Human Microbial Metagenomics: Understanding Our Microbial Selves

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Human Microbial MetagenomicsUnderstanding Our
Microbial Selves
Claire M. Fraser-Liggett, Ph.D. University of
Maryland School of Medicine Institute of Genome
Sciences
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Topics of discussion

• What is human metagenomics?
• Why are these microbial communities important?
• How are metagenomics studies different from
  traditional genomics projects?
• Summary of results from recent human metagenomics
  studies
• Looking to the future
• Challenges and opportunities
• implications for human health and disease

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The New Science of MetagenomicsNational Research
Council Report

• A new science that seeks to understand biology at
  the aggregate level, transcending the individual
  organism to focus on the genes in the community
  and how they interact to serve a collective
  function.
• It is both a set of research techniques AND a
  research field.
• It is the science of (microbial) communities.
  Metagenomics will be the systems biology of the
  biosphere.

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The Human Microbial Metagenome
Microbiome A multi-genus/species community of
bacteria that exists within a defined
environmental domain Human Microbiome The
community of bacteria that live on/in the
human host (mucosal surfaces, skin, tooth
surface, etc.) Beneficial/neutral/adversarial M
etagenomics The culture-independent study of the
genomes of many organisms simultaneously in
order to understand microbial communities as
intact systems
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The Human Microbial Metagenome
Humans are born without any microorganisms Colo
nization of skin, oral/respiratory tract,
genitourinary system and gastrointestinal tract
begins immediately at birth Our adult bodies
contain 10 times more microbial cells than human
cells Human colon contains up to 100 trillion
bacteria Numerous studies have suggested that
shifts in the populations of microbial
communities may be associated with a number of
important acute and chronic diseases
inflammatory bowel disease, obesity,
cardiovascular disease, eczema and other skin
diseases, vaginal infections This
presents an opportunity to develop new approaches
to therapy as a means of maintaining health

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The Human Microbial Metagenome

Four environments on the human body are the most
densely populated with microorganisms -gastroin
testinal tract (800 phylotypes) -oral cavity
(500 phylotypes) -vagina (200 phylotypes) -skin
(100 phylotypes)
Our current focus is on the colon, the oral
cavity, and the vagina.
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How to study the human microbiome?
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A Metagenomics Blueprint

• Surveys of community diversity
• Gene/genome content whole genome sequencing
• Functional analysis work in progress
• Mapping of community function back to single
  cells future goal

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Assays of community diversity

• Molecular fingerprinting (T-RFLP)
• Array-based approaches (Affymetrix Phylochip)
• 16S rDNA gene amplification and sequencing

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75 Caucasian
T-RFLP analysis of human vaginal microbial
communities Forney and Ravel (unpublished)
16 community types were identified among
Caucasian women
75 African-American
12 community types were identified among
African-American women
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Hybridization-based approaches

• Version 2.0 of the Affymetrix PhyloChip targets
  over 30,000 unique 16S rRNA sequences, totaling
  almost 9,000 distinct taxonomic groups

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Setting The Stage

• Our metagenome is a composite of Homo sapiens
  genes and genes present in the genomes of the
  trillions of microbes that colonize our adult
  bodies

16S rRNA sequence-based enumeration of the
adult human distal gut microbiota Eckburg et
al. (11,831 sequences) Ley and Gordon (19,653
sequences)
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16S rRNA Sequencing Human Gut Microbiome
Taxonomy 16S r DNA
Human 1

Bacteroides
Clostridium
Bifidobacterium
Thermoanaerobacter
Bacillus
Human 7
Enterococcus
Streptococcus
Treponema
Porphyromonas
Human 8
Vibrio
Listeria
Streptomyces
Fusobacterium
Lactobacillus
Geobacter
Methanosarcina
Pseudomonas
Escherichia
Oceanobacillus
Heliobacillus
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Metagenomics can theoretically access 100 of an
environment
Direct isolation of DNA from the environment
Traditional cultivation and genomics can at best
access between 1-10 of an environment
Isolation and laboratory cultivation
DNA
Traditional genomics approaches
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Metagenomics of the Human Microbiome

