Comparison of multiple whole-genome and Spike-only sequencing protocols for estimating variant frequencies via wastewater-based epidemiology (preprint)

Sequencing of SARS-CoV-2 in wastewater provides a key opportunity to monitor the prevalence of variants spatiotemporally, potentially facilitating their detection simultaneously with, or even prior to, observation through clinical testing. However, there are multiple sequencing methodologies available. This study aimed to evaluate the performance of alternative protocols for detecting SARS-CoV-2 variants. We tested the detection of two synthetic RNA SARS-CoV-2 genomes in a wide range of ratios and at two concentrations representative of those found in wastewater using whole-genome and Spike-gene-only protocols utilising Illumina and Oxford Nanopore platforms. We developed a Bayesian hierarchical model to determine the predicted frequencies of variants and the error surrounding our predictions. We found that most of the sequencing protocols detected polymorphic nucleotide frequencies at a level that would allow accurate determination of the variants present at higher concentrations. Most methodologies, including the Spike-only approach, could also predict variant frequencies with a degree of accuracy in low-concentration samples but, as expected, with higher error around the estimates. All methods were additionally confirmed to detect the same prevalent variants in a set of wastewater samples. Our results provide the first quantitative statistical comparison of a range of alternative methods that can be used successfully in the surveillance of SARS-CoV-2 variant frequencies from wastewater.

City-wide wastewater genomic surveillance through the successive emergence of SARS-CoV-2 Alpha and Delta variants (preprint)

Genomic surveillance of SARS-CoV-2 has been essential to provide an evidence base for public health decisions throughout the SARS-CoV-2 pandemic. Sequencing data from clinical cases has provided data crucial to understanding disease transmission and the detection, surveillance, and containment of outbreaks of novel variants, which continue to pose fresh challenges. However, genomic wastewater surveillance can provide important complementary information by providing estimates of variant frequencies which do not suffer from sampling bias, and capturing all variants circulating in a population. Here we show that genomic SARS-CoV-2 wastewater surveillance can detect fine-scale differences within urban centres, specifically within the city of Liverpool, UK, during the emergence of Alpha and Delta variants between November 2020 and June 2021. Overall, the correspondence between wastewater and clinical variant frequencies demonstrates the reliability of wastewater surveillance. Yet, discrepancies between the two approaches in when the Alpha variant was first detected emphasises that wastewater monitoring can also capture missing information resulting from asymptomatic cases or communities less engaged with testing programmes, as found by a simultaneous surge testing effort across the city.