SARS-CoV-2 Whole-Genome Sequencing Using Oxford Nanopore Technology for Variant Monitoring in Wastewaters

Type Article
Date 2022-06
Language English
Author(s) Barbe Laure1, Schaeffer Julien1, Besnard Alban1, Jousse Sarah1, Wurtzer Sébastien2, Moulin Laurent2, Obepine Consortium 1, Le Guyader Soizick1, Desdouits MarionORCID1
Affiliation(s) 1 : Laboratoire de Microbiologie (LSEM, Unité MASAE), IFREMER, Nantes, France
2 : R&D Laboratory, DRDQE, Eau de Paris, Ivry-sur-Seine, France
Source Frontiers In Microbiology (1664-302X) (Frontiers Media), 2022-06 , Vol. 13 , P. 889811 (14p.)
DOI 10.3389/fmicb.2022.889811
WOS© Times Cited 13
Keyword(s) SARS-CoV-2, variant of concern, wastewater-based epidemiology, next-generation sequencing, Oxford Nanopore Technology, sewage, ARTIC

Since the beginning of the Coronavirus Disease-19 (COVID-19) pandemic, multiple Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mutations have been reported and led to the emergence of variants of concern (VOC) with increased transmissibility, virulence or immune escape. In parallel, the observation of viral fecal shedding led to the quantification of SARS-CoV-2 genomes in wastewater, providing information about the dynamics of SARS-CoV-2 infections within a population including symptomatic and asymptomatic individuals. Here, we aimed to adapt a sequencing technique initially designed for clinical samples to apply it to the challenging and mixed wastewater matrix, and hence identify the circulation of VOC at the community level. Composite raw sewage sampled over 24 h in two wastewater-treatment plants (WWTPs) from a city in western France were collected weekly and SARS-CoV-2 quantified by RT-PCR. Samples collected between October 2020 and May 2021 were submitted to whole-genome sequencing (WGS) using the primers and protocol published by the ARTIC Network and a MinION Mk1C sequencer (Oxford Nanopore Technologies, Oxford, United Kingdom). The protocol was adapted to allow near-full genome coverage from sewage samples, starting from ∼5% to reach ∼90% at depth 30. This enabled us to detect multiple single-nucleotide variant (SNV) and assess the circulation of the SARS-CoV-2 VOC Alpha, Beta, Gamma, and Delta. Retrospective analysis of sewage samples shed light on the emergence of the Alpha VOC with detection of first co-occurring signature mutations in mid-November 2020 to reach predominance of this variant in early February 2021. In parallel, a mutation-specific qRT-PCR assay confirmed the spread of the Alpha VOC but detected it later than WGS. Altogether, these data show that SARS-CoV-2 sequencing in sewage can be used for early detection of an emerging VOC in a population and confirm its ability to track shifts in variant predominance.

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Barbe Laure, Schaeffer Julien, Besnard Alban, Jousse Sarah, Wurtzer Sébastien, Moulin Laurent, Obepine Consortium, Le Guyader Soizick, Desdouits Marion (2022). SARS-CoV-2 Whole-Genome Sequencing Using Oxford Nanopore Technology for Variant Monitoring in Wastewaters. Frontiers In Microbiology, 13, 889811 (14p.). Publisher's official version : , Open Access version :