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      Improved data analysis for the MinION nanopore sequencer


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          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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          Biological sequence analysis

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            MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island.

            Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.
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              Automated Forward and Reverse Ratcheting of DNA in a Nanopore at Five Angstrom Precision1

              Single-molecule techniques have been developed for commercial DNA sequencing 1,2 . One emerging strategy uses a nanopore to analyze DNA molecules as they are driven electrophoretically in single file order past a sensor 3-5 . However, uncontrolled DNA strand electrophoresis through nanopores is too fast for accurate base reads 6 . A proposed solution would employ processive enzymes to deliver DNA through the pore at a slower average rate 7 . Here, we describe forward and reverse ratcheting of DNA templates through the α–hemolysin (α-HL) nanopore controlled by wild-type phi29 DNA polymerase (phi29 DNAP). DNA strands were examined in single file order at one nucleotide spatial precision in real time. The registry error probability (either an insertion or deletion during one pass along a template strand) ranged from 10% to 24.5% absent optimization. This general strategy facilitates multiple reads of individual template strands and is transferrable to other nanopore devices for implementation of DNA sequence analysis.

                Author and article information

                Nat Methods
                Nat. Methods
                Nature methods
                6 June 2016
                16 February 2015
                April 2015
                14 June 2016
                : 12
                : 4
                : 351-356
                [1 ]University of California Santa Cruz Genomics Institute, Santa Cruz, CA USA
                [2 ]Department of Biomolecular Engineering, University of California, Santa Cruz, CA USA
                Author notes
                Correspondence: bioinformatics, benedict@ 123456soe.ucsc.edu ; nanopore technology, makeson@ 123456soe.ucsc.edu

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                Life sciences
                Life sciences


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