The trajectory of microbial single-cell sequencing


Over the past decade, it has become nearly routine to sequence genomes of individual microbial cells directly isolated from environmental samples ranging from deep-sea hydrothermal vents to insect guts, providing a powerful complement to shotgun metagenomics in microbial community studies. In this review, we address the technical aspects and challenges of single-cell genome sequencing and discuss some of the scientific endeavors that it has enabled. Specifically, we highlight newly added leaves and branches in the genomic tree of bacterial and archaeal life and illustrate the unique and exciting advantages that single-cell genomics offers over metagenomics, both now and in the near future.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Single-cell sequencing and analysis workflow for standard (untargeted) and targeted single-cell sequencing approaches.
Figure 2: Timeline of scientific milestones in single-cell microbial sequencing.
Figure 3: Single-cell sequencing links all DNA-containing elements within a cell and can also reveal tight physical associations between cells.
Figure 4: Bacterial and archaeal SSU-rRNA-gene-based phylogenetic tree.


  1. 1

    Venter, J.C. et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304, 66–74 (2004).

  2. 2

    Tyson, G.W. et al. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428, 37–43 (2004).

  3. 3

    Zhang, L. et al. Whole genome amplification from a single cell: implications for genetic analysis. Proc. Natl. Acad. Sci. USA 89, 5847–5851 (1992).

  4. 4

    Dean, F.B. et al. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. USA 99, 5261–5266 (2002).

  5. 5

    Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

  6. 6

    Zhang, D.Y., Brandwein, M., Hsuih, T. & Li, H.B. Ramification amplification: a novel isothermal DNA amplification method. Mol. Diagn. 6, 141–150 (2001).

  7. 7

    Gawad, C., Koh, W. & Quake, S.R. Single-cell genome sequencing: current state of the science. Nat. Rev. Genet. 17, 175–188 (2016).

  8. 8

    Raghunathan, A. et al. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71, 3342–3347 (2005).

  9. 9

    Zhang, K. et al. Sequencing genomes from single cells by polymerase cloning. Nat. Biotechnol. 24, 680–686 (2006).

  10. 10

    Yoon, H.S. et al. Single-cell genomics reveals organismal interactions in uncultivated marine protists. Science 332, 714–717 (2011).

  11. 11

    Stepanauskas, R. et al. Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles. Nat. Commun. 8, 84 (2017).

  12. 12

    Eren, A.M. et al. Anvi'o: an advanced analysis and visualization platform for 'omics data. PeerJ 3, e1319 (2015).

  13. 13

    Blainey, P.C. The future is now: single-cell genomics of bacteria and archaea. FEMS Microbiol. Rev. 37, 407–427 (2013).

  14. 14

    Marcy, Y. et al. Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc. Natl. Acad. Sci. USA 104, 11889–11894 (2007).

  15. 15

    Leung, K. et al. A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities. Proc. Natl. Acad. Sci. USA 109, 7665–7670 (2012).

  16. 16

    Woyke, T. et al. One bacterial cell, one complete genome. PLoS One 5, e10314 (2010).

  17. 17

    Stepanauskas, R. & Sieracki, M.E. Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time. Proc. Natl. Acad. Sci. USA 104, 9052–9057 (2007).

  18. 18

    Marcy, Y. et al. Nanoliter reactors improve multiple displacement amplification of genomes from single cells. PLoS Genet. 3, 1702–1708 (2007).

  19. 19

    Osborne, G.W. Recent advances in flow cytometric cell sorting. Methods Cell Biol. 102, 533–556 (2011).

  20. 20

    Campbell, J.H. et al. UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota. Proc. Natl. Acad. Sci. USA 110, 5540–5545 (2013).

  21. 21

    Dodsworth, J.A. et al. Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage. Nat. Commun. 4, 1854 (2013).

  22. 22

    Gole, J. et al. Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat. Biotechnol. 31, 1126–1132 (2013).

  23. 23

    Xu, L., Brito, I.L., Alm, E.J. & Blainey, P.C. Virtual microfluidics for digital quantification and single-cell sequencing. Nat. Methods 13, 759–762 (2016).

  24. 24

    Zengler, K. et al. Cultivating the uncultured. Proc. Natl. Acad. Sci. USA 99, 15681–15686 (2002).

  25. 25

    Dichosa, A.E., Daughton, A.R., Reitenga, K.G., Fitzsimons, M.S. & Han, C.S. Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes. Nat. Protoc. 9, 608–621 (2014).

