Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Published Online August 4, 2005
Science DOI: 10.1126/science.1117389

Reports

Submitted on July 4, 2005
Accepted on July 27, 2005

Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome

Jay Shendure 1*, Gregory J. Porreca 1*, Nikos B. Reppas 1, Xiaoxia Lin 1, John P. McCutcheon 2, Abraham M. Rosenbaum 1, Michael D. Wang 1, Kun Zhang 1, Robi D. Mitra 3, George M. Church 1

1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
2 Department of Genetics; Howard Hughes Medical Institute, Washington University, St. Louis, MO 63110, USA.
3 Department of Genetics

* To whom correspondence should be addressed.
Jay Shendure , E-mail: shendure{at}alumni.princeton.edu
Gregory J. Porreca , E-mail: gregory_porreca{at}student.hms.harvard.edu

We describe a new DNA sequencing technology. A commonly available, inexpensive epifluorescence microscope can be converted to rapid nonelectrophoretic DNA sequencing automation. We apply this to resequence an evolved strain of Escherichia coli at less than 1 error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that are amplified in parallel to 1-micrometer beads by emulsion PCR. Millions 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.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
LookSeq: A browser-based viewer for deep sequencing data.
H. M. Manske and D. P. Kwiatkowski (2009)
Genome Res. 19, 2125-2132
   Abstract »    Full Text »    PDF »
Studying bacterial infections through culture-independent approaches.
G. B. Rogers, M. P. Carroll, and K. D. Bruce (2009)
J. Med. Microbiol. 58, 1401-1418
   Abstract »    Full Text »    PDF »
Yeast genome analysis identifies chromosomal translocation, gene conversion events and several sites of Ty element insertion.
Y. Shibata, A. Malhotra, S. Bekiranov, and A. Dutta (2009)
Nucleic Acids Res. 37, 6454-6465
   Abstract »    Full Text »    PDF »
Sequencing Genomes: From Individuals to Populations.
K. U. Mir (2009)
Brief Funct Genomic Proteomic 8, 367-378
   Abstract »    Full Text »    PDF »
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
   Abstract »    Full Text »    PDF »
Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing.
D. Summerer, H. Wu, B. Haase, Y. Cheng, N. Schracke, C. F. Stahler, M. S. Chee, P. F. Stahler, and M. Beier (2009)
Genome Res. 19, 1616-1621
   Abstract »    Full Text »    PDF »
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
   Abstract »    Full Text »    PDF »
Large-Scale Discovery of Gene-Enriched SNPs.
M. A. Gore, M. H. Wright, E. S. Ersoz, P. Bouffard, E. S. Szekeres, T. P. Jarvie, B. L. Hurwitz, A. Narechania, T. T. Harkins, G. S. Grills, et al. (2009)
The Plant Genome 2, 121-133
   Abstract »    Full Text »    PDF »
Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses.
M. J. Fullwood, C.-L. Wei, E. T. Liu, and Y. Ruan (2009)
Genome Res. 19, 521-532
   Abstract »    Full Text »    PDF »
Male gametophyte development: a molecular perspective.
M. Borg, L. Brownfield, and D. Twell (2009)
J. Exp. Bot. 60, 1465-1478
   Abstract »    Full Text »    PDF »
Next-Generation Sequencing: From Basic Research to Diagnostics.
K. V. Voelkerding, S. A. Dames, and J. D. Durtschi (2009)
Clin. Chem. 55, 641-658
   Abstract »    Full Text »    PDF »
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
   Abstract »    Full Text »    PDF »
