Key Points
-
DNA sequencing can provide insights into organisms that are difficult to study because they are inaccessible by conventional methods such as laboratory culture.
-
Isolation and sequencing of DNA from mixed communities of organisms (metagenomics) has revealed surprising insights into diversity and evolution.
-
Genomic DNA sequence, and even complete genomes in some cases, has been generated from organisms that exist only in tight association with other organisms, including various obligate symbionts and pathogens, members of natural microbial consortia and an extinct cave bear.
-
Partially assembled or unassembled genomic sequence from complex microbial communities has revealed the existence of novel and environment-specific genes.
-
Advances in DNA sequencing technology and computational methods promise to accelerate progress in metagenomics.
Abstract
Although genomics has classically focused on pure, easy-to-obtain samples, such as microbes that grow readily in culture or large animals and plants, these organisms represent only a fraction of the living or once-living organisms of interest. Many species are difficult to study in isolation because they fail to grow in laboratory culture, depend on other organisms for critical processes, or have become extinct. Methods that are based on DNA sequencing circumvent these obstacles, as DNA can be isolated directly from living or dead cells in various contexts. Such methods have led to the emergence of a new field, which is referred to as metagenomics.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Boffelli, D., Nobrega, M. A. & Rubin, E. M. Comparative genomics at the vertebrate extremes. Nature Rev. Genet. 5, 456–465 (2004).
Shendure, J., Mitra, R. D., Varma, C. & Church, G. M. Advanced sequencing technologies: methods and goals. Nature Rev. Genet. 5, 335–344 (2004).
Somerville, C. C., Knight, I. T., Straube, W. L. & Colwell, R. R. Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl. Environ. Microbiol. 55, 548–554 (1989).
Venter, J. C. et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304, 66–74 (2004). This project to sequence the entire metagenome of the Sargasso Sea surface waters revealed unexpected community complexity and sequence diversity.
Breitbart, M. et al. Genomic analysis of uncultured marine viral communities. Proc. Natl Acad. Sci. USA 99, 14250–14255 (2002).
LaMontagne, M. G., Michel, F. C. Jr, Holden, P. A. & Reddy, C. A. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. J. Microbiol. Methods 49, 255–264 (2002).
von Wintzingerode, F., Gobel, U. B. & Stackebrandt, E. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21, 213–229 (1997).
Kolman, C. J. & Tuross, N. Ancient DNA analysis of human populations. Am. J. Phys. Anthropol. 111, 5–23 (2000).
Breitbart, M. et al. Metagenomic analyses of an uncultured viral community from human feces. J. Bacteriol. 185, 6220–6223 (2003).
Liles, M. R., Manske, B. F., Bintrim, S. B., Handelsman, J. & Goodman, R. M. A census of rRNA genes and linked genomic sequences within a soil metagenomic library. Appl. Environ. Microbiol. 69, 2684–2691 (2003).
Stein, J. L., Marsh, T. L., Wu, K. Y., Shizuya, H. & DeLong, E. F. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J. Bacteriol. 178, 591–599 (1996).
Berry, A. E., Chiocchini, C., Selby, T., Sosio, M. & Wellington, E. M. Isolation of high molecular weight DNA from soil for cloning into BAC vectors. FEMS Microbiol. Lett. 223, 15–20 (2003).
Suzuki, M. T., Beja, O., Taylor, L. T. & Delong, E. F. Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton. Environ. Microbiol. 3, 323–331 (2001).
Schmidt, T. M., DeLong, E. F. & Pace, N. R. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J. Bacteriol. 173, 4371–4378 (1991).
Beja, O. et al. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289, 1902–1906 (2000). A seminal paper in metagenomics, this study identified a novel protein on a BAC from the uncultivated SAR86 group of bacterioplankton that was later revealed to represent a previously unknown, widespread group of ecologically important light-harvesting proteins.
Beja, O. et al. Comparative genomic analysis of archaeal genotypic variants in a single population and in two different oceanic provinces. Appl. Environ. Microbiol. 68, 335–345 (2002).
