A Novel High-Throughput Nanopore-Sequencing-Based Strategy for Rapid and Automated S-Protein Typing of SARS-CoV-2 Variants

• Published in: Viruses 2021-12, Vol.13 (12), p.2548
• Main Authors: Wagner, Gabriel E, Totaro, Massimo G, Volland, André, Lipp, Michaela, Saiger, Sabine, Lichtenegger, Sabine, Forstner, Patrick, von Laer, Dorothee, Oberdorfer, Gustav, Steinmetz, Ivo
• Format: Article
• Language: English
• Subjects: Amino acids; Antibodies; Automation; Bar codes; Bioinformatics; Communication; COVID-19 - epidemiology; COVID-19 - virology; Epidemiological Monitoring; Genomes; Genotype; High-Throughput Nucleotide Sequencing - methods; Humans; Immune Evasion - genetics; Infectivity; Mutation; nanopore; Nanopore Sequencing - methods; next-generation sequencing; Pipelines; Proteins; S-protein; SARS-CoV-2; SARS-CoV-2 - genetics; SARS-CoV-2 - immunology; Severe acute respiratory syndrome coronavirus 2; Spike Glycoprotein, Coronavirus - genetics; Surveillance; vaccine escape; Vaccines; Whole genome sequencing
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v13122548

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 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.