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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

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...[Show more]

dc.contributor.authorJones, Ashley
dc.contributor.authorTorkel, Cynthia
dc.contributor.authorStanley, David
dc.contributor.authorNasim, Jamila
dc.contributor.authorBorevitz, Justin
dc.contributor.authorSchwessinger, Benjamin
dc.date.accessioned2022-06-23T05:18:40Z
dc.date.available2022-06-23T05:18:40Z
dc.identifier.issn1932-6203
dc.identifier.urihttp://hdl.handle.net/1885/267498
dc.description.abstractRapid 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.
dc.description.sponsorshipA.J. and B.S. received sequencing funds from Bioplatforms Australia, as part of the Genomics for Australian Pants initiative www. genomicsforaustralianplants.com J.B. received funds from an Australian Research Council Centre of Excellence (Plant Energy Biology) (CE140100008) and Discovery Project (DP150103591) www.arc.gov.au B.S. received funds from an Australian Research Council Future Fellowship (FT180100024) www.arc.gov.au
dc.format.mimetypeapplication/pdf
dc.language.isoen_AU
dc.publisherPublic Library of Science
dc.rights© 2021 Jones et al.
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.sourcePLOS ONE
dc.titleHigh-molecular weight DNA extraction, clean-up and size selection for long-read sequencing
dc.typeJournal article
local.description.notesImported from PLOS
local.identifier.citationvolume16
dc.date.issued2021-07-15
local.publisher.urlhttp://www.plosone.org/
local.type.statusPublished Version
local.contributor.affiliationJones, A., Research School of Biology, The Australian National University
local.contributor.affiliationTorkel, S., Research School of Biology, The Australian National University
local.contributor.affiliationStanley, D., Research School of Biology, The Australian National University
local.contributor.affiliationNasim, J., Research School of Biology, The Australian National University
local.contributor.affiliationBorevitz, J., Research School of Biology, The Australian National University
local.contributor.affiliationSchwessinger, B., Research School of Biology, The Australian National University
dc.relationhttp://purl.org/au-research/grants/arc/CE140100008
dc.relationhttp://purl.org/au-research/grants/arc/DP150103591
dc.relationhttp://purl.org/au-research/grants/arc/FT180100024
local.bibliographicCitation.issue7
local.bibliographicCitation.startpagee0253830-1
local.bibliographicCitation.lastpagee0253830-6
local.identifier.doi10.1371/journal.pone.0253830
dc.date.updated2021-07-18T10:05:30Z
dcterms.accessRightsOpen Access
dc.provenanceThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
dc.rights.licenseCreative Commons Attribution License
CollectionsANU Research Publications

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