The Human Microbiome Project

1. The Human Microbiome Project Brie Bibb David Chong Julia Cochran Brandon Crostick Nick Niland

2. What makes a human? • Human metabolic features- combo of human and microbial traits • Microbiota- microrganisms that live inside and on humans • Microbiome- the genomes of the microbial symbionts

3. Goals of HMP • To break down artificial barriers between medical microbiology and environmental microbiology • Ultimately to associates differences in communities with differences in metabolic function and/or disease

4. Possible questions that may be answered by the HMP • How stable and resilient is an individual’s microbiota throughout one day and during his/her lifespan? • How similar are microbiomes between members of a family, community or across communities in different environments? • Do all humans have an identifiable “core” microbiome and how is it acquired and transmitted? • What affects the genetic diversity of the microbiome and how does this diversity affect adaptation by the microrganism and the host to markedly different lifestyles and to various physiological or pathophysiological states?

5. Considerations • Sampling: • temporal (over course of time) scales • Biogeography: spatial scales • micrometer • centimeter • meter • Microbiomes will need to be characterized by comparing limited data types collected from a limited set of individuals

6. What do we know about the human microbiome? • From comparative metagenomics • uncovered functional attributes of the microbiome

7. Functional contributions of gut microbiota • Synthesis of vitamins and harvest of otherwise inaccessible nutrients • Metabolism of xenobiotics and other metabotypes • Renewal of gut epithelial cells • Development and activity of the immune system • Cardiac size? • Locomotion

8. Needs for success • How do you define a “healthy” individual? • How do you get rid of those darn host cells?

9. Connecting gene fragments to organisms • Classification without using phylogenetic marker genes • Markov-based model: • Uses frequency of short nucleotide sequences (relatively insensitive for short sequences and heterogeneous genomes) • Homology-based sequencing: • Accurate and provides additional advantage of placing each sequence in the context of multiple alignment and a phylogenetic tree • Combination is the best for determining functions associated with the genome

10. Key Issues • Effectiveness due to horizontal gene transfer • Better, faster, more scalable method for generating a huge phylogenetic tree that contains millions of sequences • Identify the best way to account for the affects of the genome

11. Proposals of HMP • Associate differences in communities with differences in metabolic function and/or disease • Move toward an integrated ‘system metagenomics’ approach • Functional gene arrays

12. Goals • New diagnostic biomarkers of health • Industrial application • Deeper understanding of nutritional requirements of humans • Personalized drug and diet regimen

14. 16S rRNA 1541 bases Found in bacteria and archeae Freitas and Merkle, 2004

15. Bacteroidetes and Firmicutes make up >99% of all phylotypes One prominent methanogenicarchaeon. Methanobrevibactersmithii Relman, D. 2009

17. Sequencing the microbiome • Took Venter’s whole-genome shotgun sequencing approach in studying the mixed microbial communities. • The abundance of a species is represented by the random shotgun sequence coverage of that species. • Compared shotgun rRNA sequences with PCR of rRNA sequences and analyzed metabolic pathways with known clusters of orthologous groups.

18. Experimental Procedures • Overview of Procedures: • Shotgun Sequence Stool Samples • Taxonomic Assignment using Shotgun Data ORF’s • Taxonomic Assignment Via Shotgun Data rRNA • Taxonomic Assigment using rRNA PCR • Metabolic Pathway Enrichment Analyses

19. Shotgun Sequencing • .3g fecal matter • 28 yo female • 36 yo male • One vegetarian, • One meat eater • Travel to Brazil, France • Stayed home • No antibiotics • No medical problems Venter et. al. 2001

20. Taxonomic Assigment Using Shotgun Data rRNA Sequences • Shotgun Sequence Compared with Known 16s Ribosomal Subunit Sequences Using Blastn • Using Contigs Greater Than 200 BP

21. Taxonomic Assigment Using Shotgun Sequence ORF’s • Long-Orfs program used to identify ORF’s • ORF’s then searched with BLASTP • Majority Rule for multiple assigments • Specific assigments should be viewed with caution

