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.

Keywords

MultiplexDNA sequencingBiologyComputational biologyDNAPolymerase chain reactionMultiplex polymerase chain reactionGeneticsGene

MeSH Terms

Acrylic ResinsAlgorithmsAutomationCosts and Cost AnalysisDNA LigasesDNA PrimersDNABacterialEscherichia coliEvolutionMolecularFluorescent DyesGelsGene LibraryGenomeBacterialMicroscopyFluorescenceMicrospheresMutationNucleic Acid HybridizationPoint MutationPolymerase Chain ReactionSequence AnalysisDNA

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Publication Info

Year
2005
Type
article
Volume
309
Issue
5741
Pages
1728-1732
Citations
1377
Access
Closed

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Cite This

Jay Shendure, Gregory J. Porreca, Nikos B. Reppas et al. (2005). Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome. Science , 309 (5741) , 1728-1732. https://doi.org/10.1126/science.1117389

Identifiers

DOI
10.1126/science.1117389
PMID
16081699

Data Quality

Data completeness: 86%