High-molecular-weight DNA extraction for broadleaved and conifer tree species

Abstract

Abstract Plant genome sequencing based on long reads has increasingly been applied also to tree species in recent years. A crucial step in these genome projects is the successful extraction of high-molecular-weight DNA in high quality and sufficient quantity, which is imperative for long-read sequencing. The extraction of high-molecular-weight DNA in trees has been limited and difficult to conduct. To achieve successful extraction, modification of the protocol for tree species is necessary. Here, we present a protocol for high-molecular-weight DNA extraction from the broadleaved tree species Fraxinus excelsior and from the conifer species Taxus baccata. The protocol is based on nuclei isolation and is divided into two main steps, i.e. nuclei separation based on the nuclei isolation buffer and DNA extraction using the Nanobind® plant nuclei kit by Pacific Biosciences (PacBio). The protocol can be applied to different tree species to obtain high-molecular-weight DNA in high quality, which can be used for Oxford Nanopore Technologies (ONT) and PacBio sequencing. ONT sequencing using four high-molecular-weight DNA preparations from Fraxinus excel-sior resulted in different read length N50 values (12.91-38.19 kb) and total base output (5.81-23.17 Gb), emphasizing the complex nature of the sequencing pipeline of high-molecular-weight DNA. PacBio HiFi sequencing of Taxus baccata produced circular consensus sequencing reads with an average length of 12.9 kb and an N50 of 13.59 kb. Altogether, this study presents a protocol to obtain high-molecular-weight DNA from different challenging tree species and discusses several important points that can be considered when adapting the protocol to additional species.