ナノポアシーケンシング技術とその応用

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Learn more: PMC Disclaimer | PMC Copyright Notice MedComm (2020) . 2023 Jul 10;4(4):e316. doi: 10.1002/mco2.316 Nanopore sequencing technology and its applications Peijie Zheng Peijie Zheng 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China Find articles by Peijie Zheng 1, # , Chuntao Zhou Chuntao Zhou 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China Find articles by Chuntao Zhou 1, # , Yuemin Ding Yuemin Ding 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China 2 Institute of Translational Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China 3 Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China Find articles by Yuemin Ding 1, 2, 3, # , Bin Liu Bin Liu 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China Find articles by Bin Liu 1 , Liuyi Lu Liuyi Lu 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China Find articles by Liuyi Lu 1 , Feng Zhu Feng Zhu 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China Find articles by Feng Zhu 1 , Shiwei Duan Shiwei Duan 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China 2 Institute of Translational Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China 3 Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China Find articles by Shiwei Duan 1, 2, 3, ✉ Author information Article notes Copyright and License information 1 Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China 2 Institute of Translational Medicine, School of Medicine, Zhejiang University City College, Hangzhou, China 3 Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China * Correspondence , Shiwei Duan, Department of Clinical Medicine, School of Medicine, Zhejiang University City College, Hangzhou 310015, China. Email: [email protected] # These authors contributed equally to this work. ✉ Corresponding author. Revised 2023 May 29; Received 2022 Dec 30; Accepted 2023 May 31; Collection date 2023 Aug. © 2023 The Authors. MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMC Copyright notice PMCID: PMC10333861 PMID: 37441463 Abstract Since the development of Sanger sequencing in 1977, sequencing technology has played a pivotal role in molecular biology research by enabling the interpretation of biological genetic codes. Today, nanopore sequencing is one of the leading third‐generation sequencing technologies. With its long reads, portability, and low cost, nanopore sequencing is widely used in various scientific fields including epidemic prevention and control, disease diagnosis, and animal and plant breeding. Despite initial concerns about high error rates, continuous innovation in sequencing platforms and algorithm analysis technology has effectively addressed its accuracy. During the coronavirus disease (COVID‐19) pandemic, nanopore sequencing played a critical role in detecting the severe acute respiratory syndrome coronavirus‐2 virus genome and containing the pandemic. However, a lack of understanding of this technology may limit its popularization and application. Nanopore sequencing is poised to become the mainstream choice for preventing and controlling COVID‐19 and future epidemics while creating value in other fields such as oncology and botany. This work introduces the contributions of nanopore sequencing during the COVID‐19 pandemic to promote public understanding and its use in emerging outbreaks worldwide. We discuss its application in microbial detection, cancer genomes, and plant genomes and summarize strategies to improve its accuracy. Keywords: nanopore sequencing, SARS‐CoV‐2, COVID‐19, cancer, plant, genome, mutation, pandemic Nanopore sequencing has been instrumental in managing the COVID‐19 pandemic. This review aims to increase public understanding of its role in identifying SARS‐CoV‐2 variants and tracking changes in regional epidemiology. Additionally, the review explores the use of nanopore sequencing in detecting other microbial outbreaks, as well as its applications in cancer and plant genomics. 1. INTRODUCTION The concept of nanopore sequencing, where single‐stranded nucleic acids pass through a nanopore in a membrane under an electric field, was first proposed by David Deamer in the 1980s. 1 Despite initial skepticism, technological advances eventually made nanopore sequencing a reality. 2 Proteins such as α‐hemolysin from Staphylococcus aureus 3 , 4 , 5 and Mycobacterium smegmatis porin A (MspA) 6 , 7 were shown to distinguish the four bases on single‐stranded nucleotide molecules. The use of phi29 DNA polymerase slowed down the translocation of nucleic acid molecules through the nanopore, improving the signal‐to‐noise ratio. 8 , 9 Oxford Nanopore Technologies (ONT), founded in 2005 by Oxford professor Bayley and colleagues, 10 facilitated the commercialization of nanopore sequencing with the release of their MinION sequencer in 2014. 2 Currently, ONT has established a complete sequencing system process that includes advanced library preparation techniques combined with amplicon and other technologies, as well as numerous bioinformatics methods for analyzing and mining nanopore sequencing data. 2 Since 2019, COVID‐19 caused by severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) has spread to over 200 countries worldwide. 11 As an RNA virus, SARS‐CoV‐2 continuously mutates during transmission, and the emergence of SARS‐CoV‐2 variants poses challenges for epidemic control. 12 Obtaining a complete SARS‐CoV‐2 genome is crucial for detecting mutations because sequence changes can reduce the sensitivity of SARS‐CoV‐2 detection techniques. 13 The first genome sequence of SARS‐CoV‐2 was obtained through metagenomic sequencing. 14 Nanopore sequencing has played a significant role in the COVID‐19 pandemic. 15 There is no doubt that nanopore sequencing has broad prospects, as demonstrated during the COVID‐19 pandemic. The portability, high efficiency, and low cost of nanopore sequencing make it particularly well suited for dynamically monitoring SARS‐CoV‐2 mutations and the spread of the COVID‐19 pandemic in different countries and regions. Currently, nanopore sequencing has been used for diagnostic sequencing of SARS‐CoV‐2, 16 genome sequencing, 17 and related research 18 of SARS‐CoV‐2. In the field of pathogenic microorganism detection, nanopore sequencing has been used not only for detecting SARS‐CoV‐2 but also for identifying, typing, and monitoring the transmission of newly emerging monkeypox virus (MPXV) 19 and norovirus (NoV). 20 Nanopore sequencing can also be used to detect microbial communities in human tissues such as the skin and intestinal tract, 21 , 22 as well as in environmental samples. 23 Its long single‐molecule long‐read sequencing capabilities make it widely used in cancer research 24 , 25 and clinical diagnosis and treatment. 26 , 27 Additionally, nanopore sequencing has unique value in resolving high‐quality plant genomes. 28 However, public concerns about the error rate of nanopore sequencing still greatly limit its promotion and use. In reality, nanopore sequencing is a very cost‐effective technology, 29 especially for underdeveloped regions. With the rapid iteration of ONT's sequencing platform and the development of new base‐calling algorithms, the accuracy of nanopore sequencing has greatly improved. Furthermore, its application during the COVID‐19 pandemic has proven that data obtained through nanopore sequencing is reliable. 30 In this review, we have detailed the specific advantages of nanopore sequencing technology and its contributions during the COVID‐19 pandemic, including rapid identification of SARS‐CoV‐2, genotyping, dynamic transmission monitoring, and elucidation of related mechanisms of SARS‐CoV‐2. This work introduces the use of nanopore sequencing for detecting pathogenic microorganisms other than SARS‐CoV‐2, including viruses and bacteria. It also covers the application of nanopore sequencing in cancer research and clinical practice, as well as in plant genomics. Finally, we have analyzed the reasons for the low accuracy of nanopore sequencing and strategies for improving it. By introducing the contributions of nanopore sequencing during the COVID‐19 pandemic and its potential in other fields, we hope to promote the widespread application of next‐generation sequencing (NGS) technology and contribute to controlling new pandemics, including COV