Rapid Sequencing of Multiple RNA Viruses in Their Native Form

• Published in: Frontiers in microbiology 2019-02, Vol.10, p.260-260
• Main Authors: Wongsurawat, Thidathip, Jenjaroenpun, Piroon, Taylor, Mariah K, Lee, Jasper, Tolardo, Aline Lavado, Parvathareddy, Jyothi, Kandel, Sangam, Wadley, Taylor D, Kaewnapan, Bualan, Athipanyasilp, Niracha, Skidmore, Andrew, Chung, Donghoon, Chaimayo, Chutikarn, Whitt, Michael, Kantakamalakul, Wannee, Sutthent, Ruengpung, Horthongkham, Navin, Ussery, David W, Jonsson, Colleen B, Nookaew, Intawat
• Format: Article
• Language: English
• Subjects: genome; Microbiology; nanopore sequencing; native RNA; single-stranded RNA; subgenomic mRNA; virus
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2019.00260

Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97% identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.