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.
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DOI：
10.3389/fmicb.2018.02748
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发表时间：
2018
影响因子：
5.2
通讯作者：
Maeda T
中科院分区：
文献类型：
--
作者：
Imai K;Tamura K;Tanigaki T;Takizawa M;Nakayama E;Taniguchi T;Okamoto M;Nishiyama Y;Tarumoto N;Mitsutake K;Murakami T;Maesaki S;Maeda T
引言：流感病毒的全基因组测序（WGS）对于制备疫苗和应对新出现的病毒非常重要。然而，WGS在发展中国家很难使用传统的下一代测序仪进行，那里的设施往往不足。在这项研究中，我们开发了一个高通量的WGS方法流感病毒在临床标本中的MinION便携式测序仪。方法：采用多重RT-PCR方法，从日本东京地区采集的13份临床标本中扩增甲型和B型流感病毒全基因组。通过使用定制条形码引物的巢式PCR，将用于多重MinION测序的条形码标签与每个多重RT-PCR扩增子一起添加。将所有条形码化扩增子混合，并使用具有1D 2测序试剂盒的MinION测序仪进行多重测序。此外，使用Illumina MiSeq平台对每个临床标本生成的多重RT-PCR扩增子进行测序，以验证MinION测序仪的性能。通过比较Illumina MiSeq平台和MinION测序仪中的变异识别结果，计算MinION测序的准确率、召回率和精确率。结果：多重RT-PCR成功扩增出13份临床标本中的甲型和B型流感病毒全基因组。我们使用Illumina MiSeq平台鉴定了6个样品为A型流感病毒H3 N2亚型，7个样品为B型流感病毒Yamagata谱系。MinION测序仪的总体准确率、召回率和精确率分别为99.95%、89.41%和97.88%（1D读数）和99.97%、93.28%和99.86%（1D 2读数）。结论：我们建立了一种新的甲型和B型流感病毒的WGS方法。为了更好地利用MinION测序仪实时监测基因重排和评估新出现的病毒，有必要提高读数准确性和分析工具。
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.
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影响因子：
11.8
作者：
MacFadden, Derek R.;McGeer, Allison;Hanage, William P.
通讯作者：
Hanage, William P.
影响因子：
11.8
作者：
Hoenen T;Groseth A;Rosenke K;Fischer RJ;Hoenen A;Judson SD;Martellaro C;Falzarano D;Marzi A;Squires RB;Wollenberg KR;de Wit E;Prescott J;Safronetz D;van Doremalen N;Bushmaker T;Feldmann F;McNally K;Bolay FK;Fields B;Sealy T;Rayfield M;Nichol ST;Zoon KC;Massaquoi M;Munster VJ;Feldmann H
通讯作者：
Feldmann H
影响因子：
11.8
作者：
Fedson DS
通讯作者：
Fedson DS
影响因子：
5.2
作者：
Wang J;Moore NE;Deng YM;Eccles DA;Hall RJ
通讯作者：
Hall RJ
DOI：
10.1093/infdis/jiy335
发表时间：
2018-09-22
期刊：
The Journal of infectious diseases
影响因子：
--
作者：
Houlihan CF;Frampton D;Ferns RB;Raffle J;Grant P;Reidy M;Hail L;Thomson K;Mattes F;Kozlakidis Z;Pillay D;Hayward A;Nastouli E
通讯作者：
Nastouli E