1. Genome sequencing in microfabricated high-density picolitre reactors
- Author
-
Marcel, Margulies, Michael, Egholm, William E, Altman, Said, Attiya, Joel S, Bader, Lisa A, Bemben, Jan, Berka, Michael S, Braverman, Yi-Ju, Chen, Zhoutao, Chen, Scott B, Dewell, Lei, Du, Joseph M, Fierro, Xavier V, Gomes, Brian C, Godwin, Wen, He, Scott, Helgesen, Chun Heen, Ho, Chun He, Ho, Gerard P, Irzyk, Szilveszter C, Jando, Maria L I, Alenquer, Thomas P, Jarvie, Kshama B, Jirage, Jong-Bum, Kim, James R, Knight, Janna R, Lanza, John H, Leamon, Steven M, Lefkowitz, Ming, Lei, Jing, Li, Kenton L, Lohman, Hong, Lu, Vinod B, Makhijani, Keith E, McDade, Michael P, McKenna, Eugene W, Myers, Elizabeth, Nickerson, John R, Nobile, Ramona, Plant, Bernard P, Puc, Michael T, Ronan, George T, Roth, Gary J, Sarkis, Jan Fredrik, Simons, John W, Simpson, Maithreyan, Srinivasan, Karrie R, Tartaro, Alexander, Tomasz, Kari A, Vogt, Greg A, Volkmer, Shally H, Wang, Yong, Wang, Michael P, Weiner, Pengguang, Yu, Richard F, Begley, and Jonathan M, Rothberg
- Subjects
Time Factors ,2 base encoding ,Sequence assembly ,Mycoplasma genitalium ,Hybrid genome assembly ,Computational biology ,Biology ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Article ,symbols.namesake ,Fiber Optic Technology ,Paired-end tag ,Genetics ,Sanger sequencing ,Multidisciplinary ,Massive parallel sequencing ,Shotgun sequencing ,Microchemistry ,Electrophoresis, Capillary ,Reproducibility of Results ,DNA sequencing theory ,Genomics ,Sequence Analysis, DNA ,symbols ,Emulsions ,Genome, Bacterial - 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.
- Published
- 2005