Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer
Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the h...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.875347/full |
| _version_ | 1828876579989618688 |
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| author | Yu-Chieh Liao Han-Chieh Wu Ci-Hong Liou Tsai-Ling Yang Lauderdale Tsai-Ling Yang Lauderdale I-Wen Huang Jui-Fen Lai Feng-Jui Chen Feng-Jui Chen |
| author_facet | Yu-Chieh Liao Han-Chieh Wu Ci-Hong Liou Tsai-Ling Yang Lauderdale Tsai-Ling Yang Lauderdale I-Wen Huang Jui-Fen Lai Feng-Jui Chen Feng-Jui Chen |
| author_sort | Yu-Chieh Liao |
| collection | DOAJ |
| description | Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing. |
| first_indexed | 2024-12-13T08:23:11Z |
| format | Article |
| id | doaj.art-d61c45703ae74ae394e0f6fcac2b7401 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-13T08:23:11Z |
| publishDate | 2022-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-d61c45703ae74ae394e0f6fcac2b74012022-12-21T23:53:57ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-03-011310.3389/fmicb.2022.875347875347Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION SequencerYu-Chieh Liao0Han-Chieh Wu1Ci-Hong Liou2Tsai-Ling Yang Lauderdale3Tsai-Ling Yang Lauderdale4I-Wen Huang5Jui-Fen Lai6Feng-Jui Chen7Feng-Jui Chen8Institute of Population Health Sciences, National Health Research Institutes, Zhunan Town, Miaoli County, TaiwanNational Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, TaiwanNational Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, TaiwanNational Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, TaiwanDepartment of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, TaiwanNational Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, TaiwanNational Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, TaiwanNational Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, TaiwanDepartment of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, TaiwanMolecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.https://www.frontiersin.org/articles/10.3389/fmicb.2022.875347/fullnanopore sequencingmolecular typingmultiplex polymerase chain reactionmultilocus sequence typingMinION |
| spellingShingle | Yu-Chieh Liao Han-Chieh Wu Ci-Hong Liou Tsai-Ling Yang Lauderdale Tsai-Ling Yang Lauderdale I-Wen Huang Jui-Fen Lai Feng-Jui Chen Feng-Jui Chen Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer Frontiers in Microbiology nanopore sequencing molecular typing multiplex polymerase chain reaction multilocus sequence typing MinION |
| title | Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer |
| title_full | Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer |
| title_fullStr | Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer |
| title_full_unstemmed | Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer |
| title_short | Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer |
| title_sort | rapid and routine molecular typing using multiplex polymerase chain reaction and minion sequencer |
| topic | nanopore sequencing molecular typing multiplex polymerase chain reaction multilocus sequence typing MinION |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.875347/full |
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