Simulation Appraisal of the Adequacy of Number of Background Markers for Relationship Estimation in Association Mapping.
J. Yu, Z. Zhang, C. Zhu, D. A. Tabanao, G. Pressoir, M. R. Tuinstra, S. Kresovich, R. J. Todhunter, and E. S. Buckler (2009)
The Plant Genome 2, 63-77
   Abstract »    Full Text »    PDF »
A Snapshot of the Emerging Tomato Genome Sequence.
L. A. Mueller, R. K. Lankhorst, S. D. Tanksley, J. J. Giovannoni, R. White, J. Vrebalov, Z. Fei, J. van Eck, R. Buels, A. A. Mills, et al. (2009)
The Plant Genome 2, 78-92
   Abstract »    Full Text »    PDF »
Real-Time DNA Sequencing from Single Polymerase Molecules.
J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, et al. (2009)
Science 323, 133-138
   Abstract »    Full Text »    PDF »
Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template.
K. U. Mir, H. Qi, O. Salata, and G. Scozzafava (2009)
Nucleic Acids Res. 37, e5
   Abstract »    Full Text »    PDF »
A single molecule array for digital targeted molecular analyses.
J. Goransson, C. Wahlby, M. Isaksson, W. M. Howell, J. Jarvius, and M. Nilsson (2009)
Nucleic Acids Res. 37, e7
   Abstract »    Full Text »    PDF »
Population Genetic Inference From Resequencing Data.
R. Jiang, S. Tavare, and P. Marjoram (2009)
Genetics 181, 187-197
   Abstract »    Full Text »    PDF »
Comparative Genomics of Grasses Promises a Bountiful Harvest.
A. H. Paterson, J. E. Bowers, F. A. Feltus, H. Tang, L. Lin, and X. Wang (2009)
Plant Physiology 149, 125-131
   Full Text »    PDF »
Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms.
P. A. C. 't Hoen, Y. Ariyurek, H. H. Thygesen, E. Vreugdenhil, R. H. A. M. Vossen, R. X. de Menezes, J. M. Boer, G.-J. B. van Ommen, and J. T. den Dunnen (2008)
Nucleic Acids Res. 36, e141
   Abstract »    Full Text »    PDF »
Efficient mapping of Applied Biosystems SOLiD sequence data to a reference genome for functional genomic applications.
B. D. Ondov, A. Varadarajan, K. D. Passalacqua, and N. H. Bergman (2008)
Bioinformatics 24, 2776-2777
   Abstract »    Full Text »    PDF »
Keeping Up With the Next Generation: Massively Parallel Sequencing in Clinical Diagnostics.
J. R. ten Bosch and W. W. Grody (2008)
J. Mol. Diagn. 10, 484-492
   Abstract »    Full Text »    PDF »
ZOOM! Zillions of oligos mapped.
H. Lin, Z. Zhang, M. Q. Zhang, B. Ma, and M. Li (2008)
Bioinformatics 24, 2431-2437
   Abstract »    Full Text »    PDF »
De novo DNA synthesis using single molecule PCR.
T. B. Yehezkel, G. Linshiz, H. Buaron, S. Kaplan, U. Shabi, and E. Shapiro (2008)
Nucleic Acids Res. 36, e107
   Abstract »    Full Text »    PDF »
Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.
J. Guo, N. Xu, Z. Li, S. Zhang, J. Wu, D. H. Kim, M. Sano Marma, Q. Meng, H. Cao, X. Li, et al. (2008)
PNAS 105, 9145-9150
   Abstract »    Full Text »    PDF »
Status and Prospects of Association Mapping in Plants.
C. Zhu, M. Gore, E. S. Buckler, and J. Yu (2008)
The Plant Genome 1, 5-20
   Abstract »    Full Text »    PDF »
A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning.
A. Valouev, J. Ichikawa, T. Tonthat, J. Stuart, S. Ranade, H. Peckham, K. Zeng, J. A. Malek, G. Costa, K. McKernan, et al. (2008)
Genome Res. 18, 1051-1063
   Abstract »    Full Text »    PDF »
The new paradigm of flow cell sequencing.
R. A. Holt and S. J.M. Jones (2008)
Genome Res. 18, 839-846
   Abstract »    Full Text »    PDF »
