Rapid Sequencing of Multiple RNA Viruses in Their Native Form

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, subge...

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Published inFrontiers in microbiology Vol. 10; p. 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 Journal Article
LanguageEnglish
Published Switzerland Frontiers Media S.A 25.02.2019
Subjects
genome
Microbiology
nanopore sequencing
native RNA
single-stranded RNA
subgenomic mRNA
virus
genome
nanopore sequencing
native RNA
MinION
subgenomic mRNA
rapid detection
single-stranded RNA
virus
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Summary: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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Reviewed by: Timokratis Karamitros, University of Oxford, United Kingdom; Pasquale Saldarelli, Institute for Sustainable Plant Protection (IPSP-CNR), Italy
These authors have contributed equally to this work
This article was submitted to Virology, a section of the journal Frontiers in Microbiology
Edited by: Sead Sabanadzovic, Mississippi State University, United States
ISSN:1664-302X
1664-302X
DOI:10.3389/fmicb.2019.00260