Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore sequencing

Rios, Géraldine; Lacoux, Caroline; Leclercq, Vianney; Diamant, Anna; Lebrigand, Kévin; Lazuka, Adèle; Soyeux, E.; Lacroix, Sébastien; Fassy, Julien; Couesnon, Aurélie; Thiéry, Richard; Mari, Bernard; Pradier, Christian; Waldmann, Rainer; Barbry, Pascal

doi:10.1101/2021.07.09.21257475

Cited by 17 publications

(31 citation statements)

References 20 publications

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“…We were thus able to observe a comparable distribution among patients from the whole city of Marseille and in the RS wastewater network and a comparable distribution among patients living in the district corresponding to B7 and in the B7 wastewater network. More general, very recently published national studies carried out in the Netherlands and Belgium and several nations/cities in the UK have reached the same conclusions [42,45,46]. The results using the Bio-T Kit ® SARS-CoV-2 screening kit were less precise.…”

Section: Discussionmentioning

confidence: 62%

“…In this paper, we demonstrate that the analysis of SARS-CoV-2 variants from wastewater by next-generation sequencing is a powerful tool capable of improving our understanding of the outbreak transmission dynamics and, also, of reducing the burden of COVID-19. Rios et al recently showed efficient SARS-CoV2 variant monitoring in the wastewater network of Nice, France by using Nanopore technology [45]. Herein, we demonstrated the key importance of the optimisation of storage and the pre-treatment conditions combined with a sequencing technology (NOVASEQ) providing a great depth of sequencing and, thus, being able to detect minority variants.…”

Section: Discussionmentioning

confidence: 69%

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Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France

et al. 2021

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The monitoring of SARS-CoV-2 RNA in sewage has been proposed as a simple and unbiased means of assessing epidemic evolution and the efficiency of the COVID-19 control measures. The past year has been marked by the emergence of variants that have led to a succession of epidemic waves. It thus appears that monitoring the presence of SARS-CoV-2 in wastewater alone is insufficient, and it may be important in the future to also monitor the evolution of these variants. We used a real-time RT-PCR screening test for variants in the wastewater of our city to assess the effectiveness of direct SARS-CoV-2 sequencing from the same wastewater. We compared the genome sequencing results obtained over the large RS network and the smaller B7 network with the different distributions of the variants observed by RT-PCR screening. The prevalence of the “UK variant” in the RS and B7 networks was estimated to be 70% and 8% using RT-PCR screening compared to 95% and 64% using genome sequencing, respectively. The latter values were close to the epidemiology observed in patients of the corresponding area, which were 91% and 58%, respectively. Genome sequencing in sewage identified SARS-CoV-2 of lineage B.1.525 in B7 at 27% (37% in patients), whereas it was completely missed by RT-PCR. We thus determined that direct sequencing makes it possible to observe, in wastewater, a distribution of the variants comparable to that revealed by genomic monitoring in patients and that this method is more accurate than RT-PCR. It also shows that, rather than a single large sample, it would be preferable to analyse several targeted samples if we want to more appropriately assess the geographical distribution of the different variants. In conclusion, this work supports the wider surveillance of SARS-CoV-2 variants in wastewater by genome sequencing and targeting small areas on the condition of having a sequencing capacity and, when this is not the case, to developing more precise screening tests based on the multiplexed detection of the mutations of interest.

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Section: Discussionmentioning

confidence: 62%

Section: Discussionmentioning

confidence: 69%

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France

et al. 2021

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“…Furthermore, the quality criteria in those studies were not evaluated using a defined population (Izquierdo-Lara et al, 2020;Jahn et al, 2021). Izquierdo-Lara used a minimum depth coverage of 50× and minimum AF of 10% (Izquierdo-Lara et al, 2021), while Rios et al (2021) adopted a minimum depth coverage of 100× without indicating an AF threshold. Based on the results in this study, these recommendations appear not sufficiently strict as a sequencing coverage of 100× and 250× at an AF of 20 and 10% respectively was required to observe all LFV.…”

