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Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing
Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emerg...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10104291/ https://www.ncbi.nlm.nih.gov/pubmed/37058515 http://dx.doi.org/10.1371/journal.pone.0284211 |
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author | Child, Harry T. O’Neill, Paul A. Moore, Karen Rowe, William Denise, Hubert Bass, David Wade, Matthew J. Loose, Matt Paterson, Steve van Aerle, Ronny Jeffries, Aaron R. |
author_facet | Child, Harry T. O’Neill, Paul A. Moore, Karen Rowe, William Denise, Hubert Bass, David Wade, Matthew J. Loose, Matt Paterson, Steve van Aerle, Ronny Jeffries, Aaron R. |
author_sort | Child, Harry T. |
collection | PubMed |
description | Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emergence of novel variants that may impact the course of an epidemic. Population-level surveillance of viruses through genomic sequencing of wastewater captures unbiased lineage data, including cryptic asymptomatic and undiagnosed infections, and has been shown to detect infection outbreaks and novel variant emergence before detection in clinical samples. Here, we present an optimised protocol for quantification and sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in influent wastewater, used for high-throughput genomic surveillance in England during the COVID-19 pandemic. This protocol utilises reverse compliment PCR for library preparation, enabling tiled amplification across the whole viral genome and sequencing adapter addition in a single step to enhance efficiency. Sequencing of synthetic SARS-CoV-2 RNA provided evidence validating the efficacy of this protocol, while data from high-throughput sequencing of wastewater samples demonstrated the sensitivity of this method. We also provided guidance on the quality control steps required during library preparation and data analysis. Overall, this represents an effective method for high-throughput sequencing of SARS-CoV-2 in wastewater which can be applied to other viruses and pathogens of humans and animals. |
format | Online Article Text |
id | pubmed-10104291 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-101042912023-04-15 Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing Child, Harry T. O’Neill, Paul A. Moore, Karen Rowe, William Denise, Hubert Bass, David Wade, Matthew J. Loose, Matt Paterson, Steve van Aerle, Ronny Jeffries, Aaron R. PLoS One Lab Protocol Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emergence of novel variants that may impact the course of an epidemic. Population-level surveillance of viruses through genomic sequencing of wastewater captures unbiased lineage data, including cryptic asymptomatic and undiagnosed infections, and has been shown to detect infection outbreaks and novel variant emergence before detection in clinical samples. Here, we present an optimised protocol for quantification and sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in influent wastewater, used for high-throughput genomic surveillance in England during the COVID-19 pandemic. This protocol utilises reverse compliment PCR for library preparation, enabling tiled amplification across the whole viral genome and sequencing adapter addition in a single step to enhance efficiency. Sequencing of synthetic SARS-CoV-2 RNA provided evidence validating the efficacy of this protocol, while data from high-throughput sequencing of wastewater samples demonstrated the sensitivity of this method. We also provided guidance on the quality control steps required during library preparation and data analysis. Overall, this represents an effective method for high-throughput sequencing of SARS-CoV-2 in wastewater which can be applied to other viruses and pathogens of humans and animals. Public Library of Science 2023-04-14 /pmc/articles/PMC10104291/ /pubmed/37058515 http://dx.doi.org/10.1371/journal.pone.0284211 Text en © 2023 Child 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 Child, Harry T. O’Neill, Paul A. Moore, Karen Rowe, William Denise, Hubert Bass, David Wade, Matthew J. Loose, Matt Paterson, Steve van Aerle, Ronny Jeffries, Aaron R. Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing |
title | Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing |
title_full | Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing |
title_fullStr | Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing |
title_full_unstemmed | Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing |
title_short | Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing |
title_sort | optimised protocol for monitoring sars-cov-2 in wastewater using reverse complement pcr-based whole-genome sequencing |
topic | Lab Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10104291/ https://www.ncbi.nlm.nih.gov/pubmed/37058515 http://dx.doi.org/10.1371/journal.pone.0284211 |
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