbp) that were, on average, ca. five times longer than those obtained using a commercial kit, and contaminants were removed more effectively. Conclusions: This effective HMW DNA extraction protocol can be used as a standard protocol for a diverse array of taxa, which will enhance plant genomic research.