Section: Discussionmentioning

confidence: 99%

“…The EU recommends the generation of one million reads per sample and a read length of more than 100 bp (European Commission, 2021). A few studies evaluated LFV in wastewater samples, by using local haplotype reconstruction with ShoRAH (Jahn et al, 2021) or iVar and setting up a minimum coverage of 50×, Phred score of ≥30 and a minimal allelic frequency (AF) of 10% (Izquierdo-Lara et al, 2021) or a minimum base quality filter of 20 with a minimum coverage of 100× (Rios et al, 2021). However, none of these studies evaluated their approach on well-defined populations nor determined detection thresholds for retaining LFV.…”

Section: Introductionmentioning

confidence: 99%

Strategy and Performance Evaluation of Low-Frequency Variant Calling for SARS-CoV-2 Using Targeted Deep Illumina Sequencing

et al. 2021

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The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of diagnostic and wastewater samples has been explored as an epidemiological surveillance method for the competent authorities. Currently, only the consensus genome sequence of the most abundant strain is taken into consideration for analysis, but multiple variant strains are now circulating in the population. Consequently, in diagnostic samples, potential co-infection(s) by several different variants can occur or quasispecies can develop during an infection in an individual. In wastewater samples, multiple variant strains will often be simultaneously present. Currently, quality criteria are mainly available for constructing the consensus genome sequence, and some guidelines exist for the detection of co-infections and quasispecies in diagnostic samples. The performance of detection and quantification of low-frequency variants using whole genome sequencing (WGS) of SARS-CoV-2 remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the mutations defining the SARS-CoV-2 lineage B.1.1.7 (alpha variant) as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to construct mixed samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation and lower sensitivity were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10, 5, 3, and 1%, requires at least a sequencing coverage of 250, 500, 1500, and 10,000×, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be, and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at certain allelic frequencies. This approach will be useful to detect and quantify low-frequency variants in both diagnostic (e.g., co-infections and quasispecies) and wastewater [e.g., multiple variants of concern (VOCs)] samples.

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“…As soon as viral genomes of VOCs become available within the international scientific community, e.g., via GISAID (Elbe & Buckland-Merrett, 2017), variant-specific PCR primers and probes can be developed and deployed on regularly collected wastewater samples to understand the dynamics of community disease burden caused by VOCs (Peterson et al 2022). While sequencing viral genomes from wastewater is technically feasible, either via targeted amplicon tiling protocols (Rios et al 2021; Lin et al 2021) or shotgun metagenomics (Rothman et al 2021; Pérez-Cataluña et al 2022), these comprehensive approaches are significantly more costly and time consuming than targeted RT-qPCR screening of RNA extracted from wastewater that can provide accurate data on VOCs at a fraction of the cost, and in near-real time. In Alberta wastewater is sampled, processed and analyzed in university laboratories in Calgary and Edmonton, and reported to health officials and online to the public two days after sampling.…”

Section: Introductionmentioning

confidence: 99%

Emergence and Spread of the SARS-CoV-2 Omicron Variant in Alberta Communities Revealed by Wastewater Monitoring

Hubert

Acosta

Waddell

et al. 2022

Preprint

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Wastewater monitoring of SARS-CoV-2 allows for early detection and monitoring of COVID-19 burden in communities and can track specific variants of concern. Targeted assays enabled relative proportions of SARS-CoV-2 Omicron and Delta variants to be determined across 30 municipalities covering >75% of the province of Alberta (pop. 4.5M) in Canada, from November 2021 to January 2022. Larger cities like Calgary and Edmonton exhibited a more rapid emergence of Omicron relative to smaller and more remote municipalities. Notable exceptions were Banff, a small international resort town, and Fort McMurray, a more remote northern city with a large fly-in worker population. The integrated wastewater signal revealed that the Omicron variant represented close to 100% of SARS-CoV-2 burden prior to the observed increase in newly diagnosed clinical cases throughout Alberta, which peaked two weeks later. These findings demonstrate that wastewater monitoring offers early and reliable population-level results for establishing the extent and spread of emerging pathogens including SARS-CoV-2 variants.

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Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore sequencing

Cited by 17 publications

References 20 publications

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France

Strategy and Performance Evaluation of Low-Frequency Variant Calling for SARS-CoV-2 Using Targeted Deep Illumina Sequencing

Emergence and Spread of the SARS-CoV-2 Omicron Variant in Alberta Communities Revealed by Wastewater Monitoring

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