Lopez-Garcia, P., Brochier, C., Moreira, D. & Rodriguez-Valera, F. Comparative analysis of a genome fragment of an uncultivated mesopelagic crenarchaeote reveals multiple horizontal gene transfers. Environ. Microbiol. 6, 19–34 (2004).
Quaiser, A. et al. First insight into the genome of an uncultivated crenarchaeote from soil. Environ. Microbiol. 4, 603–611 (2002).
Quaiser, A. et al. Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics. Mol. Microbiol. 50, 563–575 (2003).
Fraser, C. M. et al. Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 281, 375–388 (1998). This paper reported the first genome sequence of a microbe that could not be grown in continuous pure culture.
Andersson, S. G. et al. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396, 133–140 (1998).
Cole, S. T. et al. Massive gene decay in the leprosy bacillus. Nature 409, 1007–1011 (2001).
Bentley, S. D. et al. Sequencing and analysis of the genome of the Whipple's disease bacterium Tropheryma whipplei. Lancet 361, 637–644 (2003).
Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y. & Ishikawa, H. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407, 81–86 (2000). This paper reported the first complete genome sequence of an uncultivated intracellular symbiont and revealed significant genome reduction.
Tamas, I. et al. 50 million years of genomic stasis in endosymbiotic bacteria. Science 296, 2376–2379 (2002).
Akman, L. et al. Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia. Nature Genet. 32, 402–407 (2002).
Gil, R. et al. The genome sequence of Blochmannia floridanus: comparative analysis of reduced genomes. Proc. Natl Acad. Sci. USA 100, 9388–9393 (2003).
van Ham, R. C. et al. Reductive genome evolution in Buchnera aphidicola. Proc. Natl Acad. Sci. USA 100, 581–586 (2003).
Wu, M. et al. Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol. 2, e69 (2004).
Salzberg, S. L. et al. Serendipitous discovery of Wolbachia genomes in multiple Drosophila species. Genome Biol. 6, R23 (2005).
Foster, J. et al. The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode. PLoS Biol. 3, e121 (2005).
Oshima, K. et al. Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nature Genet. 36, 27–29 (2004).
Liu, H., Rodes, B., Chen, C. Y. & Steiner, B. New tests for syphilis: rational design of a PCR method for detection of Treponema pallidum in clinical specimens using unique regions of the DNA polymerase I gene. J. Clin. Microbiol. 39, 1941–1946 (2001).
Renesto, P. et al. Genome-based design of a cell-free culture medium for Tropheryma whipplei. Lancet 362, 447–449 (2003). Using information on T. whipplei's metabolic deficiencies revealed by its genome sequence, these investigators successfully created the first pure culture system for this organism and reduced its in vitro generation time by a factor of 15.
Fenollar, F. & Raoult, D. Molecular genetic methods for the diagnosis of fastidious microorganisms. Apmis 112, 785–807 (2004).
Ogata, H. & Claverie, J. M. Metagrowth: a new resource for the building of metabolic hypotheses in microbiology. Nucleic Acids Res. 33 (database issue), D321–D324 (2005).
Lemos, E. G., Alves, L. M. & Campanharo, J. C. Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa. FEMS Microbiol. Lett. 219, 39–45 (2003).
Hofreiter, M., Serre, D., Poinar, H. N., Kuch, M. & Paabo, S. Ancient DNA. Nature Rev. Genet. 2, 353–359 (2001).
Cooper, A. et al. Complete mitochondrial genome sequences of two extinct moas clarify ratite evolution. Nature 409, 704–707 (2001).
Hofreiter, M. et al. Evidence for reproductive isolation between cave bear populations. Curr. Biol. 14, 40–43 (2004).
Poinar, H., Kuch, M., McDonald, G., Martin, P. & Paabo, S. Nuclear gene sequences from a Late Pleistocene sloth coprolite. Curr. Biol. 13, 1150–1152 (2003).
Greenwood, A. D., Capelli, C., Possnert, G. & Paabo, S. Nuclear DNA sequences from Late Pleistocene megafauna. Mol. Biol. Evol. 16, 1466–1473 (1999).
Noonan, J. P. et al. Genomic sequencing of Pleistocene cave bears. Science 309, 597–599 (2005). The first report of a DNA sequence from an extinct species that was generated without PCR amplification.
