Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater

Abstract

Wastewater-based genomic surveillance of the SARS-CoV-2 virus shows promise to complement genomic epidemiology efforts. Multiplex tiled PCR is a desirable approach for targeted genome sequencing of SARS-CoV-2 in wastewater due to its low cost and rapid turnaround time. However, it is not clear how different multiplex tiled PCR primer schemes or wastewater sample matrices impact the resulting SARS-CoV-2 genome coverage. The objective of this work was to assess the performance of three different multiplex primer schemes, consisting of 150bp, 400bp, and 1200bp amplicons, as well as two wastewater sample matrices, influent wastewater and primary sludge, for targeted genome sequencing of SARS-CoV-2. Wastewater samples were collected weekly from five municipal wastewater treatment plants (WWTPs) in the Metro Vancouver region of British Columbia, Canada during a period of increased COVID-19 case counts from February to April, 2021. RNA extracted from clarified influent wastewater provided significantly higher genome coverage (breadth and median depth) than primary sludge samples across all primer schemes. Shorter amplicons appeared more resilient to sample RNA degradation, but were hindered by greater primer pool complexity in the 150bp scheme. The identified optimal primer scheme (400bp) and sample matrix (influent) was capable of detecting the emergence of mutations associated with genomic variants of concern, of which the daily wastewater load significantly correlated with clinical case counts. Taken together, these results provide guidance on best practices for implementing wastewater-based genomic surveillance, and demonstrate its ability to inform epidemiology efforts by detecting genomic variants of concern circulating within a geographic region.

Wastewater-based epidemiology: current uses and future opportunities as a public health surveillance tool

“…Monitoring wastewater for new genetic variants has been proposed for early identification and targeting mitigation measures where variants of concern are rising. The detection of genetic variants has been demonstrated to be possible and to correlate with clinical data and trends (Ai et al, 2021;Carcereny et al, 2021;Lin et al, 2021). The lack of sensitivity of existing test methods limits the ability to detect low numbers of variant cases before they are detected through clinical testing on the scale of a large WWTP.…”