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. Streptocarpus schliebenii (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 Streptocarpus 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. Iris pseudacorus, Pulmonaria affinis, Corytoplectus speciosus, and Ilex aquifolium. 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.