High-throughput Mutational Surveillance of the SARS-CoV-2 Spike Gene

Abstract

SARS-CoV-2 has evolved rapidly towards higher infectivity and partial immune escape over the course of the pandemic. This evolution is driven by the enormous virus population, that has infected close to 200 million people by now. Therefore, cost effective and scalable methods are needed to monitor viral evolution globally. Mutation-specific PCR approaches have become inadequate to distinguish the variety of circulating SARS-CoV-2 variants and are unable to detect novel ones. Conversely, whole genome sequencing protocols remain too labor- and cost-intensive to monitor SARS-CoV-2 at the required density. By adapting SARSeq we present a simple, fast, and scalable S-gene tiling pipeline for focused sequencing of the S-gene encoding for the spike protein. This method reports on all sequence positions with known importance for infectivity and immunity, yet scales to >20K samples per run. S-gene tiling is used for nationwide surveillance of SARS-CoV-2 at a density of 10% to 50% of all cases of infection in Austria. SARSeq S-tiling uncovered several infection clusters with variants of concern such as the biggest known cluster of Beta/B.1.351 outside Africa and successfully informed public health measures in a timely manner, allowing their successful implementation. Our close monitoring of mutations further highlighted evolutionary constraints and freedom of the spike protein ectodomain and sheds light on foreseeable evolutionary trajectories.

National-scale surveillance of emerging SARS-CoV-2 variants in wastewater

“…In the period of examination between Jan 1 st and Sep 15 th , 2021, on average 70.8% of all identified SARS-CoV-2 cases in all towns connected to any examined WWT were tested for SARS-CoV-2 mutations and variants. Sequencing based tests were performed by partial sequencing of the spike gene 94 , Sanger sequencing or WGS. However, most cases were analysed using PCRs for specific mutations, performed in the responsible diagnosis laboratory and reported to AGES.…”

“…Sequencing based tests were performed by partial sequencing of the spike gene 94 , Sanger sequencing or WGS. However, most cases were analysed using PCRs for specific mutations, performed in the responsible diagnosis laboratory and reported to AGES.…”

“…*) by August 2021 55 . Notably, the Austrian province of Tyrol harboured one of the largest clusters of the Beta (B.1.351) variant outside of South Africa from January to March 2021 56 . Such spatiotemporal dynamics of emerging viral variants coupled with a national epidemiological surveillance system 57 , including comprehensive and routine genotyping of positive cases, provided the stage to develop and validate the necessary methodology for WW-based variant monitoring by sequencing.…”

Rapid surveillance platforms for key SARS-CoV-2 mutations in Denmark

“…VOCs include the Alpha the average time from sample to genome being ~1-7 days depending on the scale of sequencing performed. This has led to the development of alternatives to WGS such as SARSeq, which is based on sequencing of the ectodomain of the SARS-CoV-2 spike protein 27 or sequencing of the whole S gene using Sanger sequencing 28 . While such approaches yield cost and reagent savings, the turnaround time, preparation effort for these and cost are still higher compared to RT-qPCR detection platforms.…”

“…However, the utility of WGS in a pandemic such as this also carries with it a significant cost in the form of reagents, equipment as well as turnaround time – the average time from sample to genome being ∼1-7 days depending on the scale of sequencing performed. This has led to the development of alternatives to WGS such as SARSeq, which is based on sequencing of the ectodomain of the SARS-CoV-2 spike protein 27 or sequencing of the whole S gene using Sanger sequencing 28 .…”