Targeted sequence capture of coxsackievirus A6 using nanopore sequencing directly from clinical specimens

Coxsackievirus A6 (CVA6) has emerged as a predominant causative agent of hand, foot, and mouth disease worldwide. Efficient genomic surveillance is crucial for understanding its evolution and spread. Nanopore sequencing presents a promising platform for rapid and cost-effective viral genome sequencing directly from clinical samples. We developed a tiling amplicon-based nanopore sequencing protocol for CVA6. Eight clinical CVA6-positive samples, spanning a wide range of Ct values (15.54-25.28), were each serially diluted to three concentrations (undiluted, 10⁻¹, and 10⁻²), generating a total of 24 libraries. These libraries were sequenced using a PromethION flow cell and a MinION flow cell on PromethION and GridION platforms, respectively. Sensitivity was assessed across the dilution series, and accuracy was benchmarked against Illumina sequencing. The protocol demonstrated high sensitivity and speed, achieving >10× average sequencing depth within 5 mins using a PromethION flow cell. High genome coverage (≥96.5%) was consistently achieved across all samples, including serial dilutions, with minimal genetic divergence: only one to five single-nucleotide variants were observed between different dilution levels of the same specimen. Complete assembly was obtained from all test samples in the 10^-2 dilutions (Ct: 21.73-32.7). Comparative genomic analysis revealed 99.81%-99.98% identity between Nanopore- and Illumina-derived consensus sequences, with sequences from the same sample clustering together phylogenetically. This study establishes a sensitive, accurate, and cost-effective nanopore sequencing workflow for CVA6 genomic surveillance directly from clinical specimens. The workflow delivers clear, data-informed implementation guidance, making it a practical tool for outbreak response and evolutionary tracking.IMPORTANCECoxsackievirus A6 (CVA6) has become a predominant cause of hand, foot, and mouth disease worldwide, necessitating rapid and accessible genomic surveillance. This study establishes a tiling amplicon-based nanopore sequencing protocol that delivers complete CVA6 genomes directly from clinical samples within hours. The workflow demonstrates high sensitivity across a wide range of Ct values, achieves >99.8% consensus accuracy compared to Illumina sequencing, and offers clear, data-driven guidelines for implementation. By enabling same-day, cost-effective genomic analysis, this approach equips public health laboratories with a practical tool for real-time outbreak response and tracking of viral evolution.