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

Kazuo Imai,Takashi Murakami,Norihito Tarumoto,Misako Okamoto,Yasumasa Nishiyama,Mari Takizawa,Kaku Tamura,Takahiko Taniguchi,Tomomi Tanigaki,Eiko Nakayama,Takuya Maeda,Kotaro Mitsutake,Shigefumi Maesaki

doi:10.3389/fmicb.2018.02748

Kazuo Imai, Takashi Murakami + Show 11 more

Open Access

https://doi.org/10.3389/fmicb.2018.02748

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Abstract

Introduction: 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.Methods: 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 1D2 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.Results: 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 1D2 reads.Conclusion: 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.

Highlights

Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses
The nested PCR assays were performed using KAPA HiFi HotStart ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, United States) and thermal cycling was carried out under the following conditions: 94◦C for 5 min, followed by 18 cycles at Multiplex RT-PCR for Clinical Samples
Based on the results of RT-PCR amplicon sequencing using the Illumina MiSeq, 6 samples were identified as influenza A H3N2 subtype and 7 samples were identified as influenza B Yamagata lineage

Summary

Introduction

Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. We developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer. The genomes of influenza viruses frequently develop genetic changes and have a high level of genetic diversity, which contributes to their ability to evade the human immune system (Grenfell et al, 2004). A simple amplification method using RT-PCR for influenza virus has been developed and is contributing to the efficient surveillance of influenza viruses via next-generation sequencing (NGS) (Zhou et al, 2009; Zhou et al, 2014; Zhao et al, 2016). It is challenging to establish facilities with an NGS platform for the surveillance of influenza viruses in developing countries (Vemula et al, 2016)

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