Single-amplicon RT-PCR and Illumina sequencing of rhinovirus and enterovirus genomes v1

This is the authors' abstract. We don't add key points for this paper.

This protocol amplifies the coding-complete genome of rhinoviruses and enteroviruses (approximately 7 kb) from extracted RNA in a single RT-PCR, using a 5′ UTR forward primer (f448) and a reverse primer that targets the tag introduced during reverse transcription with T30TAG, and prepares the product for Illumina sequencing. A visible band on an agarose gel before library preparation indicates successful amplification. In 140 rhinovirus-positive respiratory specimens, the protocol yielded genomic data from 84% of specimens, including six co-infections with two rhinovirus genotypes (of the same or different species) and one co-infection of rhinovirus with coxsackievirus. The protocol also recovered high-quality genomes of 26 enterovirus A–D types in a panel of 41 isolates.