Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights

ABSTRACT The emergence of novel SARS-CoV-2 genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using dsDNA biotin-labeled probes, and built accompanying software. The positive and negative percent agreement were defined in comparison to an orthogonal RT-PCR assay (PPA and NPA: both 96.7%). The limit of detection was established to be 800 copies/ml with an average fold-enrichment of 46,791x. We identified novel 107 mutations, including 24 in the functionally-important spike protein. Further, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and macrolide resistance in SARS-CoV-2-positive patients. This hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates. TEASER This is the first target hybridization capture-based NGS assay to detect SARS-CoV-2 genetic variants for tracking viral evolution.