1、The Human Microbiome Project,Brie Bibb David Chong Julia Cochran Brandon Crostick Nick Niland,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,Goals of HMP,To
2、break down artificial barriers between medical microbiology and environmental microbiology Ultimately to associates differences in communities with differences in metabolic function and/or disease,Possible questions that may be answered by the HMP,How stable and resilient is an individuals microbiot
3、a 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
4、 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?,Considerations,Sampling: temporal (over course of time) scales Biogeography: spatial scales micrometer centimetermete
5、r Microbiomes will need to be characterized by comparing limited data types collected from a limited set of individuals,What do we know about the human microbiome?,From comparative metagenomics uncovered functional attributes of the microbiome,Functional contributions of gut microbiota,Synthesis of
6、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,Needs for success,How do you define a “healthy” individual? How do you get rid of those darn
7、 host cells?,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 additiona
8、l advantage of placing each sequence in the context of multiple alignment and a phylogenetic treeCombination is the best for determining functions associated with the genome,Key Issues,Effectiveness due to horizontal gene transferBetter, faster, more scalable method for generating a huge phylogeneti
9、c tree that contains millions of sequencesIdentify the best way to account for the affects of the genome,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,Goals,New
10、 diagnostic biomarkers of health Industrial application Deeper understanding of nutritional requirements of humans Personalized drug and diet regimen,Freitas and Merkle, 2004,16S rRNA1541 basesFound in bacteria and archeae,Relman, D. 2009,Bacteroidetes and Firmicutes make up 99% of all phylotypesOne
11、 prominent methanogenicarchaeon. Methanobrevibactersmithii,Sequencing the microbiome,Took Venters 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
12、rRNA sequences with PCR of rRNA sequences and analyzed metabolic pathways with known clusters of orthologous groups.,Experimental Procedures,Overview of Procedures: Shotgun Sequence Stool Samples Taxonomic Assignment using Shotgun Data ORFs Taxonomic Assignment Via Shotgun Data rRNA Taxonomic Assigm
13、ent using rRNA PCR Metabolic Pathway Enrichment Analyses,Shotgun Sequencing,Venter et. al. 2001,.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,Taxonomic Assigment Using Shotgun Data rRNA Sequences,Shotgu
14、n Sequence Compared with Known 16s Ribosomal Subunit Sequences Using Blastn Using Contigs Greater Than 200 BP,Taxonomic Assigment Using Shotgun Sequence ORFs,Long-Orfs program used to identify ORFs ORFs then searched with BLASTP Majority Rule for multiple assigments Specific assigments should be vie
15、wed with caution,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 Phylotype
16、s assigned with 99% match Novel phylotypes considered uncultured,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,Metab
17、olic 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,Comparison of random metagenome reads with completed genome of B. longum and M. smithii* AMOScmp
18、 was used to identify closely related organisms to previously sequenced species,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% identityWhat does this suggest?,Methanobrevibacter smithii The d
19、ominant 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,Identified genes using blastx w/all open reading frames with 35% identityAll enzyme commissions (EC
20、s) that were highly redundant were removed for analysisKEGG: Pathway maps for metabolism and other cellular processes, as well as human diseases; manually created from published materialsCog: Each COG consists of individual proteins or groups of paralogs from at least 3 lineages and thus corresponds
21、 to an ancient conserved domain.,-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 ar
22、e not present in the human glycobiome, helps break down and metabolize glucose, galactose, fructose, arabinose, manose, xylose.,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 regul
23、ate most metabolic processes, however, the presence of certain bacteria and archaea do contribute to metabolic processes,KEGG(kyoto encycopedia of genes and genomes), COGs(clusters of orthologous groups). Used to compare function groups of genes against a baseline bacterial metabolism. And score for
24、 enrichment,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 v
25、itamins essential for human health, including B1 and B6,MEP pathway may be new avenue for anti-biotic researchSome bacteria use the MEP pathway instead of the mevanolate pathway for IPP biosynthesis This could be detrimental to gut flora and potentially the host,Gut microbiome enriched for methanoge
26、nic pathwayThis helps remove H2 from the gut via methanogenesis.,Future Research,Future research could be conducted on people with and without IBS crohns or any other gastrointestinal disorder Better coverage needed for shotgun sequencing Also new experimental approach should be created which allow
27、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 antibio
28、tics, and creation of pathogenic specific antibiotics that dont 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,Questions,Acknowledgements,Thanks to Professor Young and the Western Washington University Biology Department!,
