Tom Delmont: From the Terragenome Project to Global Metagenomic Comparisons: Implications for the...

Earth Microbiome Project and

Global Metagenomic Comparisons

Tom O. Delmont Emmanuel Prestat Pascal Simonet Timothy M. Vogel

15.16%

8.10%

Environmental Microbial Genomics GroupLaboratoire Ampère . Ecole Centrale de Lyon . Université de Lyon

Functional subsystems distribution among 77

metagenomes

Earth Microbiome Project and

Global Metagenomic Comparisons

Tom O. Delmont Emmanuel Prestat Pascal Simonet Timothy M. Vogel

15.16%

8.10%

Metasoil project(Terragenome) Global Ocean

Survey

Environmental Microbial Genomics GroupLaboratoire Ampère . Ecole Centrale de Lyon . Université de Lyon

Functional subsystems distribution among 77

metagenomes

1/ Metasoil project (Terragenome consortium)

To sequence as in depth as possible the Rothamsted soil metagenome

A 2 million fosmid library was constructed (Libragen Company)

90 Titanium pyrosequencing runs and some HiSeq are being generated by

varying different parameters

To sequence as in depth as possible the Rothamsted soil metagenome

A 2 million fosmid library was constructed (Libragen Company)

90 Titanium pyrosequencing runs and some HiSeq are being generated by

varying different parameters

-Time (years/seasons)

-Spatial variations (e.g. depth)

-Methodology (DNA extraction approaches)

To maximize the natural and methodological fluctuations of this soil metagenome (Delmont et al., 2011, AEM)

Our strategy to sequence a new environment:Five dimensions:

1 for time3 for space

1 for methodology


Seasonal \ Sampling effect Cell lysis stringency effectDepth effect

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8 Species distribution (SEED annotation) using four lyses

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Important DNA extraction biases

Concept of standard deviation of distribution

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Comparison of functional distributions among metagenomes (1million reads) using MG RAST and STAMP


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10Rothamsted soil


Species distribution

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Important methodological fluctuations

Concept of metagenomic variance

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)1/ Metasoil project (Terragenome consortium)

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12 Rothamsted soilPuerto-Rico forest soil


Species distribution

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)1/ Metasoil project (Terragenome consortium)

The other metagenomic variance is lacking

Comparison difficult

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Clustering-based subsystems

Carbohydrates

Amino Acids and Derivatives

Protein Metabolism

Cofactors, Vitamins, Prosthetic Groups, Pigments

Cell Wall and Capsule

Unclassified

Virulence

DNA Metabolism

RNA Metabolism

Respiration

Nucleosides and Nucleotides

Membrane Transport

Cell Division and Cell Cycle

Stress Response

Phosphorus Metabolism

Fatty Acids and Lipids

Motility and Chemotaxis

Sulfur Metabolism

Metabolism of Aromatic Compounds

Regulation and Cell signaling

Nitrogen Metabolism

Miscellaneous

Potassium metabolism

Photosynthesis

Macromolecular Synthesis

Secondary Metabolism

Prophage

Dormancy and Sporulation

Relative distribution in percentage36 metagenomes from the GOS (coastal and open oceans)

2/ Lessons from the Global ocean survey

Different timesDifferent locations

Only one method used

Do these datasets represent this environment?

If not DNA extraction,cells filtration effect?

*When comparing samples from the same environment:we use (in general subjectively) the same method

To summarize


*When studying a new environment: use different approaches metagenomic variance (represents a global picture)

Temporal, spatial and methodological variations

To summarize

DNA extraction dilemma

Until the inverse is proved, we should consider that DNA biases are different between and among environments

Problem when using one single method

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Number of probes

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One DNA extraction approach (<40%)

15 DNA extraction approaches (>99%)

The diversity is highly underestimated whenusing only one DNA extraction approach

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(Delmont et al., 2011, AEM)

DNA extraction dilemma alternative


1/ We cannot know how cosmopolitan are taxa with one method2/ Replicates are insufficient (biases are hidden behind strong reproducibilities)

The most protocols we use,The most species will be detected

The better the global picture will be

DNA extraction dilemma alternative


1/ We cannot know how cosmopolitan are taxa with one method2/ Replicates are insufficient (biases are hidden behind strong reproducibilities)

The most protocols we use,The most species will be detected

Proposition: MoBio for all samples (referential protocol)-Microbial ecologists send DNA samples that represent, in their

point of view, the environment they study since years

The “tricky” DNA extraction effort could be shared by laboratories involved in EMP


*When studying a new environment: use different approaches metagenomic variance (represents a global picture)

Temporal, spatial and methodological variations

*When performing global metagenomic comparisons:metagenomic variance for all environments

the experimental design depends on the environmentneed to be flexible and adapted to specific problems

To summarize

To summarize

Possible metagenomic definition of ecosystem boundaries:

When inter-environmental distribution differences are globally stronger than intra-environmental fluctuations (natural OR

methodological)

Need to define environments at the microorganism level

Global sampling grid is not coherent

Carbohydrates CELLwall Regulation Fatty Membrane dormancy

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Relative distribution of the function x among n ecosystems

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As a perspective for EMP

Carbohydrates CELLwall Regulation Fatty Membrane dormancy

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Relative distribution of the function x among n ecosystems

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As a perspective for EMP

My vision of EMP is a concerted and flexible experimental design constructed

with the expertise of all microbial ecologists to represent for the best

microbial communities

What should be the next sensational “omic” project?


Colonizing Mars and waiting for a Martian microbiome project?

Or sequencing an alien gut (with metadata of course)


Tom Delmont: From the Terragenome Project to Global Metagenomic Comparisons: Implications for the...

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