Volume 180, October 2025, 105845Author links open overlay panel, , , , , , , , , , , , , Highlights●

SpikeID is a sequence-based rapid high throughput screening assay for the accurate identification of SARS-CoV-2 variants.

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The assay is a reliable and cost-effective option to WGS for timely variant tracking in clinical and public health settings.

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SpikeID can be used for rapid SARS-CoV-2 typing in outbreak investigations and contact tracing, as part of active response.

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The sequenced-based genotyping strategy of SpikeID can be expanded to identify other endemic and emergent viral pathogens.

AbstractBackground

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) are characterized by distinct mutations in the S1 domain of the viral spike protein. This domain encompasses the N-terminal domain, the receptor-binding domain, and part of the cleavage site region. While mutations in other genomic regions of SARS-CoV-2 can impact VOC potential, the S1 domain holds particular importance for identifying variants and assessing antigenic evolution and immune escape potential.

Methods

We describe a rapid high-throughput sequencing-based assay, SpikeID, for the unbiased detection and identification of SARS-CoV-2 variants based on spike S1 amplicon sequencing. We benchmarked the SpikeID assay against Illumina whole-genome sequencing across 622 clinical biospecimens, representing lineages that circulated globally from October 2021 to January 2024.

Results

SpikeID unambiguously detected 100 % of WHO-designated VOCs and identified PANGO lineages circulating at ≥1 % prevalence in the New York City (NYC) area with 93 % accuracy in comparison to whole-genome sequencing. This reduction in accuracy was largely due to PANGO lineages that are only distinguishable by mutations outside the S1 domain.

Conclusions

We demonstrate the utility and scalability of the SpikeID assay during the emergence and subsequent surge of Omicron and Omicron-derived lineages in New York City, and show that our approach enables cost-effective, reliable, and near-real-time detection of emerging lineages.

Keywords

Long-read sequencing

Virus evolution

Genotyping

Molecular surveillance

SARS-CoV-2

Virus variants

Spike

Data availabilityThe SpikeID analysis pipeline is available at https://zenodo.org/records/15881340. Source data and code for generating the figures in this manuscript are available at https://github.com/BakelLab/manuscript_SpikeID. Nucleotide sequences are available in GenBank (Supplementary Table 5).

© 2025 The Authors. Published by Elsevier B.V.