Abstract

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

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Acknowledgements

The research leading to these results has received funding from the European Community's Seventh Framework Programme FP7- HEALTH-F4-2007-201052: Metagenomics of the Human Intestinal Tract (MetaHIT) and FP7-HEALTH-2010-261376: International Human Microbiome Standards, as well as the Novo Nordisk Foundation Center for Biosustainability. Work on the clustering concept has been supported by the OpenGPU FUI collaborative research projects, with funding from DGCIS. Researchers on the project were granted access to the HPC resources of CCRT under the allocation 2011-036707 made by GENCI (Grand Equipement National de Calcul Intensif). The company Alliance Services Plus (AS+) has provided help to scale up the process, especially, V. Arslan, D. Tello, V. Ducrot, T. Saidani and S. Monot. The authors affiliated with MGP are funded, in part, by the Metagenopolis ANR-11-DPBS-0001 grant. Ciberehd is funded by the Instituto de Salud Carlos III (Spain). M.A. was supported by a grant from the Ministère de la Recherche et de l'Education Nationale (France).

Author information

Author notes

    • H Bjørn Nielsen
    • , Mathieu Almeida
    • , H Bjørn Nielsen
    •  & Mathieu Almeida

    These authors contributed equally to this work.

Affiliations

  1. Center for Biological Sequence Analysis, Technical University of Denmark, Kongens Lyngby, Denmark.

    • H Bjørn Nielsen
    • , Agnieszka Sierakowska Juncker
    • , Simon Rasmussen
    • , Damian R Plichta
    • , Laurent Gautier
    • , Anders G Pedersen
    • , Ida Bonde
    • , Marcelo B Quintanilha dos Santos
    • , Piotr Dworzynski
    • , Ole Lund
    • , David W Ussery
    • , Agnieszka S Juncker
    • , Thomas Sicheritz-Ponten
    •  & Søren Brunak
  2. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.

    • H Bjørn Nielsen
    • , Agnieszka Sierakowska Juncker
    • , Ida Bonde
    • , Nikolaj Blom
    • , Agnieszka S Juncker
    • , Thomas Sicheritz-Ponten
    •  & Søren Brunak
  3. INRA, Institut National de la Recherche Agronomique, UMR 14121 MICALIS, Jouy en Josas, France.

    • Mathieu Almeida
    • , Emmanuelle Le Chatelier
    • , Jean-Michel Batto
    • , Fouad Boumezbeur
    • , Joël Doré
    • , Sean Kennedy
    • , Pierre Léonard
    • , Florence Levenez
    • , Bouziane Moumen
    • , Nicolas Pons
    • , Edi Prifti
    • , Pierre Leonard
    • , Pierre Renault
    • , S Dusko Ehrlich
    • , Alexandre Jamet
    • , Antonella Cultrone
    • , Christine Delorme
    • , Emmanuelle Maguin
    • , Eric Guedon
    • , Gaetana Vandemeulebrouck
    • , Ghalia Khaci
    • , Maarten van de Guchte
    • , Nicolas Sanchez
    • , Rozenn Dervyn
    • , Séverine Layec
    •  & Yohanan Winogradski
  4. INRA, Institut National de la Recherche Agronomique, US 1367 Metagenopolis, Jouy en Josas, France.

    • Mathieu Almeida
    • , Emmanuelle Le Chatelier
    • , Jean-Michel Batto
    • , Fouad Boumezbeur
    • , Joël Doré
    • , Sean Kennedy
    • , Pierre Léonard
    • , Florence Levenez
    • , Bouziane Moumen
    • , Nicolas Pons
    • , Edi Prifti
    • , S Dusko Ehrlich
    • , Benoit Quinquis
    • , Florence Haimet
    • , Hervé Blottière
    •  & Nathalie Galleron
  5. Department of Computer Science, Center for Bioinformatics and Computational Biology, University of Maryland, USA.

    • Mathieu Almeida
  6. BGI Hong Kong Research Institute, Hong Kong, China.

    • Junhua Li
    •  & Junjie Qin
  7. BGI-Shenzhen, Shenzhen, China.

    • Junhua Li
    • , Manimozhiyan Arumugam
    • , Karsten Kristiansen
    • , Junjie Qin
    •  & Jun Wang
  8. School of Bioscience and Biotechnology, South China University of Technology, Guangzhou, China.

    • Junhua Li
  9. European Molecular Biology Laboratory, Heidelberg, Germany.

    • Shinichi Sunagawa
    • , Manimozhiyan Arumugam
    • , Jens Roat Kultima
    • , Julien Tap
    • , Takuji Yamada
    •  & Peer Bork
  10. Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Institut de Génomique, Évry, France.

    • Eric Pelletier
    • , Denis Le Paslier
    • , François Artiguenave
    • , Jean Weissenbach
    •  & Thomas Bruls
  11. Centre National de la Recherche Scientifique, Évry, France.

    • Eric Pelletier
    •  & Denis Le Paslier
  12. Université d'Évry Val d'Essonne, Évry, France.

    • Eric Pelletier
    •  & Denis Le Paslier
  13. The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

    • Trine Nielsen
    • , Manimozhiyan Arumugam
    • , Kristoffer S Burgdorf
    • , Torben Hansen
    • , Oluf Pedersen
    •  & Jun Wang
  14. Digestive System Research Unit, University Hospital Vall d'Hebron, Ciberehd, Barcelona, Spain.

