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Rapid single-colony whole-genome sequencing of bacterial pathogens
OBJECTIVES: As a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to genera...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977605/ https://www.ncbi.nlm.nih.gov/pubmed/24370932 http://dx.doi.org/10.1093/jac/dkt494 |
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author | Köser, Claudio U. Fraser, Louise J. Ioannou, Avgousta Becq, Jennifer Ellington, Matthew J. Holden, Matthew T. G. Reuter, Sandra Török, M. Estée Bentley, Stephen D. Parkhill, Julian Gormley, Niall A. Smith, Geoffrey P. Peacock, Sharon J. |
author_facet | Köser, Claudio U. Fraser, Louise J. Ioannou, Avgousta Becq, Jennifer Ellington, Matthew J. Holden, Matthew T. G. Reuter, Sandra Török, M. Estée Bentley, Stephen D. Parkhill, Julian Gormley, Niall A. Smith, Geoffrey P. Peacock, Sharon J. |
author_sort | Köser, Claudio U. |
collection | PubMed |
description | OBJECTIVES: As a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to generate DNA libraries for whole-genome sequencing directly from single bacterial colonies grown on primary culture plates. METHODS: We developed our protocol using single colonies of 17 bacterial pathogens responsible for severe human infection that were grown using standard diagnostic media and incubation conditions. We then applied this method to four clinical scenarios that currently require time-consuming reference laboratory tests: full identification and genotyping of salmonellae; identification of bla(NDM-1), a highly transmissible carbapenemase resistance gene, in Klebsiella pneumoniae; detection of genes encoding staphylococcal toxins associated with specific disease syndromes; and monitoring of vaccine targets to detect vaccine escape in Neisseria meningitidis. RESULTS: We validated our single-colony whole-genome sequencing protocol for all 40 combinations of pathogen and selective, non-selective or indicator media tested in this study. Moreover, we demonstrated the clinical value of this method compared with current reference laboratory tests. CONCLUSIONS: This advance will facilitate the implementation of whole-genome sequencing into diagnostic and public health microbiology. |
format | Online Article Text |
id | pubmed-3977605 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-39776052014-04-07 Rapid single-colony whole-genome sequencing of bacterial pathogens Köser, Claudio U. Fraser, Louise J. Ioannou, Avgousta Becq, Jennifer Ellington, Matthew J. Holden, Matthew T. G. Reuter, Sandra Török, M. Estée Bentley, Stephen D. Parkhill, Julian Gormley, Niall A. Smith, Geoffrey P. Peacock, Sharon J. J Antimicrob Chemother Original Research OBJECTIVES: As a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to generate DNA libraries for whole-genome sequencing directly from single bacterial colonies grown on primary culture plates. METHODS: We developed our protocol using single colonies of 17 bacterial pathogens responsible for severe human infection that were grown using standard diagnostic media and incubation conditions. We then applied this method to four clinical scenarios that currently require time-consuming reference laboratory tests: full identification and genotyping of salmonellae; identification of bla(NDM-1), a highly transmissible carbapenemase resistance gene, in Klebsiella pneumoniae; detection of genes encoding staphylococcal toxins associated with specific disease syndromes; and monitoring of vaccine targets to detect vaccine escape in Neisseria meningitidis. RESULTS: We validated our single-colony whole-genome sequencing protocol for all 40 combinations of pathogen and selective, non-selective or indicator media tested in this study. Moreover, we demonstrated the clinical value of this method compared with current reference laboratory tests. CONCLUSIONS: This advance will facilitate the implementation of whole-genome sequencing into diagnostic and public health microbiology. Oxford University Press 2014-05 2013-12-25 /pmc/articles/PMC3977605/ /pubmed/24370932 http://dx.doi.org/10.1093/jac/dkt494 Text en © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Research Köser, Claudio U. Fraser, Louise J. Ioannou, Avgousta Becq, Jennifer Ellington, Matthew J. Holden, Matthew T. G. Reuter, Sandra Török, M. Estée Bentley, Stephen D. Parkhill, Julian Gormley, Niall A. Smith, Geoffrey P. Peacock, Sharon J. Rapid single-colony whole-genome sequencing of bacterial pathogens |
title | Rapid single-colony whole-genome sequencing of bacterial pathogens |
title_full | Rapid single-colony whole-genome sequencing of bacterial pathogens |
title_fullStr | Rapid single-colony whole-genome sequencing of bacterial pathogens |
title_full_unstemmed | Rapid single-colony whole-genome sequencing of bacterial pathogens |
title_short | Rapid single-colony whole-genome sequencing of bacterial pathogens |
title_sort | rapid single-colony whole-genome sequencing of bacterial pathogens |
topic | Original Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977605/ https://www.ncbi.nlm.nih.gov/pubmed/24370932 http://dx.doi.org/10.1093/jac/dkt494 |
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