The Human Microbiome Project

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The Human Microbiome Project

• Brie Bibb
• David Chong
• Julia Cochran
• Brandon Crostick
• Nick Niland

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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

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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

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Possible questions that may be answered by the HMP

• How stable and resilient is an individuals
  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?

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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

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What do we know about the human microbiome?

• From comparative metagenomics
• uncovered functional attributes of the microbiome

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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

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Needs for success

• How do you define a healthy individual?
• How do you get rid of those darn host cells?

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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

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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

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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

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Goals

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

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16S rRNA 1541 bases Found in bacteria and
archeae
Freitas and Merkle, 2004
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Bacteroidetes and Firmicutes make up gt99 of all
phylotypes One prominent methanogenicarchaeon.
Methanobrevibactersmithii
Relman, D. 2009
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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 rRNA sequences with PCR of rRNA
  sequences and analyzed metabolic pathways with
  known clusters of orthologous groups.

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Experimental Procedures

• Overview of Procedures
• Shotgun Sequence Stool Samples
• Taxonomic Assignment using Shotgun Data ORFs
• Taxonomic Assignment Via Shotgun Data rRNA
• Taxonomic Assigment using rRNA PCR
• Metabolic Pathway Enrichment Analyses

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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
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Taxonomic Assigment Using Shotgun Data rRNA
Sequences

• Shotgun Sequence Compared with Known 16s
  Ribosomal Subunit Sequences Using Blastn
• Using Contigs Greater Than 200 BP

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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 viewed with caution

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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

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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

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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

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• 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

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• 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?

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• 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

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• Identified genes using blastx w/all open reading
  frames with gt35 identity
• All enzyme commissions (ECs) 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.

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

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Questions
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Acknowledgements

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