1. Microbial diversity and virulence probing of five different body sites Anu Rebbapragada, Pub. Health Ontario Central Lab. Canada Wei-Jen Lin, Cal State Polytech. Univ. Pomona, USA Thomas Keller, Oregon Health &Science Univ., USA Anna Edlund, JCVI, USA

2. Questions: -What are the differences in taxonomic & functional diversity between and within body sites? -Is there a difference in the diversity of virulence genes between body sites?

3. Sample selection in IMG/HMP -Downloaded 10 metagenomes -Selected 2 metagenomes per body site Table for General Statistics Compare Genomes Genome Statistics Breakdown by selected genomes, general statistics Bar Graph for % GC Created in Excel Same person analyzed for skin and stool sample

4. Radial Tree by Class Radial Tree Compare Genomes Phylogenetic Distribution Radial Tree of 5 selected metagenomes Customize by Phyla, Class, Domain etc categories

5. Bacterial diversity at class level 14 2 4 11 5 Skin Nose Pie charts and tables created by clicking node on Radial Tree

6. Diversity of Archaea by Phyla and Class levels Pie charts and tables created by clicking node on Radial Tree. Radial tree customized to display at either Phyla or Class level.

7. Bacterial diversity behind your ear Left earRight ear Selected only 2 metagenomes (ear samples) to generate radial tree. Pie charts and tables created by clicking node on Radial Tree. Radial tree customized to display at either Phyla or Class level.

8. Functional diversity across the metagenomes COG functions Compare Genomes Genome Clustering, Correlation Matrix Select metagenomes of interest Clustering method: Correlation Matrix Function: COG Display: r2 values by pairwise comparison across all metagenomes

9. PCA 2-D COG groups Stool Tongue Skin NoseVagina Compare Genomes Genome Clustering, Correlation Matrix Select metagenomes of interest Clustering method: PCA Principle component analysis Function: COG (clustering by COG pathways) Display: 2D plot, mouse over redX to identify the metagenome identity Created figure in powerpoint to show clustering of COGs across metagenomes Can also show Hierarchical clustering by Function (COG, Pfam etc) or Taxonomy (Genus, Family etc) or Function Categories (COG, KEGG, Pfam)

10. PCA COG pathways Stool Tongue Skin Nose Vagina Compare Genomes Genome Clustering, Correlation Matrix Select metagenomes of interest Clustering method: PCA Principle component analysis Function Category: COG pathway Display: 2D plot, mouse over redX to identify the metagenome identity Created figure in powerpoint to show clustering of COGs across metagenomes Now the Nose samples don’t cluster together suggesting differences in pathways

11. Hierarchical Clustering across Metagenomes by COG Compare Genomes Genome Clustering, Correlation Matrix Select metagenomes of interest Clustering method: Hierarchical Function: COG Display: Phylogram showing distribution of metagenomes

12. Compare COG function between Metagenomes Compare genomes function comparison tool abundance profile function comparison select query genome Export table

13. Secretion Pathways Select Metagenomes of interest Find Functions Search All pathways, Select KEGG pathways only Type “secretion” as Keyword Click on Pathway OID for bacterial secretion View Map

14. Gene Products in Type III Secretion pathway Select Metagenomes of interest Find Functions, Function Search Keyword: “Secretion”, Gene Product name, Click GO Text Filter: “Type III”, click Apply Select MetaGenomes View Profile Output: Table of Function ID (gene product name) and abundance (gene counts) in each metagenome

15. Gene Products in Type Vb secretion (hemolysin) pathway Select Metagenomes of interest Find Functions, Function Search Keyword: “Secretion”, Gene Product name, Click GO Text Filter: “ShlB”, click Apply Select MetaGenomes View Profile Output: Table of Function ID (gene product name) and abundance (gene counts) in each metagenome

16. Gene Counts Across Metagenomes for Type VI Secretion Pathway

17. Distribution of antibiotic resistance genes by human body sites Nose Ears Stool TongueVagina Select Metagenomes of interest Find Functions, Function Search Keyword: individual searches for each antibiotic resistance category under Gene Product name, Click GO Output: Table of Function ID (gene product name) and abundance (gene counts) in each metagenome Exported Table, Tally of totals in spreadsheet and created PieCharts in Excel

18. Antibiotic resistance in human body sites Normalized to counts/10,000 genes

19. US Antibiotic Usage Data Google search :) www.cddep.org/ResistanceMap

20. Study Limitations & Future Analytical Directions