Novel Multiplexed Amplicon-Based Sequencing to Quantify SARS-CoV-2 RNA from Wastewater

Abstract

The application of wastewater-based epidemiology (WBE) to support the global response to the COVID-19 pandemic has shown encouraging outcomes. The accurate, sensitive, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in municipal wastewater is critical for WBE. Here, we present a novel approach based on multiplexed amplicon-based sequencing, namely the ATOPlex platform, for detecting SARS-CoV-2. The ATOPlex platform is capable of quantifying SARS-CoV-2 RNA at concentrations that are at least 1 order of magnitude lower than the detection limit of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Robust and accurate phylogenetic placement can be done at viral concentrations 4 times lower than the detection limit of RT-qPCR. We further found that the solid fraction in wastewater harbors a considerable amount of viral RNA, highlighting the need to extract viral RNA from the solid and liquid fractions of wastewater. This study delivers a highly sensitive, phylogenetically informative, and high-throughput analytical workflow that facilitates the application of WBE.

RNA in Municipal Wastewater Reveals Magnitudes of COVID-19 Outbreaks across Four Waves Driven by SARS-CoV-2 Variants of Concern

“…Allele-specific RT-qPCR is useful for screening circulating VOC lineages at the level of human populations; however, WGS can recover nearly complete genomes from potential degraded SARS-COV-2 RNA in wastewater, which could be missed by targeted clinical screening assays based on small numbers of mutations. 58 When one compares VOCs α and γ, VOC δ has a greater ratio between viral load in wastewater to number of daily new cases ( Figures 2 E and 4 B). Viral concentrations of VOC δ in oropharyngeal swabs was about 1000 times greater than those observed for SARS-CoV-2 clade 19A/19B, which makes VOC δ more contagious.…”

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works

“…They used a PEG viral concentration method for liquid influent and direct extraction for dewatered primary settled solids and found solids to liquid SARS-CoV-2 RNA concentration ratios of ∼10 3 mL g −1 . Consistent with these findings, Ni et al 17 applied amplicon sequencing to enumerate SARS-CoV-2 genomes in sewage and noted that the solid fraction contained a considerable proportion of the viral RNA.…”

“…SARS-CoV-2 RNA has been quantified in both the liquid and solid fractions of wastewater. 8,[15][16][17] Sewage can be collected from publicly owned treatment works (POTWs) or from access points in the piped sewage network including at the building scale and assayed for virus nucleic acids. 18 The solid fraction can be settled from raw sewage using Imhoff cones 19 or collected from a primary clarifier, a POTW unit process that allows solids to settle as part of the treatment train.…”

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance

“…Genomic sequencing of wastewater may not only shed light on the evolution of SARS-CoV-2 during an outbreak by identifying viral mutations, but it could also be applied as an approach for quantifying genomic fragments of SARS-CoV-2 as well. A recent study used a high-throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique and claimed that ATOPlex is capable of quantifying SARS-CoV-2 RNA in wastewater at concentrations that are at least one order of magnitude lower than RT-qPCR ( Ni et al, 2021 ). That proof-of-concept study compared the detection achieved by US CDC N1 and N2 RT-qPCR assays with ATOPlex, using a dilution series of cDNA samples generated from a commercially available SARS-CoV-2 RNA positive control, rather than seeding SARS-CoV-2 in wastewater samples.…”