Kirkness, E. F. et al. The dog genome: survey sequencing and comparative analysis. Science 301, 1898–1903 (2003).
Tyson, G. W. et al. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428, 37–43 (2004). This paper reports the first assembled genomes to emerge from shotgun sequencing of environmental samples, allowing metabolic reconstruction of community members.
Johnson, D. B. & Hallberg, K. B. The microbiology of acidic mine waters. Res. Microbiol. 154, 466–473 (2003).
Wu, J., Sunda, W., Boyle, E. A. & Karl, D. M. Phosphate depletion in the western North Atlantic Ocean. Science 289, 759–762 (2000).
McDonald, A. E. & Vanlerberghe, G. C. Alternative oxidase and plastoquinol terminal oxidase in marine prokaryotes of the Sargasso Sea. Gene 349, 15–24 (2005).
Sabehi, G., Beja, O., Suzuki, M. T., Preston, C. M. & DeLong, E. F. Different SAR86 subgroups harbour divergent proteorhodopsins. Environ. Microbiol. 6, 903–910 (2004).
Meyer, J. Miraculous catch of iron–sulfur protein sequences in the Sargasso Sea. FEBS Lett. 570, 1–6 (2004).
LeCleir, G. R., Buchan, A. & Hollibaugh, J. T. Chitinase gene sequences retrieved from diverse aquatic habitats reveal environment-specific distributions. Appl Environ Microbiol 70, 6977–83 (2004).
Tringe, S. G. et al. Comparative metagenomics of microbial communities. Science 308, 554–557 (2005). This study revealed that differences in gene content among communities are apparent even in unassembled genomic data.
Smith, C. R. & Baco, A. R. in Oceanography and Marine Biology: an Annual Review Vol. 41 (eds Gibson, R. N. & Atkinson, R. J. A.) 311–354 (Taylor & Francis, London, 2003).
Karner, M. B., DeLong, E. F. & Karl, D. M. Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409, 507–510 (2001).
Dubilier, N. et al. Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an oligochaete worm. Nature 411, 298–302 (2001).
Relman, D. A. & Falkow, S. The meaning and impact of the human genome sequence for microbiology. Trends Microbiol. 9, 206–208 (2001).
Hallam, S. J. et al. Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305, 1457–1462 (2004). A genomic analysis of uncultured Archaea from deep-sea sediments that provided evidence for a 'reverse-methanogenesis' mechanism of anaerobic methane oxidation.
Nusslein, K. & Tiedje, J. M. Characterization of the dominant and rare members of a young Hawaiian soil bacterial community with small-subunit ribosomal DNA amplified from DNA fractionated on the basis of its guanine and cytosine composition. Appl. Environ. Microbiol. 64, 1283–1289 (1998).
Waters, E. et al. The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc. Natl Acad. Sci. USA 100, 12984–12988 (2003).
Garcia-Chapa, M., Batlle, A., Rekab, D., Rosquete, M. R. & Firrao, G. PCR-mediated whole genome amplification of phytoplasmas. J. Microbiol. Methods 56, 231–242 (2004).
Dumont, M. G. & Murrell, J. C. Stable isotope probing — linking microbial identity to function. Nature Rev. Microbiol. 3, 499–504 (2005).
Bernard, L. et al. A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems. Cytometry 43, 314–321 (2001).
Park, H. S., Schumacher, R. & Kilbane, J. J. 2nd. New method to characterize microbial diversity using flow cytometry. J. Ind. Microbiol. Biotechnol. 32, 94–102 (2005).
Gu, F. et al. In situ and non-invasive detection of specific bacterial species in oral biofilms using fluorescently labeled monoclonal antibodies. J. Microbiol. Methods 62, 145–160 (2005).
Sekar, R., Fuchs, B. M., Amann, R. & Pernthaler, J. Flow sorting of marine bacterioplankton after fluorescence in situ hybridization. Appl. Environ. Microbiol. 70, 6210–6219 (2004).
Lin, Y. S., Tsai, P. J., Weng, M. F. & Chen, Y. C. Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria. Anal. Chem. 77, 1753–1760 (2005).
