Abstract
Infectious diseases caused by diverse pathogens-including viruses, bacteria, and fungi-pose a significant diagnostic challenge, especially when conventional assays fail to identify the causative agents. Next-generation sequencing (NGS), particularly hybrid-capture-based NGS, offers a culture-independent approach for comprehensive pathogen detection directly from clinical samples. In this study, we developed a hybrid-capture-based NGS panel targeting 85 respiratory pathogens (62 viruses, 23 bacteria/fungi) and evaluated its analytical and clinical performance. Analytical validation using 50 pathogens demonstrated high sensitivity, with 10× genome coverage ≥ 96% in 32 of 36 viral targets and ≥ 95% in all 14 bacterial/fungal targets. The panel maintained ≥ 80% genome coverage at Ct values up to 34. Clinical assessment of 561 respiratory specimens identified 25 pathogen species, achieving ≥ 90% genome coverage in 57.5% of virus-positive and 83.6% of bacteria-positive samples. Notably, additional pathogens were detected in high Ct value undetected by RT-PCR, and 35 cases of viral-viral and viral-bacterial co-infections were identified. This approach improves the detection performance for respiratory infections of unknown etiology and serves as a practical platform for the detection of emerging pathogens and public health preparedness. This technology has significant potential for applications in infectious disease surveillance and development of vaccines and therapeutics.