  26. 26

    Fitzsimons, M.S. et al. Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome. Genome Res. 23, 878–888 (2013).

  27. 27

    Spencer, S.J. et al. Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers. ISME J. 10, 427–436 (2016).

  28. 28

    Lan, F., Demaree, B., Ahmed, N. & Abate, A.R. Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding. Nat. Biotechnol. 35, 640–646 (2017).

  29. 29

    Blanco, L. et al. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940 (1989).

  30. 30

    Woyke, T. et al. Assembling the marine metagenome, one cell at a time. PLoS One 4, e5299 (2009).

  31. 31

    Lasken, R.S. & Stockwell, T.B. Mechanism of chimera formation during the Multiple Displacement Amplification reaction. BMC Biotechnol. 7, 19 (2007).

  32. 32

    Marshall, I.P., Blainey, P.C., Spormann, A.M. & Quake, S.R. A single-cell genome for Thiovulum sp. Appl. Environ. Microbiol. 78, 8555–8563 (2012).

  33. 33

    Rodrigue, S. et al. Whole genome amplification and de novo assembly of single bacterial cells. PLoS One 4, e6864 (2009).

  34. 34

    Chitsaz, H. et al. Efficient de novo assembly of single-cell bacterial genomes from short-read data sets. Nat. Biotechnol. 29, 915–921 (2011).

  35. 35

    Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).

  36. 36

    Peng, Y., Leung, H.C., Yiu, S.M. & Chin, F.Y. IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28, 1420–1428 (2012).

  37. 37

    Konstantinidis, K.T. & Tiedje, J.M. Genomic insights that advance the species definition for prokaryotes. Proc. Natl. Acad. Sci. USA 102, 2567–2572 (2005).

  38. 38

    Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437 (2013).

  39. 39

    Blainey, P.C., Mosier, A.C., Potanina, A., Francis, C.A. & Quake, S.R. Genome of a low-salinity ammonia-oxidizing archaeon determined by single-cell and metagenomic analysis. PLoS One 6, e16626 (2011).

  40. 40

    Clingenpeel, S., Clum, A., Schwientek, P., Rinke, C. & Woyke, T. Reconstructing each cell's genome within complex microbial communities-dream or reality? Front. Microbiol. 5, 771 (2015).

  41. 41

    Parks, D.H., Imelfort, M., Skennerton, C.T., Hugenholtz, P. & Tyson, G.W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25, 1043–1055 (2015).

  42. 42

    Povilaitis, T., Alzbutas, G., Sukackaite, R., Siurkus, J. & Skirgaila, R. In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique. Protein Eng. Des. Sel. 29, 617–628 (2016).

  43. 43

    Zong, C., Lu, S., Chapman, A.R. & Xie, X.S. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338, 1622–1626 (2012).

  44. 44

    Chen, M. et al. Comparison of multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in single-cell sequencing. PLoS One 9, e114520 (2014).

  45. 45

    de Bourcy, C.F. et al. A quantitative comparison of single-cell whole genome amplification methods. PLoS One 9, e105585 (2014).

  46. 46

    Chen, C. et al. Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI). Science 356, 189–194 (2017).

  47. 47

    Lynch, M.D. & Neufeld, J.D. Ecology and exploration of the rare biosphere. Nat. Rev. Microbiol. 13, 217–229 (2015).

  48. 48

    Eloe-Fadrosh, E.A., Ivanova, N.N., Woyke, T. & Kyrpides, N.C. Metagenomics uncovers gaps in amplicon-based detection of microbial diversity. Nat. Microbiol. 1, 15032 (2016).

  49. 49

    Eloe-Fadrosh, E.A. et al. Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs. Nat. Commun. 7, 10476 (2016).

  50. 50

    Sipos, R. et al. Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targeting bacterial community analysis. FEMS Microbiol. Ecol. 60, 341–350 (2007).

  51. 51

    Baker, G.C., Smith, J.J. & Cowan, D.A. Review and re-analysis of domain-specific 16S primers. J. Microbiol. Methods 55, 541–555 (2003).

  52. 52

    Youssef, N.H. et al. Insights into the metabolism, lifestyle and putative evolutionary history of the novel archaeal phylum 'Diapherotrites'. ISME J. 9, 447–460 (2015).

  53. 53

    Baker, B.J. et al. Lineages of acidophilic archaea revealed by community genomic analysis. Science 314, 1933–1935 (2006).

