Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome

American Association for the Advancement of Science (AAAS) - Tập 309 Số 5741 - Trang 1728-1732 - 2005
Jay Shendure1,2,3, Gregory J. Porreca1,2,3, Nikos B. Reppas1,2,3, Xiaoxia Lin1,2,3, John P. McCutcheon1,2,3, Abraham M. Rosenbaum1,2,3, Michael D. Wang1,2,3, Kun Zhang1,2,3, Robi D. Mitra1,2,3, George M. Church1,2,3
1Department of Genetics, Harvard Medical School, Boston, MA 02115 USA
2Department of Genetics, Washington University, St Louis, MO 63110, USA
3Howard Hughes Medical Institute, Washington University, St. Louis, MO 63110, USA.

Tóm tắt

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

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For advice encouragement and technical assistance we are deeply indebted to J. Zhu S. Douglas J. Chou J. Aach M. Nikku A. Lee N. Novikov and M. Wright (Church Lab); A. Blanchard G. Costa H. Ebling J. Ichikawa J. Malek P. McEwan K. McKernan A. Sheridan and D. Smith (Agencourt); S. Skiena (SUNY–Stony Brook) C. Felts (RPI); R. Fincher (Alcott); D. Focht (Bioptechs); and M. Hotfelder and J. Feng (Washington University). We thank B. Vogelstein J. Edwards and their groups for assistance with emulsion PCR. This work was supported by the National Human Genome Research Institute–Centers of Excellence in Genomic Science and U.S. Department of Energy–Genomes to Life grants.

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American Association for the Advancement of Science (AAAS)

Tập 309 Số 5741

1728-1732

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