22. Taxonomic Assigment Using PCR Data. • Broadrange primers used to amplify DNA coding 16s ribosomal subunit. • Cloned into TOPO vectors, incubated in E.coli • Sequenced using ABI 3730 sequencers • 1024 sequences aligned to in house Ribosome Database Project program. • Chimeras removed • Phylotypes assigned with 99% match • Novel phylotypes considered uncultured

23. Metabolic Pathway Enrichment • Used only identified genes • Sequences compared with NCBI Cluster of Orthologous Groups(COGs) Data. • Genes also analysed with KEGG(encyclopedia of genes) • Together metabolic assignments were made to genes

24. Metabolic Pathway Enrichment • Enrichment of metabolic pathways given a odds ratio • Determined by equation (sample metabolic level/ancestral model level) • Metabolic pathways with values over 1 considered enriched

25. Comparison of random metagenome reads with completed genome of B. longum and M. smithii • * AMOScmp was used to identify closely related organisms to previously sequenced species

26. Bifidobacterium longum – A lactic acid bacteria • 1965 Reads from (from subjects 7 and 8) 1,617,706bp of DNA • Very strong homolgy but 52% of reads less than 95% identity • What does this suggest?

27. Methanobrevibacter smithii – The dominant archaea in the gut • 3.5x coverage with 7955 reads • 8 partial 16rDNA match ups • 89% of reads 95% greater identity – suggesting? • 145/259 archaeal contigs had significantly similar identity to smithii

29. Identified genes using blastx w/all open reading frames with >35% identity • All enzyme commissions (EC’s) that were highly redundant were removed for analysis • KEGG: Pathway maps for metabolism and other cellular processes, as well as human diseases; manually created from published materials • Cog: Each COG consists of individual proteins or groups of paralogs from at least 3 lineages and thus corresponds to an ancient conserved domain.

30. -Most metabolic functions were similar between the two subjects, but there were differences in a few functional categories, possibly caused by differences in diet and lifestyle. -81 different glycoside hydrolase families were found in the microbiome, many of which are not present in the human glycobiome, helps break down and metabolize glucose, galactose, fructose, arabinose, manose, xylose.

31. The odds ratio of human genome (red), bacterial genome in KEGG (blue), and archaeal genome (yellow). The graph shows that the human distal gut metabolic functions can regulate most metabolic processes, however, the presence of certain bacteria and archaea do contribute to metabolic processes

32. KEGG(kyoto encycopedia of genes and genomes), COG’s(clusters of orthologous groups). Used to compare function groups of genes against a baseline bacterial metabolism. And score for enrichment

33. COGs representing enzymes in the MEP (2-methyl-D-erythritol 4-phosphate) pathway, used for biosynthesis of deoxyxylulose 5-phosphate (DXP) and isopenteryl pyrophosphate (IPP), are notably enriched (P G 0.0001; relative to all sequenced microbes) DXP is a precursor in the biosynthesis of vitamins essential for human health, including B1 and B6

34. MEP pathway may be new avenue for anti-biotic research Some bacteria use the MEP pathway instead of the mevanolate pathway for IPP biosynthesis This could be detrimental to gut flora and potentially the host

35. Gut microbiome enriched for methanogenic pathway This helps remove H2 from the gut via methanogenesis.

36. Future Research • Future research could be conducted on people with and without IBS crohn’s or any other gastrointestinal disorder • Better coverage needed for shotgun sequencing • Also new experimental approach should be created which allow the sequencing of the more fragile phyla of bacteria, such as bacteroitides • Analyses of horizontal gene transfers in gut microbes • Quantitation of metabolites etc, contributed and consumed by gut flora • Effects of antibiotic administration of gut flora and the host, succesion of microbes after antibiotics, and creation of pathogenic specific antibiotics that don’t effeect the gut flora or at least minimalize effects • Personalized medicine, dietary needs • I vote for the creation of synthetic butt microbes that create specific metabolites that a host may lack or could make bigger faster stronger smarter or somehow enhance with super human turd powers • Could be used in longetudinal health research

37. Questions

38. Acknowledgements • Thanks to Professor Young and the Western Washington University Biology Department!