Improved Robustness of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method

Abstract

The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for whole-genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations.

Targeted metagenomic sequencing for detection of vertebrate viruses in wastewater for public health surveillance

“…Similarly, targeted/MF increased the number of vertebrate viral families detected by more than half in both M and O WWTP samples as compared to samples that were targeted and directly extracted (targeted/DE), 80% (n= 4) and 67% (n=6), respectively (Figure S4). Similarly, Segelhurst et al, 2022 used a tangential-flow filtration method to recover 53% more complete or near complete SARS-CoV-2 genomes from wastewater samples as compared to concentration using precipitation. Still, a previous study by Hjelmsø et al, 2017 evaluated the impact of polyethylene glycol precipitation (PEG), skimmed milk flocculation, glass wool filtration, and monolithic adsorption filtration on the sequencing of viruses in wastewater.…”

Targeted Metagenomic Sequencing for Detection of Vertebrate Viruses in Wastewater for Public Health Surveillance

“…Segelhurst et al, 2022 used a tangential-flow filtration method to recover 53% more complete or near complete SARS-CoV-2 genomes from wastewater samples as compared to concentration using precipitation. 40 Still, a previous study by Hjelmsø et al, 2017 evaluated the impact of polyethylene glycol precipitation (PEG), skimmed milk flocculation, glass wool filtration, and monolithic adsorption filtration on the sequencing of viruses in wastewater. They demonstrated that concentration methods significantly impacted the detection of viruses in metagenomic sequencing reads with PEG-concentrated samples obtaining more viral-affiliated reads compared to the other methods.…”