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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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
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.
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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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DOI: https://doi.org/10.1038/nbt1214
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