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      Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

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          Abstract

          Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples’ genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.

          Most cited references13

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          Improved data analysis for the MinION nanopore sequencer

          Miten Jain, Ian T Fiddes, Karen H. Miga … (2015)
          The Oxford Nanopore MinION sequences individual DNA molecules using an array of pores that read nucleotide identities based on ionic current steps. We evaluated and optimized MinION performance using M13 genomic dsDNA. Using expectation-maximization (EM) we obtained robust maximum likelihood (ML) estimates for read insertion, deletion and substitution error rates (4.9%, 7.8%, and 5.1% respectively). We found that 99% of high-quality ‘2D’ MinION reads mapped to reference at a mean identity of 85%. We present a MinION-tailored tool for single nucleotide variant (SNV) detection that uses ML parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99%. By pairing our high-confidence alignment strategy with long MinION reads, we resolved the copy number for a cancer/testis gene family (CT47) within an unresolved region of human chromosome Xq24.
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            Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA.

            Farzin Haque, Jinghong Li, Hai-Chen Wu … (2013)
            Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed.
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              Successful test launch for nanopore sequencing.

              Nicholas J Loman, Mick Watson (2015)
              Article 0 comments Cited 68 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): G3 (Bethesda)
                Journal ID (iso-abbrev): Genetics
                Journal ID (hwp): G3: Genes, Genomes, Genetics
                Journal ID (pmc): G3: Genes, Genomes, Genetics
                Journal ID (publisher-id): G3: Genes, Genomes, Genetics
                Title: G3: Genes|Genomes|Genetics
                Publisher: Genetics Society of America
                ISSN (Electronic): 2160-1836
                Publication date (Electronic): 14 March 2018
                Publication date Collection: May 2018
                Volume: 8
                Issue: 5
                Pages: 1649-1657
                Affiliations
                [1]Department of Obstetrics and Gynecology, Columbia University Medical Center, New York, NY 10032
                Author notes
                [1 ]Corresponding authors: Zev Williams, Columbia University Medical Center. 622 West 168 th Street, PH 16-25, New York, NY 10032, E-mail: zw2421@ 123456cumc.columbia.edu ; and Shan Wei, Columbia University Medical Center. 630 West 168 th Street, P&S 16-437, New York, NY 10032 E-mail: sw3203@ 123456cumc.columbia.edu
                Article
                Publisher ID: GGG_200087
                DOI: 10.1534/g3.118.200087
                PMC ID: 5940156
                PubMed ID: 29540443
                SO-VID: 220474b5-7965-4c2b-8756-e5f0c40db380
                Copyright © Copyright © 2018 Wei et al.
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 29 January 2018
                Date accepted : 06 March 2018
                Page count
                Figures: 3, Tables: 1, Equations: 0, References: 22, Pages: 9
                Categories
                Subject: Investigation

                ScienceOpen disciplines: Genetics
                Keywords: aneuploidy detection,minion nanopore sequencing,multiplex,small dna,ultra-fast
                Data availability:
                ScienceOpen disciplines: Genetics
                Keywords: aneuploidy detection, minion nanopore sequencing, multiplex, small dna, ultra-fast

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