Sequencing genomes from single cells by polymerase cloning

Kun Zhang, Adam C. Martiny, Nikos B. Reppas, Kerrie W. Barry, Joel Malek, Sallie W. Chisholm, George M. Church

Research output: Contribution to journalArticle

271 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)680-686
Number of pages7
JournalNature Biotechnology
Volume24
Issue number6
DOIs
Publication statusPublished - Jun 2006
Externally publishedYes

Fingerprint

Cloning
Organism Cloning
Clone Cells
Genes
Genome
Amplification
DNA
Microorganisms
Prochlorococcus
Clone cells
Firearms
Mutation Rate
DNA-Directed DNA Polymerase
Cell culture
Escherichia coli
Cells
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Microbiology

Cite this

Zhang, K., Martiny, A. C., Reppas, N. B., Barry, K. W., Malek, J., Chisholm, S. W., & Church, G. M. (2006). Sequencing genomes from single cells by polymerase cloning. Nature Biotechnology, 24(6), 680-686. https://doi.org/10.1038/nbt1214

Sequencing genomes from single cells by polymerase cloning. / Zhang, Kun; Martiny, Adam C.; Reppas, Nikos B.; Barry, Kerrie W.; Malek, Joel; Chisholm, Sallie W.; Church, George M.

In: Nature Biotechnology, Vol. 24, No. 6, 06.2006, p. 680-686.

Research output: Contribution to journalArticle

Zhang, K, Martiny, AC, Reppas, NB, Barry, KW, Malek, J, Chisholm, SW & Church, GM 2006, 'Sequencing genomes from single cells by polymerase cloning', Nature Biotechnology, vol. 24, no. 6, pp. 680-686. https://doi.org/10.1038/nbt1214
Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J, Chisholm SW et al. Sequencing genomes from single cells by polymerase cloning. Nature Biotechnology. 2006 Jun;24(6):680-686. https://doi.org/10.1038/nbt1214
Zhang, Kun ; Martiny, Adam C. ; Reppas, Nikos B. ; Barry, Kerrie W. ; Malek, Joel ; Chisholm, Sallie W. ; Church, George M. / Sequencing genomes from single cells by polymerase cloning. In: Nature Biotechnology. 2006 ; Vol. 24, No. 6. pp. 680-686.
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