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 RIS Download
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