Abstract
Respiratory pathogens are a significant source of global morbidity, mortality, and economic burden, with the COVID-19 pandemic driving increased interest in and funding for respiratory disease surveillance. Syndromic panel multiplex nucleic acid amplification tests (NAATs) such as the BioFire Respiratory Panel (RP) are designed to identify the most common etiologic agents of respiratory illness. Untargeted metagenomic sequencing is a powerful tool for pathogen-agnostic detection, enabling the recovery of complete genomes for genomic epidemiology and variant tracking. In this study, we performed untargeted metagenomic sequencing of 305 samples previously negative by BioFire RP and SARS-CoV-2 testing and 26 samples that were previously positive by either of the diagnostic tests. A subset of 78 samples underwent probe-capture enrichment sequencing targeting human viruses. Using these methods, we identified human respiratory viruses in 16 of the 305 previously negative samples (5%). The most common viruses identified were Influenza C virus, Human Bocavirus, Rhinovirus A and C, and SARS-CoV-2. Consensus genomes were recovered for 14 viruses with > 90% coverage breadth, revealing closely related Bocavirus strains from neighboring counties and distinct Rhinovirus strains across samples. We also identified 21 samples with a single predominant bacterial or fungal species in the previous negative cohort. These findings underscore the challenges of identifying causal agents from multiplex NAAT-negative cases and highlight the utility of metagenomics for expanding the scope of pathogen surveillance.
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Data availability
The de-identified untargeted metagenomic and TWIST-enriched Illumina sequencing datasets are publicly available in the NCBI Sequencing Read Archive under BioProject PRJNA1296491.
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Acknowledgements
This work was supported by the Lawrence Livermore National Laboratory’s Laboratory Derived Research & Development program. We extend gratitude to Dr. Seema Jain, Dr. Jake Pry, Gail Sondermeyer Cooksey, Samuel Schildhauer, Lauren Linde, and the rest of the CDPH and local health department CalSRVSS Team. We are grateful to the CDPH VRDL Team for their work processing, managing, and testing CalSRVSS samples at CDPH including Cynthia Bernas, Brandon Brown, Alice Chen, Nick D’Angelo, Ambar Espinoza, Bianca Gonzaga, Ydelita Gonzales, Hugo Guevara, April Hatada, Blanca Molinar, Lisa Moua, Nichole Osugi, Tasha Padilla, Chao-Yang Pan, Clarence Reyes, Estela Saguar, Maria Salas, Ioana Seritan, Hilary Tamnanchit, Maria Uribe Fuentes, Chelsea Wright. The residual samples used in this study were obtained from the original community surveillance project CalSRVSS which was funded in part and by the Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Community Surveillance (grant 6 Nu50CK000539). Disclaimer The findings and conclusions in this article are those of the authors and do not necessarily represent the views or opinions of the California Department of Public Health, the California Health and Human Services Agency or Lawrence Livermore National Laboratory. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.
Funding
The residual samples used in this study were obtained from the original community surveillance project CalSRVSS which was funded in part and by the Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Community Surveillance (grant 6 Nu50CK000539).
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RSK, ACM, CRK and CJ contributed to the writing of the of the draft manuscript. RSK, CRK, ACM and JBT analyzed and interpreted the data. CJ, DAW, CM and SM acquired the samples. JBT, ACM, MB, CJ, DAW, CM and SM designed the study and experiments. ACM, JBT, DAW, SM and CM performed experiments. CJ and DAW contributed to project management. All authors contributed to reviewing and editing the final manuscript.
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Mascarenhas, A.C., Kantor, R.S., Thissen, J. et al. Metagenomic sequencing identifies potential respiratory pathogens in PCR-negative subset of surveillance samples. Sci Rep (2026). https://doi.org/10.1038/s41598-025-33917-4
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DOI: https://doi.org/10.1038/s41598-025-33917-4