Bundy, J. L. & Fenselau, C. Lectin and carbohydrate affinity capture surfaces for mass spectrometric analysis of microorganisms. Anal. Chem. 73, 751–757 (2001).
Hawkins, T. L., Detter, J. C. & Richardson, P. M. Whole genome amplification — applications and advances. Curr. Opin. Biotechnol. 13, 65–67 (2002).
Erwin, D. P. et al. Diversity of oxygenase genes from methane- and ammonia-oxidizing bacteria in the Eastern Snake River Plain aquifer. Appl. Environ. Microbiol. 71, 2016–2025 (2005).
Detter, J. C. et al. Isothermal strand-displacement amplification applications for high-throughput genomics. Genomics 80, 691–698 (2002).
Kwon, Y. M. & Cox, M. M. Improved efficacy of whole genome amplification from bacterial cells. Biotechniques 37, 40, 42, 44 (2004).
Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature (2005).
DeLong, E. F. Microbial community genomics in the ocean. Nature Rev. Microbiol. 3, 459–469 (2005).
Dehal, P. et al. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298, 2157–2167 (2002).
Holt, R. A. et al. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129–149 (2002).
Allen, E. E. & Banfield, J. F. Community genomics in microbial ecology and evolution. Nature Rev. Microbiol. 3, 489–498 (2005).
Koski, L. B. & Golding, G. B. The closest BLAST hit is often not the nearest neighbor. J. Mol. Evol. 52, 540–542 (2001).
Teeling, H., Waldmann, J., Lombardot, T., Bauer, M. & Glockner, F. O. TETRA: a web-service and a stand-alone program for the analysis and comparison of tetranucleotide usage patterns in DNA sequences. BMC Bioinformatics 5, 163 (2004).
McHardy, A. C., Goesmann, A., Puhler, A. & Meyer, F. Development of joint application strategies for two microbial gene finders. Bioinformatics 20, 1622–1631 (2004).
Overbeek, R. et al. The ERGO genome analysis and discovery system. Nucleic Acids Res. 31, 164–171 (2003).
Meyer, F. et al. GenDB — an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 31, 2187–2195 (2003).
Claudel-Renard, C., Chevalet, C., Faraut, T. & Kahn, D. Enzyme-specific profiles for genome annotation: PRIAM. Nucleic Acids Res. 31, 6633–6639 (2003).
Hugenholtz, P. Exploring prokaryotic diversity in the genomic era. Genome Biol. 3, REVIEWS0003 (2002). A provocative discussion of the problems of culture bias and the need for genomic investigation of underrepresented bacterial and archaeal phyla.
Thompson, J. R. et al. Genotypic diversity within a natural coastal bacterioplankton population. Science 307, 1311–1313 (2005).
Spencer, D. H. et al. Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa. J. Bacteriol. 185, 1316–1325 (2003).
Rocap, G., Distel, D. L., Waterbury, J. B. & Chisholm, S. W. Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S–23S ribosomal DNA internal transcribed spacer sequences. Appl. Environ. Microbiol. 68, 1180–1191 (2002).
Stahl, D. A., Lane, D. J., Olsen, G. J. & Pace, N. R. Analysis of hydrothermal vent-associated symbionts by ribosomal RNA sequences. Science 224, 409–411 (1984).
Stahl, D. A., Lane, D. J., Olsen, G. J. & Pace, N. R. Characterization of a Yellowstone hot spring microbial community by 5S rRNA sequences. Appl. Environ. Microbiol. 49, 1379–1384 (1985).
Giovannoni, S. J., Britschgi, T. B., Moyer, C. L. & Field, K. G. Genetic diversity in Sargasso Sea bacterioplankton. Nature 345, 60–63 (1990).
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703 (1991).
Amann, R. I., Ludwig, W. & Schleifer, K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169 (1995).
Theron, J. & Cloete, T. E. Molecular techniques for determining microbial diversity and community structure in natural environments. Crit. Rev. Microbiol. 26, 37–57 (2000).