ALLPATHS: De novo assembly of whole-genome shotgun microreads.
J. Butler, I. MacCallum, M. Kleber, I. A. Shlyakhter, M. K. Belmonte, E. S. Lander, C. Nusbaum, and D. B. Jaffe (2008)
Genome Res. 18, 810-820
   Abstract »    Full Text »    PDF »
Multiplex Sequencing of Pair-End-Ditags (MS-PET) for Cancer Genome Interrogation.
C. L. Wei and Y. Ruan (2008)
Am. Assoc. Cancer Res. Educ. Book 2008, 651-656
   Abstract »    Full Text »    PDF »
Single-Molecule DNA Sequencing of a Viral Genome.
T. D. Harris, P. R. Buzby, H. Babcock, E. Beer, J. Bowers, I. Braslavsky, M. Causey, J. Colonell, J. DiMeo, J. W. Efcavitch, et al. (2008)
Science 320, 106-109
   Abstract »    Full Text »    PDF »
Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis.
C. Schoen, J. Blom, H. Claus, A. Schramm-Gluck, P. Brandt, T. Muller, A. Goesmann, B. Joseph, S. Konietzny, O. Kurzai, et al. (2008)
PNAS 105, 3473-3478
   Abstract »    Full Text »    PDF »
Short read fragment assembly of bacterial genomes.
M. J. Chaisson and P. A. Pevzner (2008)
Genome Res. 18, 324-330
   Abstract »    Full Text »    PDF »
Genetic Design and Statistical Power of Nested Association Mapping in Maize.
J. Yu, J. B. Holland, M. D. McMullen, and E. S. Buckler (2008)
Genetics 178, 539-551
   Abstract »    Full Text »    PDF »
Genome-wide analysis of DNA methylation patterns.
D. Zilberman and S. Henikoff (2007)
Development 134, 3959-3965
   Abstract »    Full Text »    PDF »
3'-O-modified nucleotides as reversible terminators for pyrosequencing.
J. Wu, S. Zhang, Q. Meng, H. Cao, Z. Li, X. Li, S. Shi, D. H. Kim, L. Bi, N. J. Turro, et al. (2007)
PNAS 104, 16462-16467
   Abstract »    Full Text »    PDF »
DNA sequencing: bench to bedside and beyond.
C. A. Hutchison III (2007)
Nucleic Acids Res. 35, 6227-6237
   Abstract »    Full Text »    PDF »
Calling cards for DNA-binding proteins.
H. Wang, M. Johnston, and R. D. Mitra (2007)
Genome Res. 17, 1202-1209
   Abstract »    Full Text »    PDF »
A Perspective on DNA Microarrays in Pathology Research and Practice.
J. R. Pollack (2007)
Am. J. Pathol. 171, 375-385
   Abstract »    Full Text »    PDF »
Polony Multiplex Analysis of Gene Expression (PMAGE) in Mouse Hypertrophic Cardiomyopathy.
J. B. Kim, G. J. Porreca, L. Song, S. C. Greenway, J. M. Gorham, G. M. Church, C. E. Seidman, and J. G. Seidman (2007)
Science 316, 1481-1484
   Abstract »    Full Text »    PDF »
Fusion transcripts and transcribed retrotransposed loci discovered through comprehensive transcriptome analysis using Paired-End diTags (PETs).
Y. Ruan, H. S. Ooi, S. W. Choo, K. P. Chiu, X. D. Zhao, K.G. Srinivasan, F. Yao, C. Y. Choo, J. Liu, P. Ariyaratne, et al. (2007)
Genome Res. 17, 828-838
   Abstract »    Full Text »    PDF »
Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies.
G. M. Euskirchen, J. S. Rozowsky, C.-L. Wei, W. H. Lee, Z. D. Zhang, S. Hartman, O. Emanuelsson, V. Stolc, S. Weissman, M. B. Gerstein, et al. (2007)
Genome Res. 17, 898-909
   Abstract »    Full Text »    PDF »
Joint Estimates of Quantitative Trait Locus Effect and Frequency Using Synthetic Recombinant Populations of Drosophila melanogaster.
S. J. Macdonald and A. D. Long (2007)
Genetics 176, 1261-1281
   Abstract »    Full Text »    PDF »
Multigene amplification and massively parallel sequencing for cancer mutation discovery.
F. Dahl, J. Stenberg, S. Fredriksson, K. Welch, M. Zhang, M. Nilsson, D. Bicknell, W. F. Bodmer, R. W. Davis, and H. Ji (2007)
