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

Published version
Peer-reviewed

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Authors

Russo, Alessia 
Mayjonade, Baptiste 
Frei, Daniel 
Potente, Giacomo 
Kellenberger, Roman T 

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.

Description

Keywords

Circulomics, DNA extraction, DNA sequencing, ONT long read sequencing, PacBio, genome assembly, nanopore sequencing, plant genome

Journal Title

Front Plant Sci

Conference Name

Journal ISSN

1664-462X
1664-462X

Volume Title

13

Publisher

Frontiers Media SA