Article

Nature 437, 376-380 (15 September 2005) | doi:10.1038/nature03959; Received 6 May 2005; Accepted 10 June 2005; Published online 31 July 2005

There is a Corrigendum (26 January 2006) associated with this document.

There is a Corrigendum (4 May 2006) associated with this document.

Genome sequencing in microfabricated high-density picolitre reactors

Marcel Margulies1,5, Michael Egholm1,5, William E. Altman1, Said Attiya1, Joel S. Bader1, Lisa A. Bemben1, Jan Berka1, Michael S. Braverman1, Yi-Ju Chen1, Zhoutao Chen1, Scott B. Dewell1, Lei Du1, Joseph M. Fierro1, Xavier V. Gomes1, Brian C. Godwin1, Wen He1, Scott Helgesen1, Chun He Ho1, Gerard P. Irzyk1, Szilveszter C. Jando1, Maria L. I. Alenquer1, Thomas P. Jarvie1, Kshama B. Jirage1, Jong-Bum Kim1, James R. Knight1, Janna R. Lanza1, John H. Leamon1, Steven M. Lefkowitz1, Ming Lei1, Jing Li1, Kenton L. Lohman1, Hong Lu1, Vinod B. Makhijani1, Keith E. McDade1, Michael P. McKenna1, Eugene W. Myers2, Elizabeth Nickerson1, John R. Nobile1, Ramona Plant1, Bernard P. Puc1, Michael T. Ronan1, George T. Roth1, Gary J. Sarkis1, Jan Fredrik Simons1, John W. Simpson1, Maithreyan Srinivasan1, Karrie R. Tartaro1, Alexander Tomasz3, Kari A. Vogt1, Greg A. Volkmer1, Shally H. Wang1, Yong Wang1, Michael P. Weiner4, Pengguang Yu1, Richard F. Begley1 & Jonathan M. Rothberg1

  1. 454 Life Sciences Corp., 20 Commercial Street, Branford, Connecticut 06405, USA
  2. University of California, Berkeley, California 94720, USA
  3. Laboratory of Microbiology, The Rockefeller University, New York, New York 10021, USA
  4. The Rothberg Institute for Childhood Diseases, 530 Whitfield Street, Guilford, Connecticut 06437, USA
  5. *These authors contributed equally to this work

Correspondence to: Jonathan M. Rothberg1 Correspondence and requests for materials should be addressed to J.M.R. (Email: jrothberg@454.com). Sequences for M. genitalium and S. pneumoniae are deposited at DDBJ/EMBL/GenBank under accession numbers AAGX01000000 and AAGY01000000, respectively.

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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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