Tiled Amplicon Sequencing of HCoV
COVID-19, which is caused by SARS-CoV2, has resulted in over seven million confirmed deaths globally since late 2019, with excess mortality estimates suggesting a higher toll. As a positive-sense RNA virus in the Coronaviridae family, SARS-CoV-2 evolves through frequent mutation and recombination, specifically within the spike binding domain, which allows it to invade immune detection, increase transmissibility, and alter pathogenicity. While the clinical outcomes vary widely, evidence has shown that cross-reactive T cells that have been primed by endemic human coronaviruses–including HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43– may recognize conserved genomic regions outside of the spike protein, which could potentially reduce disease severity.
While there is widespread use of tiled amplicon whole-genome sequencing for SARS-CoV-2 surveillance, standardized approaches for endemic human coronaviruses remain limited. This gap restricts the comprehensive comparisons of genome-wide variation and the identification of conserved regions relevant to cross-reactive immunity.
This study aims to develop and optimize a rapid tiled amplicon sequencing pipeline for the detection and whole genome characterization of both SARS-CoV-2 and endemic HCoVs. Viral RNA extracted from positive clinical samples were reversed into complementary DNA, and amplified using multiplexed, overlapping primer pools spanning the entire viral genome. Sequencing is done using Oxford Nanopore sequencing to get real-time genome assembly and mutation profiling. Comparative genomic analyses will then be done to look at sequence conservation, mutation frequency, and recombination patterns across the viral genes.
We hypothesize that this approach will identify conserved genomic regions among human coronaviruses and show evolutionary patterns and limitations of human coronaviruses. These findings may provide insight into mechanisms of cross-reactive immunity and the design of broadly protective coronavirus vaccines and improved genomic surveillance strategies.