  54. 54

    Brown, C.T. et al. Unusual biology across a group comprising more than 15% of domain bacteria. Nature 523, 208–211 (2015).

  55. 55

    Woyke, T. & Rubin, E.M. Evolution. Searching for new branches on the tree of life. Science 346, 698–699 (2014).

  56. 56

    Kim, S. et al. High-throughput automated microfluidic sample preparation for accurate microbial genomics. Nat. Commun. 8, 13919 (2017).

  57. 57

    Podar, M. et al. Targeted access to the genomes of low-abundance organisms in complex microbial communities. Appl. Environ. Microbiol. 73, 3205–3214 (2007).

  58. 58

    Stepanauskas, R. Wiretapping into microbial interactions by single cell genomics. Front. Microbiol. 6, 258 (2015).

  59. 59

    Paez-Espino, D. et al. Uncovering Earth's virome. Nature 536, 425–430 (2016).

  60. 60

    Abergel, C., Legendre, M. & Claverie, J.M. The rapidly expanding universe of giant viruses: mimivirus, pandoravirus, pithovirus and mollivirus. FEMS Microbiol. Rev. 39, 779–796 (2015).

  61. 61

    Roux, S. et al. Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics. eLife 3, e03125 (2014).

  62. 62

    Labonté, J.M. et al. Single-cell genomics-based analysis of virus-host interactions in marine surface bacterioplankton. ISME J. 9, 2386–2399 (2015).

  63. 63

    Roux, S., Hallam, S.J., Woyke, T. & Sullivan, M.B. Viral dark matter and virus–host interactions resolved from publicly available microbial genomes. eLife 4, e08490 (2015).

  64. 64

    Munson-McGee, J.H. et al. Nanoarchaeota, their Sulfolobales host, and Nanoarchaeota virus distribution across Yellowstone National Park hot springs. Appl. Environ. Microbiol. 81, 7860–7868 (2015).

  65. 65

    Huber, H. et al. A new phylum of archaea represented by a nanosized hyperthermophilic symbiont. Nature 417, 63–67 (2002).

  66. 66

    He, X. et al. Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle. Proc. Natl. Acad. Sci. USA 112, 244–249 (2015).

  67. 67

    Comolli, L.R. & Banfield, J.F. Inter-species interconnections in acid mine drainage microbial communities. Front. Microbiol. 5, 367 (2014).

  68. 68

    Podar, M. et al. Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park. Biol. Direct 8, 9 (2013).

  69. 69

    Sogin, M.L. et al. Microbial diversity in the deep sea and the underexplored “rare biosphere.” Proc. Natl. Acad. Sci. USA 103, 12115–12120 (2006).

  70. 70

    Sharon, I. et al. Accurate, multi-kb reads resolve complex populations and detect rare microorganisms. Genome Res. 25, 534–543 (2015).

  71. 71

    Delmont, T.O. et al. Reconstructing rare soil microbial genomes using in situ enrichments and metagenomics. Front. Microbiol. 6, 358 (2015).

  72. 72

    Galand, P.E., Casamayor, E.O., Kirchman, D.L. & Lovejoy, C. Ecology of the rare microbial biosphere of the Arctic Ocean. Proc. Natl. Acad. Sci. USA 106, 22427–22432 (2009).

  73. 73

    Martijn, J. et al. Single-cell genomics of a rare environmental alphaproteobacterium provides unique insights into Rickettsiaceae evolution. ISME J. 9, 2373–2385 (2015).

  74. 74

    Magdanova, L.A. & Goliasnaia, N.V. Heterogeneity as an adaptive trait of the bacterial community. Mikrobiologiia 82, 3–13 (2013).

  75. 75

    Pamp, S.J., Harrington, E.D., Quake, S.R., Relman, D.A. & Blainey, P.C. Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB). Genome Res. 22, 1107–1119 (2012).

  76. 76

    Engel, P., Stepanauskas, R. & Moran, N.A. Hidden diversity in honey bee gut symbionts detected by single-cell genomics. PLoS Genet. 10, e1004596 (2014).

  77. 77

    Kashtan, N. et al. Single-cell genomics reveals hundreds of coexisting subpopulations in wild Prochlorococcus. Science 344, 416–420 (2014).

  78. 78

    Locey, K.J. & Lennon, J.T. Scaling laws predict global microbial diversity. Proc. Natl. Acad. Sci. USA 113, 5970–5975 (2016).

