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We present single-molecule, real-time sequencing data obtainedfrom a DNA polymerase performing uninterrupted template-directedsynthesis using four distinguishable fluorescently labeled deoxyribonucleosidetriphosphates (dNTPs). We detected the temporal order of theirenzymatic incorporation into a growing DNA strand with zero-modewaveguide nanostructure arrays, which provide optical observationvolume confinement and enable parallel, simultaneous detectionof thousands of single-molecule sequencing reactions. Conjugationof fluorophores to the terminal phosphate moiety of the dNTPsallows continuous observation of DNA synthesis over thousandsof bases without steric hindrance. The data report directlyon polymerase dynamics, revealing distinct polymerization statesand pause sites corresponding to DNA secondary structure. Sequencedata were aligned with the known reference sequence to assaybiophysical parameters of polymerization for each template position.Consensus sequences were generated from the single-moleculereads at 15-fold coverage, showing a median accuracy of 99.3%,with no systematic error beyond fluorophore-dependent errorrates.
Pacific Biosciences, 1505 Adams Drive, Menlo Park, CA 94025, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: jkorlach{at}pacificbiosciences.com (J.K.); sturner{at}pacificbiosciences.com (S.T.)
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