Journal Article

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

Wu, Han-Chieh, Lee, Kuan-Lin, Chiu, Yueh-Tzu, Lin, En-Ju, Chen, Feng-Jui, Chi, Chia-Yu, Jiang, Ni-Rong, Cheng, Shu-Hsing, Huang, Chih-Ting, Tien, Chih-Feng, Yu, Guann-Yi, Su, Yu-Siang, Tsai, Wan-Ting, Lee, Chien-Yu, Tsai, Wei-Hsiang, Liao, Yu-Chieh, Kuo, Yi-Ping

2025

Overview

Background 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. Methods 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. Conclusions 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. Clinical Trial Not applicable.

• Publisher: BioMed Central,BioMed Central Ltd,BMC
• Subject: Adenovirus / Analysis / Antigens / Care and treatment / Coinfection - diagnosis / Coinfection - virology / Diagnosis / DNA sequencing / Female / Genetic aspects / High-Throughput Nucleotide Sequencing - methods / Human parainfluenza virus / Humans / Infectious Diseases / Internal Medicine / Medical Microbiology / Medicine / Medicine & Public Health / Metagenomics - methods / Multiplex Polymerase Chain Reaction - methods / Nanopore sequencing / Nanopore Sequencing - methods / Nucleotide sequencing / Parasitology / Phylogeny / Respiratory syncytial virus / Sapporovirus / Surveillance / Testing / Tropical Medicine / Virus diseases / Virus Diseases - diagnosis / Virus Diseases - virology / Viruses - classification / Viruses - genetics / Viruses - isolation & purification