| Autor: |
Wagner GE; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria., Totaro MG; Department of Biochemistry, Graz University of Technology, 8010 Graz, Austria., Volland A; Department of Hygiene, Microbiology and Public Health, Institute of Virology, Medical University of Innsbruck, 6020 Innsbruck, Austria., Lipp M; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria., Saiger S; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria., Lichtenegger S; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria., Forstner P; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria., von Laer D; Department of Hygiene, Microbiology and Public Health, Institute of Virology, Medical University of Innsbruck, 6020 Innsbruck, Austria., Oberdorfer G; Department of Biochemistry, Graz University of Technology, 8010 Graz, Austria., Steinmetz I; Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria. |
| Abstrakt: |
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. |
