Genome Sequencing in Microfabricated High-Density Picolitre Reactors

Nature. 2005 Sep 15;437(7057):376-80. doi: 10.1038/nature03959. Epub 2005 Jul 31.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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

Substances

  • Emulsions