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Genome sequencing in microfabricated high-density picolitre reactors.

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.

Pubmed ID: 16056220

Authors

  • Margulies M
  • Egholm M
  • Altman WE
  • Attiya S
  • Bader JS
  • Bemben LA
  • Berka J
  • Braverman MS
  • Chen YJ
  • Chen Z
  • Dewell SB
  • Du L
  • Fierro JM
  • Gomes XV
  • Godwin BC
  • He W
  • Helgesen S
  • Ho CH
  • Ho CH
  • Irzyk GP
  • Jando SC
  • Alenquer ML
  • Jarvie TP
  • Jirage KB
  • Kim JB
  • Knight JR
  • Lanza JR
  • Leamon JH
  • Lefkowitz SM
  • Lei M
  • Li J
  • Lohman KL
  • Lu H
  • Makhijani VB
  • McDade KE
  • McKenna MP
  • Myers EW
  • Nickerson E
  • Nobile JR
  • Plant R
  • Puc BP
  • Ronan MT
  • Roth GT
  • Sarkis GJ
  • Simons JF
  • Simpson JW
  • Srinivasan M
  • Tartaro KR
  • Tomasz A
  • Vogt KA
  • Volkmer GA
  • Wang SH
  • Wang Y
  • Weiner MP
  • Yu P
  • Begley RF
  • Rothberg JM

Journal

Nature

Publication Data

September 15, 2005

Associated Grants

  • Agency: NHGRI NIH HHS, Id: P01 HG003022-020002
  • Agency: NHGRI NIH HHS, Id: R01 HG003562-02

Mesh Terms

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