Tweet
Front Microbiol. 2018 Nov 13;9:2748. doi: 10.3389/fmicb.2018.02748. eCollection 2018.
Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study.
Imai K1,2,3, Tamura K3, Tanigaki T4, Takizawa M5, Nakayama E5, Taniguchi T5, Okamoto M5, Nishiyama Y4, Tarumoto N1,2, Mitsutake K2,6, Murakami T2,7, Maesaki S1,2, Maeda T2,7.
Author information
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.
KEYWORDS:
Japan; MiSeq; MinION; influenza virus; whole genome sequencing
PMID: 30483243 PMCID: PMC6243006 DOI: 10.3389/fmicb.2018.02748
Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study.
Imai K1,2,3, Tamura K3, Tanigaki T4, Takizawa M5, Nakayama E5, Taniguchi T5, Okamoto M5, Nishiyama Y4, Tarumoto N1,2, Mitsutake K2,6, Murakami T2,7, Maesaki S1,2, Maeda T2,7.
Author information
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.
KEYWORDS:
Japan; MiSeq; MinION; influenza virus; whole genome sequencing
PMID: 30483243 PMCID: PMC6243006 DOI: 10.3389/fmicb.2018.02748