Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Jones, Ashley, Torkel, Cynthia, Stanley, David, Nasim, Jamila, Borevitz, Justin, Schwessinger, Benjamin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282028/
https://www.ncbi.nlm.nih.gov/pubmed/34264958
http://dx.doi.org/10.1371/journal.pone.0253830
_version_ 1783722932138147840
author Jones, Ashley
Torkel, Cynthia
Stanley, David
Nasim, Jamila
Borevitz, Justin
Schwessinger, Benjamin
author_facet Jones, Ashley
Torkel, Cynthia
Stanley, David
Nasim, Jamila
Borevitz, Justin
Schwessinger, Benjamin
author_sort Jones, Ashley
collection PubMed
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 Online
Article
Text
id pubmed-8282028
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-82820282021-07-28 High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing Jones, Ashley Torkel, Cynthia Stanley, David Nasim, Jamila Borevitz, Justin Schwessinger, Benjamin PLoS One Lab Protocol 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. Public Library of Science 2021-07-15 /pmc/articles/PMC8282028/ /pubmed/34264958 http://dx.doi.org/10.1371/journal.pone.0253830 Text en © 2021 Jones et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Lab Protocol
Jones, Ashley
Torkel, Cynthia
Stanley, David
Nasim, Jamila
Borevitz, Justin
Schwessinger, Benjamin
High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing
title 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_short 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
topic Lab Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282028/
https://www.ncbi.nlm.nih.gov/pubmed/34264958
http://dx.doi.org/10.1371/journal.pone.0253830
work_keys_str_mv AT jonesashley highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing
AT torkelcynthia highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing
AT stanleydavid highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing
AT nasimjamila highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing
AT borevitzjustin highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing
AT schwessingerbenjamin highmolecularweightdnaextractioncleanupandsizeselectionforlongreadsequencing