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

Alessia Russo; Baptiste Mayjonade; Daniel Frei; Giacomo Potente; Roman T Kellenberger; Léa Frachon; Dario Copetti; Bruno Studer; Jürg E Frey; Ueli Grossniklaus; Philipp M Schlüter

doi:10.3389/fpls.2022.883897

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

Front Plant Sci. 2022 May 19:13:883897. doi: 10.3389/fpls.2022.883897. eCollection 2022.

Authors

Alessia Russo^{1

2

3}, Baptiste Mayjonade⁴, Daniel Frei⁵, Giacomo Potente³, Roman T Kellenberger⁶, Léa Frachon³, Dario Copetti⁷, Bruno Studer⁷, Jürg E Frey⁵, Ueli Grossniklaus¹, Philipp M Schlüter^{2

3}

Affiliations

¹ Department of Plant and Microbial Biology and Zurich-Basel Plant Science Centre, University of Zurich, Zurich, Switzerland.
² Department of Plant Evolutionary Biology, Institute of Biology, University of Hohenheim, Stuttgart, Germany.
³ Department of Systematic and Evolutionary Botany and Zurich-Basel Plant Science Centre, University of Zurich, Zurich, Switzerland.
⁴ Laboratoire des Interactions Plantes Microbes Environnement (LIPME), INRAE, Toulouse, France.
⁵ Department of Method Development and Analytics, Agroscope, Wädenswil, Switzerland.
⁶ Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom.
⁷ Institute of Agricultural Sciences and Zurich-Basel Plant Science Centre, ETH Zürich, Zurich, Switzerland.

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.