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      A complete bacterial genome assembled de novo using only nanopore sequencing data.

      Author(s): 1 , 1 , 2
      Publication date (Electronic):
      Journal: Nature methods

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          Abstract

          We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.

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          Fast algorithms for large-scale genome alignment and comparison.

          Arthur L. Delcher, Adam Phillippy, Jane Carlton … (2002)
          We describe a suffix-tree algorithm that can align the entire genome sequences of eukaryotic and prokaryotic organisms with minimal use of computer time and memory. The new system, MUMmer 2, runs three times faster while using one-third as much memory as the original MUMmer system. It has been used successfully to align the entire human and mouse genomes to each other, and to align numerous smaller eukaryotic and prokaryotic genomes. A new module permits the alignment of multiple DNA sequence fragments, which has proven valuable in the comparison of incomplete genome sequences. We also describe a method to align more distantly related genomes by detecting protein sequence homology. This extension to MUMmer aligns two genomes after translating the sequence in all six reading frames, extracts all matching protein sequences and then clusters together matches. This method has been applied to both incomplete and complete genome sequences in order to detect regions of conserved synteny, in which multiple proteins from one organism are found in the same order and orientation in another. The system code is being made freely available by the authors.
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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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              One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly.

              Sergey Koren, Adam Phillippy (2015)
              Like a jigsaw puzzle with large pieces, a genome sequenced with long reads is easier to assemble. However, recent sequencing technologies have favored lowering per-base cost at the expense of read length. This has dramatically reduced sequencing cost, but resulted in fragmented assemblies, which negatively affect downstream analyses and hinder the creation of finished (gapless, high-quality) genomes. In contrast, emerging long-read sequencing technologies can now produce reads tens of kilobases in length, enabling the automated finishing of microbial genomes for under $1000. This promises to improve the quality of reference databases and facilitate new studies of chromosomal structure and variation. We present an overview of these new technologies and the methods used to assemble long reads into complete genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
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                Author and article information

                Journal
                Journal ID (iso-abbrev): Nat. Methods
                Title: Nature methods
                ISSN (Electronic): 1548-7105
                ISSN (Print): 1548-7091
                Publication date (Electronic): Aug 2015
                Volume: 12
                Issue: 8
                Affiliations
                [1 ] Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK.
                [2 ] 1] Ontario Institute for Cancer Research, Toronto, Ontario, Canada. [2] Department of Computer Science, University of Toronto, Toronto, Ontario, Canada.
                Article
                Publisher Item ID: nmeth.3444
                DOI: 10.1038/nmeth.3444
                PubMed ID: 26076426
                SO-VID: 41e7b85c-d1dd-46f9-8b2c-0e8b61971943
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