Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology
The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spr...
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description | The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants. |
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Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants.</description><identifier>ISSN: 2073-4441</identifier><identifier>EISSN: 2073-4441</identifier><identifier>DOI: 10.3390/w13182503</identifier><language>eng</language><publisher>Basel: MDPI AG</publisher><subject>Clustering ; Coronaviruses ; COVID-19 ; Health risks ; Laboratories ; Mutation ; Next-generation sequencing ; Polyethylene glycol ; Pools ; Public health ; Severe acute respiratory syndrome ; Severe acute respiratory syndrome coronavirus 2 ; Sewage ; Spike protein ; Technology ; Wastewater ; Wastewater collection</subject><ispartof>Water (Basel), 2021-09, Vol.13 (18), p.2503</ispartof><rights>2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c358t-1c58759d1f757c027d7510bd0c46a1fff5bcddc4043eefd74c693de804909e5c3</citedby><cites>FETCH-LOGICAL-c358t-1c58759d1f757c027d7510bd0c46a1fff5bcddc4043eefd74c693de804909e5c3</cites><orcidid>0000-0002-8395-0591 ; 0000-0001-7605-0539 ; 0000-0002-4187-4471 ; 0000-0002-1685-9982 ; 0000-0002-2657-100X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids></links><search><creatorcontrib>La Rosa, Giuseppina</creatorcontrib><creatorcontrib>Brandtner, David</creatorcontrib><creatorcontrib>Mancini, Pamela</creatorcontrib><creatorcontrib>Veneri, Carolina</creatorcontrib><creatorcontrib>Bonanno Ferraro, Giusy</creatorcontrib><creatorcontrib>Bonadonna, Lucia</creatorcontrib><creatorcontrib>Lucentini, Luca</creatorcontrib><creatorcontrib>Suffredini, Elisabetta</creatorcontrib><title>Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology</title><title>Water (Basel)</title><description>The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants.</description><subject>Clustering</subject><subject>Coronaviruses</subject><subject>COVID-19</subject><subject>Health risks</subject><subject>Laboratories</subject><subject>Mutation</subject><subject>Next-generation sequencing</subject><subject>Polyethylene glycol</subject><subject>Pools</subject><subject>Public health</subject><subject>Severe acute respiratory syndrome</subject><subject>Severe acute respiratory syndrome coronavirus 2</subject><subject>Sewage</subject><subject>Spike protein</subject><subject>Technology</subject><subject>Wastewater</subject><subject>Wastewater collection</subject><issn>2073-4441</issn><issn>2073-4441</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><recordid>eNpNUV1LxDAQLKKgqA_-gwWfBKtJk1zbx_M8P_BU8Px4LHvJ9qz0kprkkP4a_6oVRdyXWYZhdthJkgPOToQo2ekHF7zIFBMbyU7GcpFKKfnmv3072Q_hjQ0jy6JQbCf5vKEe5uOHeTpxz2kGt-uIsXE2gKth3HaveAyXuFoNgNbANCI8o2_QxgDnFElHMtBYePILtPCCIdIHRvLhm7yO2Paw6GHm7DJ9IDQwXnVto52FOb2vyerGLuEMw2AycHdoXec8wSPpV-tat-z3kq0a20D7v7ibPF1MHydX6ez-8noynqVaqCKmXKsiV6Xhda5yzbLc5IqzhWFajpDXda0W2hgtmRREtcmlHpXCUDH8gZWktNhNDn98O--GYCFWb27t7XCyylQ-UiKTZTaojn5U2rsQPNVV55sV-r7irPquoPqrQHwB5B14Mw</recordid><startdate>20210913</startdate><enddate>20210913</enddate><creator>La Rosa, Giuseppina</creator><creator>Brandtner, David</creator><creator>Mancini, Pamela</creator><creator>Veneri, Carolina</creator><creator>Bonanno Ferraro, Giusy</creator><creator>Bonadonna, Lucia</creator><creator>Lucentini, Luca</creator><creator>Suffredini, Elisabetta</creator><general>MDPI AG</general><scope>AAYXX</scope><scope>CITATION</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>COVID</scope><scope>DWQXO</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><orcidid>https://orcid.org/0000-0002-8395-0591</orcidid><orcidid>https://orcid.org/0000-0001-7605-0539</orcidid><orcidid>https://orcid.org/0000-0002-4187-4471</orcidid><orcidid>https://orcid.org/0000-0002-1685-9982</orcidid><orcidid>https://orcid.org/0000-0002-2657-100X</orcidid></search><sort><creationdate>20210913</creationdate><title>Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology</title><author>La Rosa, Giuseppina ; 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Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. 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subjects | Clustering Coronaviruses COVID-19 Health risks Laboratories Mutation Next-generation sequencing Polyethylene glycol Pools Public health Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Sewage Spike protein Technology Wastewater Wastewater collection |
title | Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology |
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