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      ALLPATHS 2: small genomes assembled accurately and with high continuity from short paired reads

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          Abstract

          Allpaths2, a method for accurately assembling small genomes with high continuity using short paired reads.

          Abstract

          We demonstrate that genome sequences approaching finished quality can be generated from short paired reads. Using 36 base (fragment) and 26 base (jumping) reads from five microbial genomes of varied GC composition and sizes up to 40 Mb, ALLPATHS2 generated assemblies with long, accurate contigs and scaffolds. Velvet and EULER-SR were less accurate. For example, for Escherichia coli, the fraction of 10-kb stretches that were perfect was 99.8% (ALLPATHS2), 68.7% (Velvet), and 42.1% (EULER-SR).

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          Most cited references13

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          A large genome center's improvements to the Illumina sequencing system.

          The Wellcome Trust Sanger Institute is one of the world's largest genome centers, and a substantial amount of our sequencing is performed with 'next-generation' massively parallel sequencing technologies: in June 2008 the quantity of purity-filtered sequence data generated by our Genome Analyzer (Illumina) platforms reached 1 terabase, and our average weekly Illumina production output is currently 64 gigabases. Here we describe a set of improvements we have made to the standard Illumina protocols to make the library preparation more reliable in a high-throughput environment, to reduce bias, tighten insert size distribution and reliably obtain high yields of data.
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            Accurate multiplex polony sequencing of an evolved bacterial genome.

            We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.
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              Single-molecule DNA sequencing of a viral genome.

              The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.
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                Author and article information

                Journal
                Genome Biol
                Genome Biology
                BioMed Central
                1465-6906
                1465-6914
                2009
                1 October 2009
                : 10
                : 10
                : R103
                Affiliations
                [1 ]Broad Institute of MIT and Harvard, Charles Street, Cambridge, MA 02141, USA
                [2 ]Life Technologies, Cummings Center, Beverly, MA 01915, USA
                [3 ]Current address: Department of Medical Genetics, Weill Cornell Medical College in Qatar, 24144 Al-Rayaan St., Doha, Qatar
                [4 ]Current address: Pacific Biosciences, Adams Drive, Menlo Park, CA 94025, USA
                Article
                gb-2009-10-10-r103
                10.1186/gb-2009-10-10-r103
                2784318
                19796385
                402401ce-1706-459c-9564-9e7352c00ca3
                Copyright ©2009 MacCallum et al.; licensee BioMed Central Ltd.

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 19 June 2009
                : 20 August 2009
                : 1 October 2009
                Categories
                Method

                Genetics
                Genetics

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