Nature Biotechnology 24, 680 - 686 (2006)
Published online: 28 May 2006; | doi:10.1038/nbt1214
Sequencing genomes from single cells by polymerase cloningKun Zhang1, Adam C Martiny2, Nikos B Reppas1, Kerrie W Barry3, Joel Malek4, Sallie W Chisholm2
& George M Church11
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 2
Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 15 Vassar Street, Cambridge, Massachusetts 02139, USA. 3
United States Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598, USA. 4
Agencourt Bioscience, 500 Cummings Center, Suite 2450, Beverly, Massachusetts 01915, USA.
Correspondence should be addressed to Kun Zhang kzhang@genetics.med.harvard.edu or George M Church http://arep.med.harvard.edu/gmc/email.html 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.
MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.
|