Acknowledgements
This work was carried out under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program and the by the University of California, Lawrence Livermore National Laboratory, Lawrence Berkeley National Laboratory and Los Alamos National Laboratory. S.G.T. was supported by a National Institutes of Health National Research Service Award Training and Fellowship grant. We would like to thank P. Hugenholtz and T. Woyke for helpful comments on the manuscript.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Related links
Glossary
- METHANOGENS
-
A group of hydrogen-consuming Archaea that generate methane by reduction of carbon dioxide.
- BIOFILM
-
A layered aggregate of microorganisms.
- NORMAL FILTRATION
-
A process in which particles that are above a certain size are removed from a fluid by forcing the solution through a membrane containing pores of a defined size.
- TANGENTAL FLOW FILTRATION
-
A process in which a fluid is pumped tangentially along the surface of a porous membrane and an applied pressure forces some of the fluid, as well as dissolved particles of sufficiently small size, across the membrane.
- GRAM-POSITIVE BACTERIA
-
Members of the phyla Actinobacteria and Firmicutes, which have a single membrane and a thick cell wall that is made of crosslinked peptidoglycan and therefore can be stained with the Gram staining procedure.
- PHYLOGENETIC ANCHORING
-
A technique that involves screening large-insert libraries made from environmental DNA for clones that contain phylogenetic marker genes, and sequencing those clones in their entirety.
- HORIZONTAL GENE TRANSFER
-
The transfer of genetic material between the genomes of two organisms that does not occur through parent–progeny routes.
- AXENIC
-
A pure culture of a single species of microorganism.
- DIFFERENTIAL LYSIS
-
A technique that uses conditions that will only lyse certain cells so that the DNA from those cells can be isolated from other cells in a community.
- PULSED-FIELD GEL ELECTROPHORESIS
-
The use of pulsed electrical fields of alternating polarity to separate large fragments of DNA.
- METAGENOMIC
-
A term used to describe techniques that characterize the genomes of whole communities of organisms rather than individual species.
- WHOLE-GENOME SHOTGUN
-
An approach to genomic sequencing that involves breaking the DNA into small pieces and cloning them into vectors, followed by sequencing the clones at random.
- DENSITY GRADIENT
-
This occurs in a solution in which the concentration of the solute is lowest at the top and gradually becomes more dense towards the bottom.
- STABLE ISOTOPE PROBING
-
A technique that relies on the incorporation of a substrate that is enriched in a stable isotope, such as 13C, to identify microorganisms that can metabolize that substrate.
- FLOW CYTOMETRY
-
A technique that measures the fluorescence of individual cells as they pass through a laser beam in an individual stream.
- AFFINITY PURIFICATION
-
A technique for purifying cells or molecules that is based on specific binding to a protein or other molecule that has been immobilized on a solid substrate, such as beads or a column.
- ISOTHERMAL STRAND DISPLACEMENT
-
A DNA amplification technique that uses rolling-circle amplification with φ29 DNA polymerase to generate large quantities of DNA without thermal cycling.
- PYROSEQUENCING
-
A DNA sequencing technique that relies on detection of pyrophosphate release on nucleotide incorporation rather than chain termination with dideoxynucleotides.
- CONTIG
-
A continuous stretch of DNA sequence that is assembled from multiple independent sequencing reads.
Rights and permissions
About this article
Cite this article
Tringe, S., Rubin, E. Metagenomics: DNA sequencing of environmental samples. Nat Rev Genet 6, 805–814 (2005). https://doi.org/10.1038/nrg1709
Published:
Issue Date:
DOI: https://doi.org/10.1038/nrg1709
This article is cited by
-
Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains
Nature Microbiology (2023)
-
Depression and fatigue in active IBD from a microbiome perspective—a Bayesian approach to faecal metagenomics
BMC Medicine (2022)
-
Environmental biomonitoring of reef fish community structure with eDNA metabarcoding in the Coral Triangle
Environmental Biology of Fishes (2021)
-
Benchmarking the MinION: Evaluating long reads for microbial profiling
Scientific Reports (2020)
-
Metagenomics and microscope revealed T. trichiura and other intestinal parasites in a cesspit of an Italian nineteenth century aristocratic palace
Scientific Reports (2020)