Assessing Multiplex Tiling PCR Sequencing Approaches for Detecting Genomic Variants of SARS-CoV-2 in Municipal Wastewater

• DOI: 10.1128/msystems.01068-21
• Publication Date: 2021-10-19T16:38:44Z
• Authors: Xuan Lin; Melissa Glier; Kevin Kuchinski; Tenysha Ross-Van ...; David McVea; John R. Tyson; Natalie Prystajecky; Ryan M. Ziels
• Keywords: Amplicon; Multiplex; Amplicon sequencing; Primer (cosmetics)

Wastewater-based genomic surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus shows promise to complement epidemiology efforts. Multiplex tiling PCR is a desirable approach for targeted genome sequencing SARS-CoV-2 in wastewater due its low cost and rapid turnaround time. However, it not clear how different multiplex primer schemes or sample matrices impact resulting coverage. The objective this work was assess performance three schemes, consisting 150-bp, 400-bp, 1,200-bp amplicons, as well two matrices, influent primary sludge, SARS-CoV-2. Wastewater samples were collected weekly from five municipal treatment plants (WWTPs) Metro Vancouver region British Columbia, Canada during period increased disease 19 (COVID-19) case counts February April 2021. RNA extracted clarified provided significantly higher coverage (breadth median depth) than sludge across all schemes. Shorter amplicons appeared be more resilient degradation but hindered by greater pool complexity 150-bp scheme. identified optimal scheme (400 bp) matrix (influent) capable detecting emergence mutations associated with variants concern, which daily load correlated clinical counts. Taken together, these results provide guidance on best practices implementing wastewater-based demonstrate ability inform efforts concern circulating within geographic region. IMPORTANCE Monitoring characteristics population can shed important insights into epidemiological aspects COVID-19 outbreak. Sequencing every patient highly populous area difficult feat, thus offers great augment characterizing pooled matrix, particularly an escalating Here, we approaches wastewater. We that strong correlations data province Columbia. These on, further support for, application tool current