Rapid Sequencing of Multiple RNA Viruses in their Native Form

25th February 2019 - Frontiers in Microbiology

A direct RNA sequencing protocol on the MinION was established for real-time, simultaneous detection and characterisation of multiple RNA viruses. The protocol described has the potential to be used for rapid genome/subgenome sequencing of other RNA viruses. Direct RNA sequencing with Oxford Nanopore Technologies brings the added benefit of detecting RNA base modifications alongside nucleotide sequence.

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.

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Authors

Thidathip Wongsurawat, Piroon Jenjaroenpun, Mariah K Taylor, Jasper Lee, Aline Lavado Tolardo, Jothi Parvathareddy, Sangam Kandel, Taylor D Wadley, Bualan Kaewnapan, Niracha Athipanyasilp, Andrew M Skidmore, Donghoon Chung, Chutikarn Chaimayo, Michael Whitt, Wannee Kantakamalakul, Ruengpung Sutthent, Navin Horthongkham, David W Ussery, Colleen B Jonsson, Intawat Nookaew.