Correspondence and requests for materials should be addressed to J. M. Rothberg (moc.454@grebhtorj). Sequences for M. genitalium and S. pneumoniae were deposited at DDBJ/EMBL/GenBank under accession numbers AAGX01000000 and AAGY01000000 respectively.

^{These authors contributed equally to the work.}

Abstract

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 60×60 mm^{fibreoptic slide containing 1,600,000 individual wells and is able to sequence 25 million bases, at 99% or better accuracy (}phred 20), in a 4 hour run. To provide sequencing templates, we clonally amplify DNA fragments on beads in the droplets of an emulsion. The template-carrying beads are loaded into the wells to convert each into a picoliter-scale sequencing reactor. We perform sequencing by synthesis using a pyrosequencing protocol optimized for solid support and the small dimension of the open reactors. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembling the Mycoplasma genitalium genome with 96% coverage at 99.96 % accuracy in one run of the machine.

Abstract

DNA sequencing has dramatically changed the nature of biomedical research and medicine. Reductions in the cost, complexity and time required to sequence large amount of DNA, including improvements in the ability to sequence bacterial and eukaryotic genomes will have significant scientific, economic and cultural impact. Large scale sequencing projects, including whole genome sequencing, have usually required the cloning of DNA fragments into bacterial vectors, amplification and purification of individual templates, followed by Sanger sequencing ¹ using fluorescent chain-terminating nucleotide analogues ² and either slab gel or capillary electrophoresis. Current estimates put the cost of sequencing a human genome between $10 and $25 million ³. Alternative sequencing methods have been described ⁴5678 however, no technology has displaced the use of bacterial vectors and Sanger sequencing as the main generators of sequence information.

In this paper we describe an integrated system whose throughput routinely enables applications requiring millions of bases of sequence information, including whole genome sequencing Our focus has been on the co-development of an emulsion-based method ⁹1011 to isolate and amplify DNA fragments in vitro, and of a fabricated substrate and instrument that performs pyrophosphate-based sequencing (“pyrosequencing” ⁵12) in picoliter-sized wells.

In a typical run we generate over 25 million bases with a phred 20 or better quality score (predicted to have an accuracy of 99% or higher). While this phred 20 quality throughput is significantly higher than that of Sanger sequencing by capillary electrophoresis, it is currently at the cost of substantially shorter reads and lower average individual read accuracy ¹³. We further characterize the performance of the system, and demonstrate that it is possible to assemble bacterial genomes de novo from relatively short reads, by sequencing a known bacterial genome, Mycoplasma genitalium (580 kbp), and comparing our shotgun sequencing and de novo assembly with the results originally obtained for this genome ¹⁴. The results of shotgun sequencing and de novo assembly of a larger bacterial genome, Streptococcus pneumoniae¹⁵ (2.1 Mbp), are presented in Supplementary Table 4.

Footnotes

Supplementary Information accompanies the paper on www.nature.com/nature.

Footnotes

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