Definition of a concentration and RNA extraction protocol for optimal whole genome sequencing of SARS-CoV-2 in wastewater (ANRS0160)

Ahlam Chaqroun (1, 2) , Ghina El Soufi (3, 4, 2) , Zuzana Gerber (5) , Julie Loutreul (6) , Nicolas Cluzel (7, 2) , Damien Delafoy (5) , Florian Sandron (5) , Léo Di Jorio (3, 4, 2) , Stéphanie Raffestin (8, 2) , Vincent Maréchal (9, 2) , Christophe Gantzer (1, 2) , Robert Olaso (5) , Jean-François Deleuze (5) , Olivier Rohr (3, 4, 2) , Nicolas Boudaud (6, 2) , Clémentine Wallet (3, 4, 2) , Isabelle Paule Bertrand (1, 2)

1 LCPME - Laboratoire de Chimie Physique et Microbiologie pour les Matériaux et l'Environnement
2 GIS OBEPINE - OBEPINE OBservatoire ÉPIdémiologique daNs les Eaux usées - Wastewater-based Epidemiology [Paris, France]
3 ARN - Architecture et Réactivité de l'ARN
4 IUT Louis Pasteur [Schiltigheim]
5 CNRGH - Centre National de Recherche en Génomique Humaine
6 Actalia [Saint-Lô]
7 SUMMIT - Sorbonne Université Maison des Modélisations Ingénieries et Technologies
8 Institut Pasteur de la Guyane
9 CRSA - Centre de Recherche Saint-Antoine

Monitoring the presence of RNA from emerging pathogenic viruses, such as SARS-CoV-2, in wastewater (WW) samples requires suitable methods to ensure an effective response. Genome sequencing of WW is one of the crucial methods, but it requires high-quality RNA in sufficient quantities, especially for monitoring emerging variants. Consequently, methods for viral concentration and RNA extraction from WW samples have to be optimized before sequencing. The purpose of this study was to achieve high coverage (≥ 90 %) and sequencing depth (at least ≥200×) even for low initial RNA concentrations (< 105 genome copies (GC)/L) in WW. A further objective was to determine the range of SARS-CoV-2 RNA concentrations that allow high-quality sequencing, and the optimal sample volume for analysis. Ultrafiltration (UF) methods were used to concentrate viral particles from large influent samples (up to 500 mL). An RNA extraction protocol using silica beads, neutral phenol-chloroform treatment, and a PCR inhibitor removal kit was chosen for its effectiveness in extracting RNA and eliminating PCR inhibitors, as well as its adaptability for use with large influent samples. Recovery rates ranged from 24 % to 63 % (N = 17) for SARS-CoV-2 naturally present in WW samples. 200 mL WW samples can be enough for UF concentration, as they showed high quality sequencing analyses with between 5 × 104 GC/L and 6 × 103 GC/L. Below 6 × 103 GC/L, high-quality sequencing was also achieved for ∼40 % of the samples using 500 mL of WW. Sequencing analysis for variant detection was performed on 200 mL WW samples with coverage of >95 % and sequencing depth of >1000×. Analyses revealed the predominance of variant EG.5, known as Eris (66 %–100 %). The use of UF methods in combination with a suitable RNA extraction protocol appear promising for sequencing enveloped viruses in WW in a context of viral emergence.

Mots clés: concentration method; SARS-CoV-2; ultrafiltration; wastewater; whol genome sequencing

Domaines: Virologie

Ahlam Chaqroun, Ghina El Soufi, Zuzana Gerber, Julie Loutreul, Nicolas Cluzel, et al.. Definition of a concentration and RNA extraction protocol for optimal whole genome sequencing of SARS-CoV-2 in wastewater (ANRS0160). Science of the Total Environment, 2024, 952, pp.175823. ⟨10.1016/j.scitotenv.2024.175823⟩. ⟨hal-04764746⟩