Reports

Real-Time DNA Sequencing from Single Polymerase Molecules

John Eid,^* Adrian Fehr,^* Jeremy Gray,^* Khai Luong,^* John Lyle,^* Geoff Otto,^* Paul Peluso,^* David Rank,^* Primo Baybayan, Brad Bettman, Arkadiusz Bibillo, Keith Bjornson, Bidhan Chaudhuri, Frederick Christians, Ronald Cicero, Sonya Clark, Ravindra Dalal, Alex deWinter, John Dixon, Mathieu Foquet, Alfred Gaertner, Paul Hardenbol, Cheryl Heiner, Kevin Hester, David Holden, Gregory Kearns, Xiangxu Kong, Ronald Kuse, Yves Lacroix, Steven Lin, Paul Lundquist, Congcong Ma, Patrick Marks, Mark Maxham, Devon Murphy, Insil Park, Thang Pham, Michael Phillips, Joy Roy, Robert Sebra, Gene Shen, Jon Sorenson, Austin Tomaney, Kevin Travers, Mark Trulson, John Vieceli, Jeffrey Wegener, Dawn Wu, Alicia Yang, Denis Zaccarin, Peter Zhao, Frank Zhong, Jonas Korlach, Stephen Turner

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

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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Genome 10K: A Proposal to Obtain Whole-Genome Sequence for 10 000 Vertebrate Species.

Genome 10K Community of Scientists (2009)
J. Hered.
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Annotating non-coding transcription using functional genomics strategies.

A. R. R. Forrest, R. F. Abdelhamid, and P. Carninci (2009)
Brief Funct Genomic Proteomic 8, 437-443
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De novo sequencing of plant genomes using second-generation technologies.

M. Imelfort and D. Edwards (2009)
Brief Bioinform 10, 609-618
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DUF1220 Domains, Cognitive Disease, and Human Brain Evolution.

L. Dumas and J.M. Sikela (2009)
Cold Spring Harb Symp Quant Biol
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Sequencing Genomes: From Individuals to Populations.

K. U. Mir (2009)
Brief Funct Genomic Proteomic 8, 367-378
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Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.

K. J. McKernan, H. E. Peckham, G. L. Costa, S. F. McLaughlin, Y. Fu, E. F. Tsung, C. R. Clouser, C. Duncan, J. K. Ichikawa, C. C. Lee, et al. (2009)
Genome Res. 19, 1527-1541
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Opportunities for microfluidic technologies in synthetic biology.

S. Gulati, V. Rouilly, X. Niu, J. Chappell, R. I. Kitney, J. B. Edel, P. S. Freemont, and A. J. deMello (2009)
J R Soc Interface 6, S493-S506
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Drug Discovery and Natural Products: End of an Era or an Endless Frontier?.

J. W.-H. Li and J. C. Vederas (2009)
Science 325, 161-165
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DNA Sudoku--harnessing high-throughput sequencing for multiplexed specimen analysis.

Y. Erlich, K. Chang, A. Gordon, R. Ronen, O. Navon, M. Rooks, and G. J. Hannon (2009)
Genome Res. 19, 1243-1253
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Genomic approaches for designing durum wheat ready for climate change with a focus on drought.

D. Z. Habash, Z. Kehel, and M. Nachit (2009)
J. Exp. Bot. 60, 2805-2815
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A practical algorithm for finding maximal exact matches in large sequence datasets using sparse suffix arrays.

Z. Khan, J. S. Bloom, L. Kruglyak, and M. Singh (2009)
Bioinformatics 25, 1609-1616
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Advantages of next-generation sequencing versus the microarray in epigenetic research.

P. J. Hurd and C. J. Nelson (2009)
Brief Funct Genomic Proteomic
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Effects of Experimental Choices and Analysis Noise on Surveys of the "Rare Biosphere".

T. J. Hamp, W. J. Jones, and A. A. Fodor (2009)
Appl. Envir. Microbiol. 75, 3263-3270
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From the Cover: Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore.

D. Stoddart, A. J. Heron, E. Mikhailova, G. Maglia, and H. Bayley (2009)
PNAS 106, 7702-7707
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Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy.

Z. Zhang, D. Alpert, R. Francis, B. Chatterjee, Q. Yu, T. Tansey, S. L. Sabol, C. Cui, Y. Bai, M. Koriabine, et al. (2009)
PNAS 106, 3219-3224
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