© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nature 499 (2013): 431–437, doi:10.1038/nature12352. ; Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called ‘microbial dark matter’. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet. ; The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. We also thank the CeBiTec Bioinformatics Resource Facility, which is supported byBMBF grant 031A190. B.P.H. and J.A.D. were supported by the NASA Exobiology grant EXO-NNX11AR78GandNSFOISE 096842and B.P.H. by a generous contribution from G. Fullmer through the UNLV Foundation. S.M.S was supported by NSF grants OCE-0452333 and OCE-1136727, and the WHOI’s Andrew W. Mellon Fund for Innovative Research; and S.J.H. by the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund, the National Sciences and Engineering Research Council (NSERC) of Canada and the TULA foundation funded Centre for Microbial Diversity and Evolution (CMDE), and the Canadian Institute for Advanced Research (CIFAR). R.S. was supported by NSF grants DEB-841933, EF-826924, OCE-1232982, OCE-821374 and OCE-1136488, and the Deep Life I grant by the Alfred P. Sloan Foundation. P.H.was supported by a Discovery Outstanding Researcher Award (DORA) from the Australian Research Council, grant DP120103498.