Efficient high-quality and high molecular weight plant DNA extraction protocol using Percoll™

Next generation long-read sequencing is a powerful approach to generate de novo genome assemblies, however it requires high-quality and high molecular weight (HMW) DNA to reach near-chromosome level assemblies. Some plants are reported as recalcitrant to high-quality HMW DNA extraction due to high levels of secondary metabolites. <i>Streptocarpus schliebenii</i> (Gesneriaceae) is one of those highly recalcitrant plants and its DNA extraction failed repeatedly with previously published protocols. Percoll™ is a silica-based colloid coated with polyvinylpyrrolidone and utilized for various aspects in phase separation including plants' nuclei isolation for Hi-C library and DNA extraction to generate bacterial artificial chromosome clones. In this study, we developed a HMW DNA extraction protocol for long-read sequencing that included a Percoll™ gradient step. To establish a stable protocol, we examined and modified buffers and steps of several previous Percoll™ gradient protocols. Instead of the previously used agarose plug method for Percoll™ DNA extraction, CTAB lysis followed by Qiagen Genomic-Tips was employed. Three <i>Streptocarpus</i> species generated optimal quality and HMW DNA. The method was further tested in 12 species across a wide range of plant lineages. The results were species specific. While HMW DNA was obtained from seven species, HMW and high-quality DNA were obtained from four species, i.e. <i>Iris pseudacorus</i>, <i>Pulmonaria affinis</i>, <i>Corytoplectus speciosus</i>, and <i>Ilex aquifolium</i>. This indicates the wide applicability of this protocol for plants. This protocol provides a useful resource for those who are working on de novo plant genome projects of recalcitrant material to obtain optimal DNA for long-read sequencing.