Low-Input High-Molecular-Weight DNA Extraction for Long-Read Sequencing From Plants of Diverse Families

Abstract

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION^® platform, with and without the use of a short fragment depletion kit.

Keywords: Circulomics; DNA extraction; DNA sequencing; ONT long read sequencing; PacBio; genome assembly; nanopore sequencing; plant genome.

FIGURE 1

Schematic overview of the DNA extraction method. The individual steps are described in detail in the see section “Methods” and in the accompanying online protocol.

FIGURE 2

Comparison of DNA extraction performance for different extraction methods. (A–C) Output from a NanoDrop spectrophotometer. (A) DNA extraction results with the Mayjonade et al. (2016) protocol, without β-ME and phenol:chloroform purification step. (B) DNA extraction with lysis buffer as described by Schalamun et al. (2019). (C) DNA extracted with the protocol described in this study (sample OPH_3). (D) TapeStation results showing the fragment size distribution and the DNA Integrity Number (DIN), for six O. sphegodes samples.

FIGURE 3

Fragment length profiles of extracted HMW DNA. (A–C) Femto Pulse profiles showing integrity and size of gDNA. (A) O. sphegodes sample OPH_3 with peaks at 110 and 153 kbp. (B) L. multiflorum (sample RAB_2) with a peak at 167 kbp. (C) B. incana (sample BRI_1) showing a broader fragment size distribution centred at 50 kbp. (D) Gel pictures for G. diffusa DNA extracts (GOR_1-4, from a pool of two initial extractions).

FIGURE 4

Nanopore sequencing read length distributions. Read length distribution for O. sphegodes OPH_3 (blue; without Circulomics kit) and L. multiflorum RAB_2_Circ (red; after Circulomics kit), showing (A) the entire normalised histogram of log-transformed read lengths and (B) a zoom-in into the section showing the longest reads (OPH_3 = 1.7 Mbp; RAB_2_Circ = 464 kbp).