Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we develope...

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Published in:Frontiers in microbiology Vol. 9; p. 2748
Main Authors: Imai, Kazuo, Tamura, Kaku, Tanigaki, Tomomi, Takizawa, Mari, Nakayama, Eiko, Taniguchi, Takahiko, Okamoto, Misako, Nishiyama, Yasumasa, Tarumoto, Norihito, Mitsutake, Kotaro, Murakami, Takashi, Maesaki, Shigefumi, Maeda, Takuya
Format: Journal Article
Language:English
Published: Switzerland Frontiers Media S.A 13-11-2018
Subjects:
influenza virus
Japan
Microbiology
MinION
MiSeq
whole genome sequencing
whole genome sequencing
influenza virus
MinION
MiSeq
Japan
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Summary:Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer. Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer. Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D reads. We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses.
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Edited by: Leonard Peruski, Centers for Disease Control and Prevention (CDC), United States
This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology
Reviewed by: Alfonso Benítez-Páez, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Spain; Zheng Wang, Yale University, United States
ISSN:1664-302X
1664-302X
DOI:10.3389/fmicb.2018.02748