PNAS 104, 9387-9392
   Abstract »    Full Text »    PDF »
Advanced sequencing technologies and their wider impact in microbiology.
N. Hall (2007)
J. Exp. Biol. 210, 1518-1525
   Abstract »    Full Text »    PDF »
A single-molecule barcoding system using nanoslits for DNA analysis.
K. Jo, D. M. Dhingra, T. Odijk, J. J. de Pablo, M. D. Graham, R. Runnheim, D. Forrest, and D. C. Schwartz (2007)
PNAS 104, 2673-2678
   Abstract »    Full Text »    PDF »
From the Cover: Synthetic cooperation in engineered yeast populations.
W. Shou, S. Ram, and J. M. G. Vilar (2007)
PNAS 104, 1877-1882
   Abstract »    Full Text »    PDF »
Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators.
J. Ju, D. H. Kim, L. Bi, Q. Meng, X. Bai, Z. Li, X. Li, M. S. Marma, S. Shi, J. Wu, et al. (2006)
PNAS 103, 19635-19640
   Abstract »    Full Text »    PDF »
Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes.
P. Ng, J. J.S. Tan, H. S. Ooi, Y. L. Lee, K. P. Chiu, M. J. Fullwood, K. G. Srinivasan, C. Perbost, L. Du, W.-K. Sung, et al. (2006)
Nucleic Acids Res. 34, e84
   Abstract »    Full Text »    PDF »
Tests of parallel molecular evolution in a long-term experiment with Escherichia coli.
R. Woods, D. Schneider, C. L. Winkworth, M. A. Riley, and R. E. Lenski (2006)
PNAS 103, 9107-9112
   Abstract »    Full Text »    PDF »
Comprehensive mutation identification in an evolved bacterial cooperator and its cheating ancestor.
G. J. Velicer, G. Raddatz, H. Keller, S. Deiss, C. Lanz, I. Dinkelacker, and S. C. Schuster (2006)
PNAS 103, 8107-8112
   Abstract »    Full Text »    PDF »
From the Cover: Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing.
R. G. Blazej, P. Kumaresan, and R. A. Mathies (2006)
PNAS 103, 7240-7245
   Abstract »    Full Text »    PDF »
Environmental whole-genome amplification to access microbial populations in contaminated sediments..
C. B. Abulencia, D. L. Wyborski, J. A. Garcia, M. Podar, W. Chen, S. H. Chang, H. W. Chang, D. Watson, E. L. Brodie, T. C. Hazen, et al. (2006)
Appl. Envir. Microbiol. 72, 3291-3301
   Abstract »    Full Text »    PDF »
Non-coding RNA..
J. S. Mattick and I. V. Makunin (2006)
Hum. Mol. Genet. 15, R17-R29
   Abstract »    Full Text »    PDF »
Genomic Approaches to Surgical Diseases: 21st Annual Samuel Jason Mixter Lecture.
P. K. Donahoe (2006)
Arch Surg 141, 409-413
   Full Text »    PDF »
Genome-Wide Detection of Polymorphisms at Nucleotide Resolution with a Single DNA Microarray.
D. Gresham, D. M. Ruderfer, S. C. Pratt, J. Schacherer, M. J. Dunham, D. Botstein, and L. Kruglyak (2006)
Science 311, 1932-1936
   Abstract »    Full Text »    PDF »
BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies.
M. Fedurco, A. Romieu, S. Williams, I. Lawrence, and G. Turcatti (2006)
Nucleic Acids Res. 34, e22
   Abstract »    Full Text »    PDF »
Genome update: distribution of two-component transduction systems in 250 bacterial genomes.
K. Kiil, J. B. Ferchaud, C. David, T. T. Binnewies, H. Wu, T. Sicheritz-Ponten, H. Willenbrock, and D. W. Ussery (2005)
Microbiology 151, 3447-3452
   Full Text »    PDF »
Shotgun haplotyping: a novel method for surveying allelic sequence variation.
S. J. Lindsay, J. K. Bonfield, and M. E. Hurles (2005)
Nucleic Acids Res. 33, e152
   Abstract »    Full Text »    PDF »


To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)