title: Accurate multiplex polony sequencing of an evolved bacterial genome.
abstract: 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.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. shendure@alumni.princeton.edu
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Porreca
authors.forename: Nikos B
authors.initials: NB
authors.lastname: Reppas
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Lin
authors.forename: John P
authors.initials: JP
authors.lastname: McCutcheon
authors.forename: Abraham M
authors.initials: AM
authors.lastname: Rosenbaum
authors.forename: Michael D
authors.initials: MD
authors.lastname: Wang
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Robi D
authors.initials: RD
authors.lastname: Mitra
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D000180:Acrylic Resins; D000465:Algorithms; D001331:Automation; D003365:Costs and Cost Analysis; D011088:DNA Ligases / Q000378:metabolism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D019143:Evolution, Molecular; D005456:Fluorescent Dyes; D005782:Gels; D015723:Gene Library; D016680:Genome, Bacterial; D008856:Microscopy, Fluorescence; D008863:Microspheres; D009154:Mutation; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000191:economics / Q000295:instrumentation / Q000379:methods*
chemical_list: D000180:Acrylic Resins; D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D005782:Gels; C016680:polyacrylamide gels; D011088:DNA Ligases