Accurate multiplex polony sequencing of an evolved bacterial genome.
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.
Shendure
Jay
J
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. shendure@alumni.princeton.edu
Porreca
Gregory J
GJ
Reppas
Nikos B
NB
Lin
Xiaoxia
X
McCutcheon
John P
JP
Rosenbaum
Abraham M
AM
Wang
Michael D
MD
Zhang
Kun
K
Mitra
Robi D
RD
Church
George M
GM
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*
D000180:Acrylic Resins; D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D005782:Gels; C016680:polyacrylamide gels; D011088:DNA Ligases