High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is...

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Autores principales:	Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4949e29638a040c6885bfe07f4b3b235
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spelling	oai:doaj.org-article:4949e29638a040c6885bfe07f4b3b2352021-12-02T20:06:59ZHigh-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.1932-620310.1371/journal.pone.0253830https://doaj.org/article/4949e29638a040c6885bfe07f4b3b2352021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0253830https://doaj.org/toc/1932-6203Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.Ashley JonesCynthia TorkelDavid StanleyJamila NasimJustin BorevitzBenjamin SchwessingerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 7, p e0253830 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ashley Jones Cynthia Torkel David Stanley Jamila Nasim Justin Borevitz Benjamin Schwessinger High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.
description	Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.
format	article
author	Ashley Jones Cynthia Torkel David Stanley Jamila Nasim Justin Borevitz Benjamin Schwessinger
author_facet	Ashley Jones Cynthia Torkel David Stanley Jamila Nasim Justin Borevitz Benjamin Schwessinger
author_sort	Ashley Jones
title	High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.
title_short	High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.
title_full	High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.
title_fullStr	High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.
title_full_unstemmed	High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.
title_sort	high-molecular weight dna extraction, clean-up and size selection for long-read sequencing.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/4949e29638a040c6885bfe07f4b3b235
work_keys_str_mv	AT ashleyjones highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing AT cynthiatorkel highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing AT davidstanley highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing AT jamilanasim highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing AT justinborevitz highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing AT benjaminschwessinger highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing
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High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.

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