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.
【저자키워드】 SARS-CoV-2, Nanopore, Surveillance, Next-generation sequencing, vaccine escape, S-protein, typing, 【초록키워드】 whole-genome sequencing, Mutation, protocol, VoC, Sequencing, bioinformatics, variant, SARS-CoV-2 variant, diagnostic, variants, amplification, validation, Immune escape, Rapid, automated, molecular, Amino acid, Analysis, S-gene, approaches, restriction, sequence, primer, offer, immunodominant, whole-genome, respiratory specimen, identify, subsequent, required, reduced, amplify, primer pair, bottleneck, flow cell, region of interest, S-protein variant, sequenced sample, 【제목키워드】 Strategy, Rapid, novel,