High throughput method for monitoring SARS-CoV-2 variants in wastewater by Nanopore sequencing

Early in the COVID-19 pandemic, it was hypothesized that wastewater-based surveillance could be used to monitor SARS-CoV-2 like it has been used to monitor poliovirus for decades. We present a high throughput laboratory procedure to quantify a mixed population of SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI) by amplicon-based sequencing of RNA purified from wastewater. RNA was purified from wastewater samples and analyzed using RT-qPCR. A subset of SARS-CoV-2 positive samples was subjected to sequencing. Here, a 1049 bp region of the spike gene was amplified and prepared for sequencing through a series of three PCR reactions. The fragments were sequenced using Oxford Nanopore Technology. The resulting reads were mapped to a set of references consisting of corresponding sequences from all VOCs and VOIs at the time of analysis. We demonstrate that it is possible to amplify and sequence a fragment of the spike gene long enough to hold multiple variant-defining mutations enabling direct reference mapping. We show that by using this high throughput method, it is possible to estimate the proportion of different SARS-CoV-2 variants in a population from 10,528 wastewater samples within a period of 15 months. This is especially useful in settings with limited capacity for clinical sampling and sequencing.