1. FACE Soil Metagenome Comparisons in IMG Melissa Dsouza, Peter Hallin, Craig Herbold, Rima Upchurch, & Paul Wilkinson

2. FACE Site Overview Free-air and Carbon Dioxide Enrichment Elevated levels of CO2 expected in future decades Metagenome data in IMG/M Forested sites Duke (Sweetgum forest) Oak Ridge (Loblolly pine) Nevada sites Soil crust Cresote Maryland Estuary

3. Metagenomes Overview Soil samples Standardized extraction across all sites Cheryl Kuske (LANL) FastDNA Spin kit 2007-2008 sampling Soil samples Extracted using FastDNA Spin kit 454 Titanium sequencing at JGI

4. Metagenomes Overview Similar GC Similar sequencing effort Scaffold data 65-75% - 1 gene Length of scaffolds varied low as 100 to 630 450-550 median Basically unassembled reads

5. Questions about FACE Question 1: Are there differences in the taxonomic and functional distributions across all sites? Question 2: Are there are any differences between taxonomy within each elevated and ambient sites? Question 3: Are there any functional differences within each elevated and ambient sites?

6. Taxonomy across sites Similar phyla present overall, but there were a few differences between sites Actinobacteria and Proteobacteria top phyla Swapped top or second between sites Other top groups shifted between sites Firmicutes, Bacteroidetes, Cyanobacteria, Acidobacteria and Verrucomicrobia

7. Functional overview across sites

8. Nitrogen fixation Added KEGG pathway (ko00910) to “Function Card” and compared in Metagenome samples Output is matrix containing gene counts Using R (Sorry IMG team!) we created a 2-dimensional clustering. Clearly, samples cluster according to site, rather than CO2 level.

9. Maryland Estuary CO +/- lights up

10. Clustering of sites PCA of all COGs in all sites Axes 1 and 2 explain ~90% of the variation Largest variation in COGs appeared related to site rather than treatment Remaining analyses done by site to examine differences by treatment Desert Estuary Forests

11. Site Function Summary Did not see significant differences in 3 of 5 sites Maryland Estuary Membrane biogenesis- related functions Soil crust Did not see a pattern Was there overlap in COG functions One: a transposase in both COGPfamTIGRfam Forest Oak Ridge000 North Carolina000 Estuar y Maryland500 Desert Soil crust782 Creosote000

12. Site Function Summary Did not see significant differences in 3 of 5 sites Maryland Estuary Membrane biogenesis- related functions Soil crust Did not see a pattern Was there overlap in COG functions One: a transposase in both COGPfamTIGRfam Forest Oak Ridge000 North Carolina000 Estuar y Maryland500 Desert Soil crust782 Creosote000

13. Transposase Maryland Estuary Dominant group was Alphaproteobacteria Nevada soil crust Dominant group was Cyanobacteria Reflective of dominant members of each site

14. Maryland Estuary COGs that were significant Uncharacterized conserved protein Transposase Glycoltransferase Serine/Theronine protein kinase Membrane-fusion protein

15. Maryland Estuary COGs that were significant Uncharacterized conserved protein Transposase Glycoltransferase Serine/Theronine protein kinase Membrane-fusion protein Cell membrane biogenesis Studied phylogenetic distribution of the genes within these COGs revealed…

16. Maryland Estuary Deltaproteobacteria were the dominant group for the phylogenetic distribution This group might be the driving force between ambient and elevated within this site Conclusion

17. Nevada Desert Soil Crust COGs that were significant Acetyltransferases Predicted hydrolases or acyltransferases Transposase Cytochrome b subunit of the bc complex Predicted ATPase FOG: PAS/PAC domain Molybdenum cofactor biosynthesis enzyme Pfams COX1 SBP_bac_5 HAMP Hydrolase MoeA_C DUF349 Radical_SAM SH3_3 TIGRfam YD repeat (two copies) delta-60 repeat domain

18. Nevada Desert Soil Crust COGs that were significant Acetyltransferases Predicted hydrolases or acyltransferases Transposase Cytochrome b subunit of the bc complex Predicted ATPase FOG: PAS/PAC domain Molybdenum cofactor biosynthesis enzyme Pfams COX1 SBP_bac_5 HAMP Hydrolase MoeA_C DUF349 Radical_SAM SH3_3 TIGRfam YD repeat (two copies) delta-60 repeat domain

19. Nevada Desert Soil Crust Actinobacteria, Alphaproteobacteria and Cyanobacteria were the most dominate in the distribution of functional categories In one case Chloroflexi (shown right) were most dominate Conclusions Predicted ATPase

20. Final Conclusions Impact of elevated carbon dioxide Did not see any specific function change Taxon played a major part in our analyses for determining significance in functions Sequence quality Our datasets were mainly unassembled sequences Low quality (shorter reads) affects annotation Frame shift questions for 454 sequencing Not seeing the “real” picture of these sites