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Sequencing genomes from single cells by polymerase cloning

Nature Biotechnology volume 24, pages 680–686 (2006)Cite this article

Abstract

Genome sequencing currently requires DNA from pools of numerous nearly identical cells (clones), leaving the genome sequences of many difficult-to-culture microorganisms unattainable. We report a sequencing strategy that eliminates culturing of microorganisms by using real-time isothermal amplification to form polymerase clones (plones) from the DNA of single cells. Two Escherichia coli plones, analyzed by Affymetrix chip hybridization, demonstrate that plonal amplification is specific and the bias is randomly distributed. Whole-genome shotgun sequencing of Prochlorococcus MIT9312 plones showed 62% coverage of the genome from one plone at a sequencing depth of 3.5×, and 66% coverage from a second plone at a depth of 4.7 ×. Genomic regions not revealed in the initial round of sequencing are recovered by sequencing PCR amplicons derived from plonal DNA. The mutation rate in single-cell amplification is <2 × 105, better than that of current genome sequencing standards. Polymerase cloning should provide a critical tool for systematic characterization of genome diversity in the biosphere.

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Figure 1: Ploning on E. coli single cells.
Figure 2: Characterization of plones.
Figure 3: Resolving hyperbranched DNA structure for sequencing library construction.

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References

  1. Shendure, J., Mitra, R.D., Varma, C. & Church, G.M. Advanced sequencing technologies: methods and goals. Nat. Rev. Genet. 5, 335–344 (2004).

    Article  CAS  Google Scholar 

  2. Kyrpides, N.C. Genomes OnLine Database (GOLD 1.0): a monitor of complete and ongoing genome projects world-wide. Bioinformatics 15, 773–774 (1999).

    Article  CAS  Google Scholar 

  3. Moreira, D. & Lopez-Garcia, P. The molecular ecology of microbial eukaryotes unveils a hidden world. Trends Microbiol. 10, 31–38 (2002).

    Article  CAS  Google Scholar 

  4. Falkowski, P.G. & de Vargas, C. Genomics and evolution. Shotgun sequencing in the sea: a blast from the past? Science 304, 58–60 (2004).

    Article  Google Scholar 

  5. Tyson, G.W. et al. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428, 37–43 (2004).

    Article  CAS  Google Scholar 

  6. Venter, J.C. et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304, 66–74 (2004).

    Article  CAS  Google Scholar 

  7. DeLong, E.F. Microbial community genomics in the ocean. Nat. Rev. Microbiol. 3, 459–469 (2005).

    Article  CAS  Google Scholar 

  8. Beja, O. et al. Comparative genomic analysis of archaeal genotypic variants in a single population and in two different oceanic provinces. Appl. Environ. Microbiol. 68, 335–345 (2002).

    Article  CAS  Google Scholar 

  9. Tringe, S.G. et al. Comparative metagenomics of microbial communities. Science 308, 554–557 (2005).

    Article  CAS  Google Scholar 

  10. Riesenfeld, C.S., Schloss, P.D. & Handelsman, J. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38, 525–552 (2004).

    Article  CAS  Google Scholar 

  11. Rodriguez-Valera, F. Environmental genomics, the big picture? FEMS Microbiol. Lett. 231, 153–158 (2004).

    Article  CAS  Google Scholar 

  12. Dean, F.B. et al. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. USA 99, 5261–5266 (2002).

    Article  CAS  Google Scholar 

  13. Hosono, S. et al. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954–964 (2003).

    Article  CAS  Google Scholar 

  14. Paez, J.G. et al. Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acids Res. 32, e71 (2004).

    Article  Google Scholar 

  15. Telenius, H. et al. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13, 718–725 (1992).

    Article  CAS  Google Scholar 

  16. Zhang, L. et al. Whole genome amplification from a single cell: implications for genetic analysis. Proc. Natl. Acad. Sci. USA 89, 5847–5851 (1992).

    Article  CAS  Google Scholar 

  17. Dietmaier, W. et al. Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am. J. Pathol. 154, 83–95 (1999).

    Article  CAS  Google Scholar 

  18. Nelson, J.R. et al. TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing. Biotechniques Suppl., 44–47 (2002).

  19. Lage, J.M. et al. Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH. Genome Res. 13, 294–307 (2003).

    Article  CAS  Google Scholar 

  20. Handyside, A.H. et al. Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. Mol. Hum. Reprod. 10, 767–772 (2004).

    Article  CAS  Google Scholar 

  21. Hellani, A. et al. Multiple displacement amplification on single cell and possible PGD applications. Mol. Hum. Reprod. 10, 847–852 (2004).

    Article  CAS  Google Scholar 

  22. Sorensen, K.J., Turteltaub, K., Vrankovich, G., Williams, J. & Christian, A.T. Whole-genome amplification of DNA from residual cells left by incidental contact. Anal. Biochem. 324, 312–314 (2004).

    Article  CAS  Google Scholar 

  23. Jiang, Z., Zhang, X., Deka, R. & Jin, L. Genome amplification of single sperm using multiple displacement amplification. Nucleic Acids Res. 33, e91 (2005).

    Article  Google Scholar 

  24. Detter, J.C. et al. Isothermal strand-displacement amplification applications for high-throughput genomics. Genomics 80, 691–698 (2002).

    Article  CAS  Google Scholar 

  25. Raghunathan, A. et al. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71, 3342–3347 (2005).

    Article  CAS  Google Scholar 

  26. Hutchison, C.A., III, Smith, H.O., Pfannkoch, C. & Venter, J.C. Cell-free cloning using {phi}29 DNA polymerase. Proc. Natl. Acad. Sci. USA 102, 17332–17336 (2005).

