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