Multiplex metagenomic sequencing for rapid viral pathogen identification and surveillance in clinical specimens

<h4>Background</h4>Rapid and accurate viral detection is essential for clinical diagnosis and effective outbreak surveillance. Traditional methods, including culture-based isolation and antigen tests, are time-consuming and limited by tissue tropism. Multiplex PCR panels, although faster, are constrained by predefined targets, limiting their ability to detect novel or unexpected viral strains.<h4>Methods</h4>We applied Oxford Nanopore Technology sequencing (ONT-Seq), a long-read, real-time, and multiplex metagenomic platform, to 85 clinical specimens using a sequence-independent, single-primer amplification (SISPA) workflow. Sequencing results were compared with routine clinical diagnostics for concordance and for identification of co-infections RESULTS: ONT-Seq achieved 80% concordance with clinical diagnostics and identified co-infections in 7% of cases missed by routine testing, including influenza C virus (ICV), and Sapporovirus. Among 58 adenovirus-positive cases, 31 samples with over 80% genome coverage at 20× depth were used for phylogenetic analysis, revealing adenovirus B3 as the predominant circulating strain.<h4>Conclusions</h4>ONT-based metagenomic sequencing enhances the detection of both known and emerging viruses in clinical specimens. Its ability to provide real-time, unbiased data supports its utility in improving diagnostic accuracy and viral surveillance.<h4>Clinical trial</h4>Not applicable.