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Rapid Whole-genome Sequencing of Zika Viruses using Direct RNA Sequencing

Journal of Bacteriology and Virology 2019년 49권 3호 p.115 ~ 123
 ( Kim Jung-Heon ) - Seoul National University Medical Research Center Institute of Endemic Diseases

 ( Kim Ji-Yeon ) - Seoul National University College of Medicine Department of Microbiology and Immunology
 ( Koo Bon-Sang ) - Korea Research Institute of Bioscience and Biotechnology National Primate Research Center
 ( Oh Han-Seul ) - Korea Research Institute of Bioscience and Biotechnology National Primate Research Center
 ( Hong Jung-Joo ) - Korea Research Institute of Bioscience and Biotechnology National Primate Research Center
 ( Hwang Eung-Soo ) - Seoul National University College of Medicine Department of Microbiology and Immunology

Abstract


Zika virus (ZIKV) is one of the pathogens which is transmitted world widely, but there are no effective drugs and vaccines. Whole genome sequencing (WGS) of viruses could be applied to viral pathogen characterization, diagnosis, molecular surveillance, and even finding novel pathogens. We established an improved method using direct RNA sequencing with Nanopore technology to obtain WGS of ZIKV, after adding poly (A) tails to viral RNA. This established method does not require specific primers, complimentary DNA (cDNA) synthesis, and polymerase chain reaction (PCR)-based enrichment, resulting in the reduction of biases as well as of the ability to find novel RNA viruses. Nanopore technology also allows to read long sequences. It makes WGS easier and faster with long-read assembly. In this study, we obtained WGS of two strains of ZIKV following the established protocol. The sequenced reads resulted in 99% and 100% genome coverage with 63.5X and 21,136X, for the ZIKV PRVABC59 and MR 766 strains, respectively. The sequence identities of the ZIKV PRVABC59 and MR 766 strains for each reference genomes were 98.76% and 99.72%, respectively. We also found that the maximum length of reads was 10,311 bp which is almost the whole genome size of ZIKV. These long-reads could make overall structure of whole genome easily, and WGS faster and easier. The protocol in this study could provide rapid and efficient WGS that could be applied to study the biology of RNA viruses including identification, characterization, and global surveillance.

키워드

Whole genome sequencing (WGS); Zika virus (ZIKV); Direct RNA sequencing
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