  79. 79

    Schloss, P.D., Girard, R.A., Martin, T., Edwards, J. & Thrash, J.C. Status of the archaeal and bacterial census: an update. MBio 7, e00201–e00216 (2016).

  80. 80

    Pruesse, E. et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196 (2007).

  81. 81

    Miller, C.S., Baker, B.J., Thomas, B.C., Singer, S.W. & Banfield, J.F. EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data. Genome Biol. 12, R44 (2011).

  82. 82

    Hug, L.A. et al. A new view of the tree of life. Nat. Microbiol. 1, 16048 (2016).

  83. 83

    Spang, A. et al. Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature 521, 173–179 (2015).

  84. 84

    Lasken, R.S. & McLean, J.S. Recent advances in genomic DNA sequencing of microbial species from single cells. Nat. Rev. Genet. 15, 577–584 (2014).

  85. 85

    Siegl, A. et al. Single-cell genomics reveals the lifestyle of Poribacteria, a candidate phylum symbiotically associated with marine sponges. ISME J. 5, 61–70 (2011).

  86. 86

    Wu, D. et al. A phylogeny-driven genomic encyclopaedia of bacteria and archaea. Nature 462, 1056–1060 (2009).

  87. 87

    Wilson, M.C. et al. An environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 506, 58–62 (2014).

  88. 88

    Lloyd, K.G. et al. Predominant archaea in marine sediments degrade detrital proteins. Nature 496, 215–218 (2013).

  89. 89

    Chen, I.A. et al. IMG/M: integrated genome and metagenome comparative data analysis system. Nucleic Acids Res. 45, D507–D516 (2016).

  90. 90

    Haroon, M.F. et al. In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery. Methods Enzymol. 531, 3–19 (2013).

  91. 91

    Yilmaz, S., Haroon, M.F., Rabkin, B.A., Tyson, G.W. & Hugenholtz, P. Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations. ISME J. 4, 1352–1356 (2010).

  92. 92

    Yamaguchi, T. et al. Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH). Syst. Appl. Microbiol. 38, 400–405 (2015).

  93. 93

    Woyke, T. & Jarett, J. Function-driven single-cell genomics. Microb. Biotechnol. 8, 38–39 (2015).

  94. 94

    Doud, D.F.R. & Woyke, T. Novel approaches in function-driven single-cell genomics. FEMS Microbiol. Rev. 41, 538–548 (2017).

  95. 95

    Berry, D. et al. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells. Proc. Natl. Acad. Sci. USA 112, E194–E203 (2015).

  96. 96

    Hatzenpichler, R. et al. Visualizing in situ translational activity for identifying and sorting slow-growing archaeal-bacterial consortia. Proc. Natl. Acad. Sci. USA 113, E4069–E4078 (2016).

  97. 97

    Martinez-Garcia, M. et al. Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia. PLoS One 7, e35314 (2012).

  98. 98

    Reintjes, G., Arnosti, C., Fuchs, B.M. & Amann, R. An alternative polysaccharide uptake mechanism of marine bacteria. ISME J. 11, 1640–1650 (2017).

  99. 99

    Hess, M. et al. Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331, 463–467 (2011).

  100. 100

    Dupont, C.L. et al. Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. 6, 1186–1199 (2012).

  101. 101

    Rinke, C. et al. Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics. Nat. Protoc. 9, 1038–1048 (2014).

  102. 102

    Clingenpeel, S., Schwientek, P., Hugenholtz, P. & Woyke, T. Effects of sample treatments on genome recovery via single-cell genomics. ISME J. 8, 2546–2549 (2014).

  103. 103

    Stepanauskas, R. Single cell genomics: an individual look at microbes. Curr. Opin. Microbiol. 15, 613–620 (2012).

  104. 104

    Wurch, L. et al. Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment. Nat. Commun. 7, 12115 (2016).

Download references


This work was conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, under contract no. DE-AC02-05CH11231. T.W. and D.F.R.D. were also supported under the LBNL Microbes to Biomes LDRD entitled “Tackling microbial-mediated plant carbon decomposition using function-driven genomics.”

Author information




T.W., D.F.R.D. and F.S. constructed the figures and wrote the article.

Corresponding author

Correspondence to Tanja Woyke.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Woyke, T., Doud, D. & Schulz, F. The trajectory of microbial single-cell sequencing. Nat Methods 14, 1045–1054 (2017).

Download citation

Further reading