Variant abundance estimation for SARS-CoV-2 in wastewater using RNA-Seq quantification

Abstract

Effectively monitoring the spread of SARS-CoV-2 variants is essential to efforts to counter the ongoing pandemic. Wastewater monitoring of SARS-CoV-2 RNA has proven an effective and efficient technique to approximate COVID-19 case rates in the population. Predicting variant abundances from wastewater, however, is technically challenging. Here we show that by sequencing SARS-CoV-2 RNA in wastewater and applying computational techniques initially used for RNA-Seq quantification, we can estimate the abundance of variants in wastewater samples. We show by sequencing samples from wastewater and clinical isolates in Connecticut U.S.A. between January and April 2021 that the temporal dynamics of variant strains broadly correspond. We further show that this technique can be used with other wastewater sequencing techniques by expanding to samples taken across the United States in a similar timeframe. We find high variability in signal among individual samples, and limited ability to detect the presence of variants with clinical frequencies <10%; nevertheless, the overall trends match what we observed from sequencing clinical samples. Thus, while clinical sequencing remains a more sensitive technique for population surveillance, wastewater sequencing can be used to monitor trends in variant prevalence in situations where clinical sequencing is unavailable or impractical.

Optimization and Application of a Multiplex Digital PCR Assay for the Detection of SARS-CoV-2 Variants of Concern in Belgian Influent Wastewater

“…WBE could potentially also be applied to provide a snapshot of the occurrence of different genetic lineages and their diversity at the population level [ 25 , 26 , 27 , 28 , 29 ]. However, the detection of different VOC in IWW samples presents also some challenges, including low concentrations of the RNA and the presence of PCR-inhibiting compounds in IWW [ 30 , 31 ].…”

“…Furthermore, the fecal shedding rate could be a variable for the different variants circulating in the general population [ 32 , 33 ]. For this reason, surveillance of the different SARS-CoV-2 VOC in IWW is complicated by tracking combinations of multiple characteristics and often co-occurring amino acid mutations [ 29 ].…”

“…This bioanalytical approach was applied to IWW samples from different locations in Belgium to retrospectively monitor the emergence of each VOC at a population scale. To our knowledge, most pre-existing WBE applications on SARS-CoV-2 variants mainly focus on sequencing [ 29 , 34 , 35 , 36 , 37 ] and RT-qPCR [ 26 , 28 , 38 , 39 ]. Only a limited number of studies have applied dPCR to estimate the VOC circulation in IWW [ 25 , 27 , 40 , 41 ].…”

Detection of prevalent SARS-CoV-2 variant lineages in wastewater and clinical sequences from cities in Québec, Canada

“…Wastewater is a complex mixture of fragmented viral RNA, which can be difficult to sequence and confidently identify mutations. Recent studies in the United States have shown that the dominant SARS-CoV-2 variants observed in clinical samples are largely mirrored in wastewater sequences (Crits-Christoph et al 2021; Baaijens et al 2021). Wastewater also has the potential to capture mutations and variant lineages not observed in human clinical samples, potentially including animal reservoirs (Smyth et al 2021).…”

Wastewater sequencing uncovers early, cryptic SARS-CoV-2 variant transmission

“…However, wastewater genomic surveillance is technically challenging 9 . Low viral loads, heavily fragmented RNA, and PCR inhibitors in complex environmental samples lead to poor sequencing coverage/quality 11 . Additionally, tools for SARS-CoV-2 lineage classification, such as pangolin 12 and UShER 13 , were designed for clinical samples containing a single dominant variant, and cannot estimate relative abundances of multiple SARS-CoV-2 lineages in samples with virus mixtures such as wastewater.…”