    Article  CAS  Google Scholar 

  27. Hafner, G.J., Yang, I.C., Wolter, L.C., Stafford, M.R. & Giffard, P.M. Isothermal amplification and multimerization of DNA by Bst DNA polymerase. Biotechniques 30, 852–856, 858, 860 passim (2001).

    Article  CAS  Google Scholar 

  28. Gray, J.W. et al. High-speed chromosome sorting. Science 238, 323–329 (1987).

    Article  CAS  Google Scholar 

  29. Chisholm, S.W., Olson, R.J., Zettler, E.R. & Goericke, R. A novel free-living prochlorophyte abundant in the oceanic euphotic zone. Nature 334, 340–343 (1988).

    Article  Google Scholar 

  30. Partensky, F., Hess, W.R. & Vaulot, D. Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol. Mol. Biol. Rev. 63, 106–127 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Rocap, G. et al. Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424, 1042–1047 (2003).

    Article  CAS  Google Scholar 

  32. Dufresne, A. et al. Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimal oxyphototrophic genome. Proc. Natl. Acad. Sci. USA 100, 10020–10025 (2003).

    Article  CAS  Google Scholar 

  33. Urbach, E. & Chisholm, S.W. Genetic diversity in Prochlorococcus Populations flow-cytometrically sorted from the Sargasso Sea and Gulf Stream. Limnol. Oceanogr. 43, 1615–1630 (1998).

    Article  CAS  Google Scholar 

  34. Panelli, S., Damiani, G., Espen, L. & Sgaramella, V. Ligation overcomes terminal underrepresentation in multiple displacement amplification of linear DNA. Biotechniques 39 174, 176, 178 passim (2005).

    Article  CAS  Google Scholar 

  35. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

    Article  CAS  Google Scholar 

  36. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

    Article  CAS  Google Scholar 

  37. Thompson, J.R. et al. Genotypic diversity within a natural coastal bacterioplankton population. Science 307, 1311–1313 (2005).

    Article  CAS  Google Scholar 

  38. Acinas, S.G. et al. Fine-scale phylogenetic architecture of a complex bacterial community. Nature 430, 551–554 (2004).

    Article  CAS  Google Scholar 

  39. Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

    Article  CAS  Google Scholar 

  40. Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Shendure, M. Umbarger and M. Wright for critical comments on the manuscript; C. Detter (DOE-JGI) for technical assistance on sequencing library construction. We would also like to thank the US Department of Energy for Genomes-to-Life Center support (G.M.C. and S.W.C.), and the National Science Foundation and the Moore Foundation for additional support (S.W.C.).

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Kun Zhang, Nikos B Reppas & George M Church

  2. Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 15 Vassar Street, Cambridge, 02139, Massachusetts, USA

    Adam C Martiny & Sallie W Chisholm

  3. United States Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, 94598, California, USA

    Kerrie W Barry

  4. Agencourt Bioscience, 500 Cummings Center, Suite 2450, Beverly, 01915, Massachusetts, USA

    Joel Malek

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Contributions

A.C.M. contributed to Prochlorococcus single-cell amplification, data analyses and manuscript writing, N.B.R. contributed to E. coli single-cell amplification, Affymetrix gene-chip analyses and writing, K.W.B. contributed to genome assembly, J.M. contributed to the development of sequencing library construction protocol, S.W.C. contributed to the design of the project and writing. K.Z. contributed to the development of the real-time amplification method, polymerase cloning, characterization of plones by Affymetrix gene-chip analyses and genome sequencing, development of the library construction protocol, data analyses and manuscript writing. S.W.C. and G.M.C. contributed to the planning and design of the project, and manuscript writing.

Note: Supplementary information is available on the Nature Biotechnology website.

Corresponding author

Correspondence to Kun Zhang.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Quantification of amplification background with real-time MDA. (PDF 98 kb)

Supplementary Fig. 2

Achieving ultra-low background amplification. (PDF 87 kb)

Supplementary Fig. 3

The effect of primer concentration and enzyme amount on the dynamics of isothermal amplification. (PDF 82 kb)

Supplementary Fig. 4

E. coli plones amplified with a restricted-randomized hexamer D6. (PDF 85 kb)

Supplementary Fig. 5

“Dips” of ratio profiles corresponds to genomic regions with low copy numbers. (PDF 72 kb)

Supplementary Fig. 6

Breaking chimeric sequences improved genome assembly. (PDF 86 kb)

Supplementary Fig. 7

Investigation of the source of chimera. (PDF 79 kb)

Supplementary Fig. 8

Genome coverage as function of genome sequencing effort. (PDF 63 kb)

Supplementary Fig. 9

Uneven read depth due to amplification bias. (PDF 163 kb)

Supplementary Fig. 10

Recovering sequences within gaps with PCR. (PDF 85 kb)

Supplementary Fig. 11

Preliminary characterization of two Prochlorococcus plones from an ocean sample. (PDF 100 kb)

Supplementary Table 1

Amplification dynamics and endogenous background of seven primers. (PDF 74 kb)

Supplementary Note

Achieving ultra-low background amplification on single cells. (PDF 107 kb)

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Zhang, K., Martiny, A., Reppas, N. et al. Sequencing genomes from single cells by polymerase cloning. Nat Biotechnol 24, 680–686 (2006). https://doi.org/10.1038/nbt1214

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