A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

Abstract Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous differen...

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Autores principales:	Kathy E. Raven, Sophia T. Girgis, Asha Akram, Beth Blane, Danielle Leek, Nicholas Brown, Sharon J. Peacock
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/524e6a03bd824fdd928c746c74026658
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spelling	oai:doaj.org-article:524e6a03bd824fdd928c746c740266582021-12-02T15:08:24ZA common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing10.1038/s41598-020-80031-82045-2322https://doaj.org/article/524e6a03bd824fdd928c746c740266582021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80031-8https://doaj.org/toc/2045-2322Abstract Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.Kathy E. RavenSophia T. GirgisAsha AkramBeth BlaneDanielle LeekNicholas BrownSharon J. PeacockNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kathy E. Raven Sophia T. Girgis Asha Akram Beth Blane Danielle Leek Nicholas Brown Sharon J. Peacock A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
description	Abstract Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.
format	article
author	Kathy E. Raven Sophia T. Girgis Asha Akram Beth Blane Danielle Leek Nicholas Brown Sharon J. Peacock
author_facet	Kathy E. Raven Sophia T. Girgis Asha Akram Beth Blane Danielle Leek Nicholas Brown Sharon J. Peacock
author_sort	Kathy E. Raven
title	A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
title_short	A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
title_full	A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
title_fullStr	A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
title_full_unstemmed	A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
title_sort	common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/524e6a03bd824fdd928c746c74026658
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A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

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