This document discusses challenges in comparing metagenomic data from different environments and studies. It argues that when exploring a new environment, multiple methodological approaches should be used to capture natural and methodological variations. When performing global comparisons, methodological variations should be considered for all environments. Defining ecosystems precisely at the microorganism level is important. The author's vision is for projects like the Earth Microbiome Project to use flexible experimental designs informed by different experts to best represent microbial communities.

1. Environmental Microbial Genomics Group Laboratoire Ampère . EcoleCentrale de Lyon . Université de Lyon Earth Microbiome Project andGlobal Metagenomic Comparisons 8.10% Functional subsystems distribution among 77 metagenomes 15.16% Tom O. Delmont Emmanuel Prestat Pascal Simonet Timothy M. Vogel

2. Environmental Microbial Genomics Group Laboratoire Ampère . EcoleCentrale de Lyon . Université de Lyon Earth Microbiome Project andGlobal Metagenomic Comparisons 8.10% Functional subsystems distribution among 77 metagenomes Metasoil project (Terragenome) Global Ocean Survey 15.16% Tom O. Delmont Emmanuel Prestat Pascal Simonet Timothy M. Vogel

3. 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

4. 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 -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 time 3 for space 1 for methodology

5. 1/ Metasoil project (Terragenome consortium) Seasonal Samplingeffect Cell lysis stringencyeffect Deptheffect Relative functional distribution (%) Species distribution (SEED annotation) using four lyses Comparison of functional distributions among metagenomes (1million reads) using MG RAST and STAMP Important DNA extraction biases Concept of standard deviation of distribution Relative distribution (%) Species

6. 1/ Metasoil project (Terragenome consortium) Seasonal Samplingeffect Cell lysis stringencyeffect Deptheffect Relative functional distribution (%) Species distribution Important methodological fluctuations Concept of metagenomic variance Relative distribution (%) Species

7. 1/ Metasoil project (Terragenome consortium) Seasonal Samplingeffect Cell lysis stringencyeffect Deptheffect Relative functional distribution (%) Species distribution The other metagenomic variance is lacking Comparison difficult Relative distribution (%) Species

8. 2/ Lessons from the Global ocean survey 36 metagenomes from the GOS (coastal and open oceans) Relative distribution in percentage Different times Different locations Only one method used Do these datasets represent this environment? If not DNA extraction, cells filtration effect?

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

10. To summarize *When comparing samples from the same environment: we use (in general subjectively) the same method *When studying a new environment: use different approaches metagenomic variance (represents a global picture) Temporal, spatial and methodological variations

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

12. Problem when using one single method 15 DNA extraction approaches (>99%) % of positive phylogenetic probes One DNA extraction approach (<40%) (Delmont et al., 2011, AEM) The diversityishighlyunderestimatedwhen usingonly one DNA extraction approach

13. DNA extraction dilemma alternative Until the inverse is proved, we should consider that DNA biases are different between and among environments 1/ We cannot know how cosmopolitan are taxa with one method 2/ 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

14. DNA extraction dilemma alternative Until the inverse is proved, we should consider that DNA biases are different between and among environments 1/ We cannot know how cosmopolitan are taxa with one method 2/ 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

15. To summarize *When comparing samples from the same environment: we use (in general subjectively) the same method *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 environment need to be flexible and adapted to specific problems

16. To summarize Need to define environments at the microorganism level Global sampling grid is not coherent Possible metagenomic definition of ecosystem boundaries: When inter-environmental distribution differences are globally stronger than intra-environmental fluctuations (natural OR methodological)

17. As a perspective for EMP Relative distribution of the function x among n ecosystems Distribution in percentage

18. As a perspective for EMP Relative distribution of the function x among n ecosystems 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 Distribution in percentage

19. What should be the next sensational “omic” project?

20. What should be the next sensational “omic” project? Colonizing Mars and waiting for a Martian microbiome project?

21. What should be the next sensational “omic” project? Or sequencing an alien gut (with metadata of course)