Goya, S.; Longley, P. C.; Mauer, C.; Greninger, A. L.

Rhinoviruses and enteroviruses are among the most frequently detected respiratory viruses, yet routine diagnostics do not report species or genotype, and standard VP1 genotyping cannot resolve transmission chains or recombination. Recovering sequence beyond VP1 currently requires probe-based enrichment, deep metagenomic sequencing, or several overlapping amplicons. We developed a single-amplicon PCR and sequencing workflow that generates coding-complete genomes from both enteroviruses and rhinoviruses in a single reaction. We evaluated the protocol on 140 rhinovirus-positive respiratory specimens and a panel of 41 isolates spanning 26 enterovirus A-D types, comparing it with hybridization capture sequencing and relating its performance to viral load measured by RT-qPCR and droplet digital RT-PCR. Single-amplicon sequencing recovered 116 high-quality rhinovirus genomes, across 57 rhinovirus genotypes, compared with 113 from hybridization capture. Single-amplicon sequencing was associated with higher fidelity and more uniform sequencing depth compared to hybridization capture. The same primer configuration also recovered two unexpected coxsackieviruses (A6 and A16) from rhinovirus-positive clinical specimens, and the expected polyprotein-coding sequences from all 40 enterovirus isolates tested. Recovery increased with viral abundance, and a visible amplicon band predicted sequencing success. Single-amplicon sequencing reagents cost approximately three-fold less per sample than hybridization capture ($30 versus $85) and required one-third as much time. These findings support single-amplicon sequencing as a simple, cost-effective approach for genomic surveillance of enteroviruses and rhinoviruses from clinical specimens.