Detection of Mutations Associated with Variants of Concern Via High Throughput Sequencing of SARS-CoV-2 Isolated from NYC Wastewater

Abstract

Monitoring SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To date, most data on SARS-CoV-2 genetic diversity has come from the sequencing of clinical samples, but such studies may suffer limitations due to costs and throughput. Wastewater-based epidemiology may provide an alternative and complementary approach for monitoring communities for novel variants. Given that SARS-CoV-2 can infect the cells of the human gut and is found in high concentrations in feces, wastewater may be a valuable source of SARS-CoV-2 RNA, which can be deep sequenced to provide information on the circulating variants in a community. Here we describe a safe, affordable protocol for the sequencing of SARS-CoV-2 RNA using high-throughput Illumina sequencing technology. Our targeted sequencing approach revealed the presence of mutations associated with several Variants of Concern at appreciable frequencies. Our work demonstrates that wastewater-based SARS-CoV-2 sequencing can inform surveillance efforts monitoring the community spread of SARS-CoV-2 Variants of Concern and detect the appearance of novel emerging variants more cheaply, safely, and efficiently than the sequencing of individual clinical samples.

Monitoring SARS-CoV-2 Populations in Wastewater by Amplicon Sequencing and Using the Novel Program SAM Refiner

“…Especially as new SARS-CoV-2 variants emerge that have altered viral fitness and/or pathogenesis, it is important for health professionals and policy makers to have up-to-date information on the viral populations present in communities. Surveillance of wastewater by high-throughput sequencing has proven to be a cost effective and reliable method to obtain such information [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Sequencing of wastewater for SARS-CoV-2 relies on whole-genome sequencing, targeted amplicon sequencing, or some intermediate of the two; each approach has advantages and disadvantages.…”

“…However, early in the SARS-CoV-2 pandemic, wastewater was used to track community levels and spread of SARS-CoV-2 by Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) methods [ 4 , 5 ]. Investigators have also used high-throughput sequencing on wastewater samples to obtain full or partial SARS-CoV-2 genomic sequences which have been used for metagenomic and epidemiologic analysis [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Sequences identified in wastewater samples may reflect known lineages as well as lineages not reported from clinical samples.…”

Molecular Epidemiology of SARS-CoV-2 in Diverse Environmental Samples Globally

“…The only exception was found in Liechtenstein where only one human was found to be infected with this emerging variant that has been found in several environmental samples. Outside Europe, this variant of concern has also been detected in wastewater and from humans in New York, USA [ 47 , 48 ]. However, detection of the variant of concern, B.1.1.7, in environmental samples from various European countries implies the need for strict quarantine of human, appropriate disinfection strategies for wastewater.…”

Alcov: Estimating Variant of Concern Abundance from SARS-CoV-2 Wastewater Sequencing Data

“…The second sample analyzed was collected in New York City (NYC) in March 2021 [12]. Only a 332-nucleotide region of the receptor binding domain was sequenced which covered six known mutations present in VOCs (S:L452R, S:S477N, S:T478K, S:E484K/Q, and S:N501Y).…”