Rapid Sequencing of Multiple RNA Viruses in Their Native Form

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

doi:10.3389/fmicb.2019.00260

Rapid Sequencing of Multiple RNA Viruses in Their Native Form

Front Microbiol. 2019 Feb 25:10:260. doi: 10.3389/fmicb.2019.00260. eCollection 2019.

Authors

Thidathip Wongsurawat¹, Piroon Jenjaroenpun¹, Mariah K Taylor², Jasper Lee², Aline Lavado Tolardo³, Jyothi Parvathareddy⁴, Sangam Kandel^{1

5}, Taylor D Wadley¹, Bualan Kaewnapan⁶, Niracha Athipanyasilp⁶, Andrew Skidmore⁷, Donghoon Chung⁷, Chutikarn Chaimayo⁶, Michael Whitt², Wannee Kantakamalakul⁶, Ruengpung Sutthent⁶, Navin Horthongkham⁶, David W Ussery^{1

8}, Colleen B Jonsson², Intawat Nookaew^{1

8}

Affiliations

¹ Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
² Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, TN, United States.
³ Virology Research Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.
⁴ Regional Biocontainment Laboratory, University of Tennessee Health Science Center, Memphis, TN, United States.
⁵ Department of Bioinformatics, University of Arkansas at Little Rock, Little Rock, AR, United States.
⁶ Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
⁷ Department of Microbiology and Immunology, University of Louisville, Louisville, KY, United States.
⁸ Department of Physiology and Biophysics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States.

Abstract

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.

Keywords: MinION; genome; nanopore sequencing; native RNA; rapid detection; single-stranded RNA; subgenomic mRNA; virus.

Abstract

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