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Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

Nature Biotechnology volume 32, pages 822–828 (2014)Cite this article

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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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Figure 1: Overview of co-abundance clustering and the MGS-augmented assembly.
Figure 2: Size distributions of co-abundance gene groups (CAGs).
Figure 3: Benchmarking sensitivity and specificity of the co-abundance clustering across a range of sequencing depths or sample numbers.
Figure 4: Comparison of the MGS:337 augmented assembly and the B. animalis reference genome.
Figure 5: Dependency associations among MGS and CAGs.
Figure 6: Gut persistence probability for B. adolescentis.

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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

  1. H Bjørn Nielsen, Mathieu Almeida, H Bjørn Nielsen, Mathieu Almeida, Julian Parkhill and Keith Turner: These authors contributed equally to this work.

Authors and 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, H Bjørn Nielsen, Agnieszka S 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, Thomas Sicheritz-Ponten, Søren Brunak, 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, H Bjørn Nielsen, Agnieszka S Juncker, Ida Bonde, Nikolaj Blom, Thomas Sicheritz-Ponten, Søren Brunak, 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, Mathieu Almeida, Emmanuelle Le Chatelier, Jean-Michel Batto, Fouad Boumezbeur, Joël Doré, Sean Kennedy, Pierre Leonard, Florence Levenez, Bouziane Moumen, Nicolas Pons, Edi Prifti, 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, Pierre Renault & S Dusko Ehrlich

  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, Mathieu Almeida, Emmanuelle Le Chatelier, Jean-Michel Batto, Fouad Boumezbeur, Joël Doré, Sean Kennedy, Florence Levenez, Bouziane Moumen, Nicolas Pons, Edi Prifti, S Dusko Ehrlich, Benoit Quinquis, Florence Haimet, Hervé Blottière, Nathalie Galleron & S Dusko Ehrlich

  5. Department of Computer Science, Center for Bioinformatics and Computational Biology, University of Maryland, USA

    Mathieu Almeida & Mathieu Almeida

  6. BGI Hong Kong Research Institute, Hong Kong, China

    Junhua Li, Junjie Qin, Junhua Li & Junjie Qin

  7. BGI-Shenzhen, Shenzhen, China

    Junhua Li, Manimozhiyan Arumugam, Karsten Kristiansen, Junjie Qin, Junhua Li, Junjie Qin, Jun Wang & Jun Wang

  8. School of Bioscience and Biotechnology, South China University of Technology, Guangzhou, China

    Junhua Li & Junhua Li

  9. European Molecular Biology Laboratory, Heidelberg, Germany

    Shinichi Sunagawa, Manimozhiyan Arumugam, Jens Roat Kultima, Julien Tap, Takuji Yamada, Shinichi Sunagawa, Manimozhiyan Arumugam, Jens Roat Kultima, Julien Tap, Takuji Yamada, Peer Bork & Peer Bork

  10. Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Institut de Génomique, Évry, France

    Eric Pelletier, Denis Le Paslier, 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, Eric Pelletier & Denis Le Paslier

  12. Université d'Évry Val d'Essonne, Évry, France

    Eric Pelletier, Denis Le Paslier, 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, Trine Nielsen, Kristoffer S Burgdorf, Torben Hansen, Oluf Pedersen, Jun Wang & Jun Wang

  14. Digestive System Research Unit, University Hospital Vall d'Hebron, Ciberehd, Barcelona, Spain

    Chaysavanh Manichanh, Natalia Borruel, Francesc Casellas, Francisco Guarner, 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 & Torben Hansen

  16. Department of Structural Biology, VIB, Brussels, Belgium

    Falk Hildebrand, Falk Hildebrand & Falony Gwen

  17. Department of Bioscience Engineering, Vrije Universiteit, Brussels, Belgium

    Falk Hildebrand, Jeroen Raes, Falk Hildebrand & Jeroen Raes

  18. Division for Epidemiology and Microbial Genomics, National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark

    Rolf S Kaas & Rolf S Kaas

  19. Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Karsten Kristiansen, Karsten Kristiansen, Jun Wang & Jun Wang

  20. Hagedorn Research Institute, Gentofte, Denmark

    Oluf Pedersen & Oluf Pedersen

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

    Oluf Pedersen, Oluf Pedersen & Niels Grarup

  22. Faculty of Health, Aarhus University, Aarhus, Denmark

    Oluf Pedersen & Oluf Pedersen

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

    Jeroen Raes & Jeroen Raes

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

    Jeroen Raes & Jeroen Raes

  25. Department of Biology, Section of Microbiology, University of Copenhagen, Copenhagen, Denmark

    Søren Sørensen & Søren Sørensen

  26. Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands

    Sebastian Tims, 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 & Takuji Yamada

