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Published Online August 4, 2005
Science DOI: 10.1126/science.1117389

Reports

Submitted on July 4, 2005
Accepted on July 27, 2005

Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome

Jay Shendure 1*, Gregory J. Porreca 1*, Nikos B. Reppas 1, Xiaoxia Lin 1, John P. McCutcheon 2, Abraham M. Rosenbaum 1, Michael D. Wang 1, Kun Zhang 1, Robi D. Mitra 3, George M. Church 1

1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
2 Department of Genetics; Howard Hughes Medical Institute, Washington University, St. Louis, MO 63110, USA.
3 Department of Genetics

* To whom correspondence should be addressed.
Jay Shendure , E-mail: shendure{at}alumni.princeton.edu
Gregory J. Porreca , E-mail: gregory_porreca{at}student.hms.harvard.edu

We describe a new DNA sequencing technology. A commonly available, inexpensive epifluorescence microscope can be converted to rapid nonelectrophoretic DNA sequencing automation. We apply this to resequence an evolved strain of Escherichia coli at less than 1 error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that are amplified in parallel to 1-micrometer beads by emulsion PCR. Millions 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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