Child, Harry T, O'Neill, Paul A, Moore, Karen, Rowe, William, Denise, Hubert, Bass, David, Wade, Matthew J, Loose, Matt, Paterson, Steve 
ORCID: 0000-0002-1307-2981, van Aerle, Ronny et al (show 1 more authors)
(2023)
Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing.
PLOS ONE, 18 (4).
e0284211-.
Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | Animals, Humans, RNA, Viral, Polymerase Chain Reaction, Complement System Proteins, Pandemics, COVID-19, SARS-CoV-2, COVID-19 Testing, Wastewater |
| Divisions: | Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 18 Apr 2023 07:31 |
| Last Modified: | 27 Oct 2023 11:15 |
| DOI: | 10.1371/journal.pone.0284211 |
| Open Access URL: | https://doi.org/10.1371/journal.pone.0284211 |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3169635 |
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