  28. Max Delbrück Centre for Molecular Medicine, Berlin, Germany

    Peer Bork & Peer Bork

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

    Jun Wang & Jun Wang

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

    S Dusko Ehrlich & 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. H Bjørn Nielsen
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  2. Mathieu Almeida
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  52. S Dusko Ehrlich
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Consortia

MetaHIT Consortium

  • H Bjørn Nielsen
  • , Mathieu Almeida
  • , Agnieszka S Juncker
  • , Simon Rasmussen
  • , Junhua Li
  • , Shinichi Sunagawa
  • , Damian R Plichta
  • , Laurent Gautier
  • , Anders G Pedersen
  • , Emmanuelle Le Chatelier
  • , Eric Pelletier
  • , Ida Bonde
  • , Trine Nielsen
  • , Chaysavanh Manichanh
  • , Manimozhiyan Arumugam
  • , Jean-Michel Batto
  • , Marcelo B Quintanilha dos Santos
  • , Nikolaj Blom
  • , Natalia Borruel
  • , Kristoffer S Burgdorf
  • , Fouad Boumezbeur
  • , Francesc Casellas
  • , Joël Doré
  • , Piotr Dworzynski
  • , Francisco Guarner
  • , Torben Hansen
  • , Falk Hildebrand
  • , Rolf S Kaas
  • , Sean Kennedy
  • , Karsten Kristiansen
  • , Jens Roat Kultima
  • , Pierre Leonard
  • , Florence Levenez
  • , Ole Lund
  • , Bouziane Moumen
  • , Denis Le Paslier
  • , Nicolas Pons
  • , Oluf Pedersen
  • , Edi Prifti
  • , Junjie Qin
  • , Jeroen Raes
  • , Søren Sørensen
  • , Julien Tap
  • , Sebastian Tims
  • , David W Ussery
  • , Takuji Yamada
  • , Pierre Renault
  • , Thomas Sicheritz-Ponten
  • , Peer Bork
  • , Jun Wang
  • , Søren Brunak
  • , S Dusko Ehrlich
  • , Alexandre Jamet
  • , Alexandre Mérieux
  • , Antonella Cultrone
  • , Antonio Torrejon
  • , Benoit Quinquis
  • , Christian Brechot
  • , Christine Delorme
  • , Christine M'Rini
  • , Willem M de Vos
  • , Emmanuelle Maguin
  • , Encarna Varela
  • , Eric Guedon
  • , Falony Gwen
  • , Florence Haimet
  • , François Artiguenave
  • , Gaetana Vandemeulebrouck
  • , Gérard Denariaz
  • , Ghalia Khaci
  • , Hervé Blottière
  • , Jan Knol
  • , Jean Weissenbach
  • , Johan E T van Hylckama Vlieg
  • , Jørgensen Torben
  • , Julian Parkhill
  • , Keith Turner
  • , Maarten van de Guchte
  • , Maria Antolin
  • , Maria Rescigno
  • , Michiel Kleerebezem
  • , Muriel Derrien
  • , Nathalie Galleron
  • , Nicolas Sanchez
  • , Niels Grarup
  • , Patrick Veiga
  • , Raish Oozeer
  • , Rozenn Dervyn
  • , Séverine Layec
  • , Thomas Bruls
  • , Yohanan Winogradski
  •  & Zoetendal Erwin G

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.

Corresponding authors

Correspondence to Søren Brunak or S Dusko Ehrlich.

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Competing interests

The authors declare no competing financial interests.

Additional information

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

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17 and Supplementary Notes 1–9 (PDF 4213 kb)

Supplementary Data 1

Sample description (XLS 259 kb)

Supplementary Data 2

MGS taxonomical statistics (XLS 264 kb)

Supplementary Data 3

MGS augmented assembly statistics (XLS 266 kb)

Supplementary Data 4

MGS augmented assemblies comparison to reference genomes (XLS 31 kb)

Supplementary Data 5

Summary information on the 6640 small CAGs (XLS 1156 kb)

Supplementary Data 6

Dependency-association network (XLS 251 kb)

Supplementary Data 7

MGS:4 + dependency-associated CAG assembly statistics (XLS 37 kb)

Supplementary Data 8

eggNOG prevalent in frequently observed MGS (XLS 36 kb)

Supplementary Data 9

Gene catalogue comparison (XLS 10 kb)

Supplementary Data 10

Bacillus subtilis essential COG list (XLS 61 kb)

Supplementary Data 11

Dependency-associations with or without companion species (XLS 37 kb)

Supplementary Software

Source code for canopy-clustering algorithm (ZIP 40 kb)

Source data

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Nielsen, H., Almeida, M., Juncker, A. et al. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes. Nat Biotechnol 32, 822–828 (2014). https://doi.org/10.1038/nbt.2939

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

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