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Genome sequencing in microfabricated high-density picolitre reactors.

Nature

Electrophoresis, Capillary, Emulsions, Fiber Optic Technology, Genome, Bacterial, Genomics, economics, instrumentation, Microchemistry, Mycoplasma genitalium, genetics, Polymerase Chain Reaction, Reproducibility of Results, Sensitivity and Specificity, Sequence Analysis, DNA, Time Factors

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      Abstract

      The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

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      Most cited references 18

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      DNA sequencing with chain-terminating inhibitors

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        Base-Calling of Automated Sequencer Traces UsingPhred. I. Accuracy Assessment

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          Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

          The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.
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            Author and article information

            Journal
            1464427
            16056220
            10.1038/nature03959

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