Zhang Kun, Martiny Adam C, Reppas Nikos B, Barry Kerrie W, Malek Joel, Chisholm Sallie W, Church George M
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Nat Biotechnol. 2006 Jun;24(6):680-6. doi: 10.1038/nbt1214. Epub 2006 May 28.
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 3.5x, and 66% coverage from a second plone at a depth of 4.7x. 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 x 10(5), better than that of current genome sequencing standards. Polymerase cloning should provide a critical tool for systematic characterization of genome diversity in the biosphere.
目前，基因组测序需要从大量近乎相同的细胞（克隆）群体中获取DNA，这使得许多难以培养的微生物的基因组序列无法获得。我们报告了一种测序策略，该策略通过实时等温扩增从单细胞DNA中形成聚合酶克隆（plones），从而无需培养微生物。通过Affymetrix芯片杂交分析的两个大肠杆菌plones表明，克隆扩增具有特异性，且偏差是随机分布的。对原绿球藻MIT9312 plones进行全基因组鸟枪法测序，在测序深度为3.5倍时，一个plone的基因组覆盖率为62%，在深度为4.7倍时，第二个plone的基因组覆盖率为66%。通过对源自克隆DNA的PCR扩增子进行测序，可以找回第一轮测序中未揭示的基因组区域。单细胞扩增中的突变率<2×10⁻⁵，优于当前的基因组测序标准。聚合酶克隆应为系统表征生物圈中的基因组多样性提供关键工具。
