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Wagner, GE; Totaro, MG; Volland, A; Lipp, M; Saiger, S; Lichtenegger, S; Forstner, P; von, Laer, D; Oberdorfer, G; Steinmetz, I.
A Novel High-Throughput Nanopore-Sequencing-Based Strategy for Rapid and Automated S-Protein Typing of SARS-CoV-2 Variants.
Viruses. 2021; 13(12): Doi: 10.3390/v13122548 [OPEN ACCESS]
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Leading authors Med Uni Graz: Wagner-Lichtenegger Gabriel
Co-authors Med Uni Graz: Forstner Patrick; Lipp Michaela; Saiger Sabine; Steinmetz Ivo; Wagner-Lichtenegger Sabine
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Abstract:: Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated "S-Protein-Typer" tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad C_t range showed successful amplification for 29/30 samples. Restriction to the region of interest freed sequencing capacity by a factor of 12-13, compared with whole-genome sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy reduced the time required to identify SARS-CoV-2 variants accordingly. The S-Protein-Typer tool identified all mutations correctly when challenged with our sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our proposed S-protein variant screening offers a simple, more rapid, and low-cost entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome approaches.
Find related publications in this database (Keywords): SARS-CoV-2; next-generation sequencing; vaccine escape; S-protein; nanopore; surveillance; typing