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

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.

Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

“…Alternative approaches to generate sequence data include amplicon-based sequencing of the influenza virus genome (51,52). However, this approach detects only the target pathogen, requiring multiple assays or more complex multiplex primer schemes to add targets and capture diverse strains of the original target.…”

Recent advances in the detection of respiratory virus infection in humans

“…Recently, a high‐throughput whole genome sequencing (WGS) method with the MinION portable sequencer was developed to test influenza A and B viruses, as subsequently validated by the Illumina MiSeq platform. The overall accuracy, precision, as well as recall rates, were 99.95%, 97.88%, and 89.41%, respectively, from 1D reads and 99.97%, 99.86%, and 93.28%, respectively, from 1D 2 reads 14 …”

Efficient whole genome sequencing of influenza A viruses

“…Since then, HTS has been successfully used to obtain whole genome sequences directly from clinical samples, after cell culture adaptation, or directly after RT-PCR amplification of individual genome segments (namely HA and NA) [15][16][17][18][19][20] . Multisegment reverse transcription-PCR reaction (M-RTPCR) is often used to amplify the genetic material to be used for preparation of sequencing libraries [21][22][23][24] . However, the quality and coverage of the viral sequences obtained can vary greatly, ranging from a high genome coverage with > 100x depth, to only a few reads per sample 15,18,[20][21][22][23][24] .…”