biostudies-literatureUnknown25(1)Su YS<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 wiBMC infectious diseases1531https://www.ebi.ac.uk/biostudies/studies/S-EPMC12604265biostudies-literatureMultiplex metagenomic sequencing for rapid viral pathogen identification and surveillance in clinical specimens.PMC12604265Jiang NRWu HCTsai WHLee CYChi CYKuo YPChen FJYu GYLiao YCCheng SHHuang CTTsai WTLee KLChiu YTLin EJSu YSTien CFfalseMultiplex 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 wi2025-01-01T00:00:00Z2025 Nov2026-07-15T13:32:02.064Z2026-07-05T03:08:40.465ZS-EPMC126042654121454610.1186/s12879-025-11952-w