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      From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy

      review-article
      , ,
      Genome Biology
      BioMed Central

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          Abstract

          Nanopore sequencing is a rapidly maturing technology delivering long reads in real time on a portable instrument at low cost. Not surprisingly, the community has rapidly taken up this new way of sequencing and has used it successfully for a variety of research applications. A major limitation of nanopore sequencing is its high error rate, which despite recent improvements to the nanopore chemistry and computational tools still ranges between 5% and 15%. Here, we review computational approaches determining the nanopore sequencing error rate. Furthermore, we outline strategies for translation of raw sequencing data into base calls for detection of base modifications and for obtaining consensus sequences.

          Electronic supplementary material

          The online version of this article (10.1186/s13059-018-1462-9) contains supplementary material, which is available to authorized users.

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          Most cited references28

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          Biological sequence analysis

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            Improved data analysis for the MinION nanopore sequencer

            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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              Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.

              Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.
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                Author and article information

                Contributors
                W.Kloosterman@umcutrecht.nl
                J.deridder-4@umcutrecht.nl
                Journal
                Genome Biol
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1474-7596
                1474-760X
                13 July 2018
                13 July 2018
                2018
                : 19
                : 90
                Affiliations
                Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3584 CG Utrecht, The Netherlands
                Author information
                http://orcid.org/0000-0002-0828-3477
                Article
                1462
                10.1186/s13059-018-1462-9
                6045860
                30005597
                a7fab92a-7016-4e88-8ee9-8797150f2904
                © The Author(s). 2018

                Open AccessThis article is 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 you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100003246, Nederlandse Organisatie voor Wetenschappelijk Onderzoek;
                Award ID: 639.072.715
                Award Recipient :
                Funded by: Universiteit Utrecht (NL)
                Award ID: NA
                Categories
                Review
                Custom metadata
                © The Author(s) 2018

                Genetics
                Genetics

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