    • Chaysavanh Manichanh
    • , Natalia Borruel
    • , Francesc Casellas
    • , Francisco Guarner
    • , Antonio Torrejon
    • , Encarna Varela
    •  & Maria Antolin
  15. Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark.

    • Torben Hansen
  16. Department of Structural Biology, VIB, Brussels, Belgium.

    • Falk Hildebrand
    •  & Falony Gwen
  17. Department of Bioscience Engineering, Vrije Universiteit, Brussels, Belgium.

    • Falk Hildebrand
    •  & Jeroen Raes
  18. National Food Institute, Division for Epidemiology and Microbial Genomics, Technical University of Denmark, Kongens Lyngby, Denmark.

    • Rolf S Kaas
  19. Department of Biology, University of Copenhagen, Copenhagen, Denmark.

    • Karsten Kristiansen
    •  & Jun Wang
  20. Hagedorn Research Institute, Gentofte, Denmark.

    • Oluf Pedersen
  21. Institute of Biomedical Science, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

    • Oluf Pedersen
    •  & Niels Grarup
  22. Faculty of Health, Aarhus University, Aarhus, Denmark.

    • Oluf Pedersen
  23. Department of Microbiology and Immunology, Rega Institute, KU Leuven, Belgium.

    • Jeroen Raes
  24. VIB Center for the Biology of Disease, Leuven, Belgium.

    • Jeroen Raes
  25. Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark.

    • Søren Sørensen
  26. Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.

    • Sebastian Tims
    • , Willem M de Vos
    • , Jørgensen Torben
    • , Michiel Kleerebezem
    •  & Zoetendal Erwin G
  27. Department of Biological Information, Tokyo Institute of Technology, Yokohama, Japan.

    • Takuji Yamada
  28. Max Delbrück Centre for Molecular Medicine, Berlin, Germany.

    • Peer Bork
  29. Princess Al Jawhara Center of Excellence in the Research of Hereditary Disorders, King Abdulaziz University, Jeddah, Saudi Arabia.

    • Jun Wang
  30. King's College London, Centre for Host-Microbiome Interactions, Dental Institute Central Office, Guy's Hospital, United Kingdom.

    • S Dusko Ehrlich
  31. Institut Mérieux, Lyon, France.

    • Alexandre Mérieux
    • , Christian Brechot
    •  & Christine M'Rini
  32. Danone Research, Palaiseau, France.

    • Gérard Denariaz
    • , Johan E T van Hylckama Vlieg
    • , Muriel Derrien
    •  & Patrick Veiga
  33. Gut Biology & Microbiology, Danone Research, Center for Specialized Nutrition, Wageningen, the Netherlands.

    • Jan Knol
    •  & Raish Oozeer
  34. The Wellcome Trust Sanger Institute, Hinxton, Cambridge, U.K.

    • Julian Parkhill
    •  & Keith Turner
  35. Istituto Europeo di Oncologia, Milan, Italy.

    • Maria Rescigno

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  1. MetaHIT Consortium

    A full list of members and affiliations appears at the end of the paper.

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Contributions

All authors are members of the Metagenomics of the Human Intestinal Tract (MetaHIT) Consortium. S.D.E. and S.B. managed the project. F.C., N.B., F.G., T.H., K.S.B. and T.N. performed clinical sampling. F.L. and C.M. performed DNA extraction. J.L., E.P. and D.L.P. performed sequencing. S.D.E., H.B.N., M.A., A.S.J., S.R., P.R. and P.B. designed the analyses. H.B.N., A.S.J., S.R., M.A., A.G.P., D.R.P., L.G., I.B., M.B., M.B.Q.d.S., M.A., J.L., J.T., S.S., T.Y., E.P., D.L.P. and R.S.K. performed the data analyses. H.B.N., S.B., A.S.J., S.R., A.G.P. and M.A. wrote the manuscript. H.B.N., S.B., S.D.E., D.R.P., I.B., P.B., E.P., O.P. and D.W.U. revised the manuscript. The MetaHIT Consortium members contributed to the design and execution of the study.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Søren Brunak or S Dusko Ehrlich.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–17 and Supplementary Notes 1–9

Excel files

  1. 1.

    Supplementary Data 1

    Sample description

  2. 2.

    Supplementary Data 2

    MGS taxonomical statistics

  3. 3.

    Supplementary Data 3

    MGS augmented assembly statistics

  4. 4.

    Supplementary Data 4

    MGS augmented assemblies comparison to reference genomes

  5. 5.

    Supplementary Data 5

    Summary information on the 6640 small CAGs

  6. 6.

    Supplementary Data 6

    Dependency-association network

  7. 7.

    Supplementary Data 7

    MGS:4 + dependency-associated CAG assembly statistics

  8. 8.

    Supplementary Data 8

    eggNOG prevalent in frequently observed MGS

  9. 9.

    Supplementary Data 9

    Gene catalogue comparison

  10. 10.

    Supplementary Data 10

    Bacillus subtilis essential COG list

  11. 11.

    Supplementary Data 11

    Dependency-associations with or without companion species

Zip files

  1. 1.

    Supplementary Software

    Source code for canopy-clustering algorithm

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DOI

https://doi.org/10.1038/nbt.2939

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