Prior to the emergence of SARS-CoV-2 in 2019, Alphacoronaviruses 229E and NL63 and Betacoronaviruses OC43 and HKU1 were already established endemic common cold viral infections. Despite their collective contribution towards global respiratory morbidity and mortality and potential to inform the future trajectory of SARS-CoV-2 endemicity, they are infrequently sequenced. We therefore developed a 1200bp amplicon-based whole genome sequencing scheme targeting all four seasonal coronaviruses and SARS-CoV-2. The Vivaldi method was applied retrospectively and prospectively using Oxford Nanopore Technology to approximately 400 seasonal coronavirus infections diagnosed in Nottingham, UK, from February 2016 to July 2023. We demonstrate that the amplicon multiplex strategy can be applied agnostically to determine complete genomes of five different species from two coronaviral genera. 304 unique seasonal coronavirus genomes of greater than 95% coverage were achieved: 64 for 229E, 85 for NL63, 128 for OC43 and 27 for HKU1. They collectively indicated a dynamic seasonal coronavirus genomic landscape, with co-circulation of multiple variants emerging and declining over the UK winter respiratory infection season, with further geographical distinction when compared to a global dataset. Prolonged infection with concomitant intra-host evolution was also observed for both Alpha- (NL63) and Betacoronaviruses (OC43). This data represents the largest single cohort of seasonal coronavirus genomes to date and also a novel amplicon scheme for their future global surveillance suitable for widespread and easy adoption in the post-SARS-CoV-2 era of viral genomics.

The authors have declared no competing interest.

The study was funded by COG-UK (COVID-19 Genomics UK Consortium). COG-UK was supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute

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The COG-UK consortium work was approved by the PHE Research Ethics and Governance Group (R&D NR0195). Use of pre-pandemic residual diagnostic nucleic acids and associated anonymized patient information was covered by ethical approval granted to the Nottingham Health Science Biobank Research Tissue Bank, by the North West - Greater Manchester Central Research Ethics Committee, UK, reference 15/NW/0685. Nottingham University Hospitals National Health Service Trust (Nottingham, UK) further approved investigation of diagnosed coronavirus positive samples under clinical audit number 23-078C.

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All data produced in the present study are available upon reasonable request to the authors