Genomics
Sequence and assemble
amp
plasmid
insert
amp
plasmid
amp
Diversity 1
plasmid
4-10 kb insert
insert
Metagenomics
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• 28 year-old female 37 year-old male, one a
  vegetarian no antibiotics in the previous year
• 65,959 and 74,462 reads from random libraries of
  fecal DNA
• Science 312, 13551359 (2006)

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Amount of overlap shared between 19,866 unique
blastx database matches for the two random
libraries
Human-7
Human-8
7,593
5,700
6,573
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Metagenomics of the Human Colon Microbiome
Glycan metabolism The plant polysaccharides we
consume are rich in xylan-, pectin- and
arabinose-containing carbohydrate structures.
The human genome lacks most of the enzymes
required for degrading these glycans. At least
twenty six different glycoside hydrolase families
are encoded in the microbiome, many of which are
not present in the human glycobiome.


Enrichment for genes in the starch metabolism
pathway in the human colonic microbiome. The left
and right sides of each boxed EC number indicate
whether the microbial gene product is present in
human colonic samples 7 and 8, respectively, and
to what extent (color scale white no hits red
17 hits).
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COG analysis reveals enrichment for archaeal
metabolism. (A) Five archaeal COGs are
significantly enriched (plt1e-5). (B) STRING
protein map of COGs (colored) and predicted
interactors (grey). (C) Location and role of
each enzyme in methanogenesis. STRING lines
indicate neighborhood (green) and co-occurence
(blue) connections.
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Human Gut Microbiome Initiative

• Goal Deep draft assemblies of 100 cultured
  representatives of the divisions represented in
  the adult microbiota Scaffolds to help interpret
  metagenomic datasets
• Approach Initial selection based on cultured
  representatives of phylotypes in 16S rRNA
  datasets public input on choices strain
  collections
• Development of ways for faster/cheaper sequencing
  (multiple platforms) new assembly tools (hybrid
  assemblies) improved annotation schemes
  creation of integrated databases

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Metagenomics of the Human Colon Microbiome
Genome diversity Pan-microbial genomes?

Bifidobacterium longum
Methanobrevibacter smithii
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Genomic diversity
genomic halo(strain-specific)
core
core
non essential
non essential
CLOSED Pan-genomeConsistent with more
isolated lifestyle and limited access to global
microbial gene pool
OPEN Pan-genomeConsistent with colonization of
multiple environments and opportunity for
multiple mechanisms of DNA exchange
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Human Gut Microbiome and Energy Balance

• Collaboration with Jeffrey Gordon (Washington
  University) and Robin Knight (University of
  Colorado Boulder)
• Study subjects will be lean and obese Caucasian
  and African-American twin pairs and their mothers
• New insights about the role of the gut microbiome
  in regulating energy balance in humans
• New strategies for targeting the microbiome for
  intentional manipulation to treat obesity

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Evidence for a link between gut microbial
communities and adiposity

• Colonization of adult germ-free mice with a gut
  microbial community from conventionally-raised
  mice produces a rapid and marked increase in
  adiposity without an increase in food consumption
• Equivalent response in males and females from
  several inbred lines
• Does not require a functional innate or adaptive
  immune system
• Mechanism increased fermentation of otherwise
  indigestible dietary polysaccharides microbial
  regulation of host genes that regulate storage of
  extracted calories in adipocytes

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Identification of a linkage between adiposity
and microbial ecology in mice
Genetic (ob/ob)
Diet-induced
Genetic and diet-induced mouse models of obesity
both exhibit increased abundance of Firmicutes
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Linkage between gut ecology and adiposity in
humans

• Following an initial loss of 5 body weight,
  there is a progressive, statistically
  significant, division-wide shift towards more
  Bacteroidetes and fewer Firmicutes as more weight
  is lost
• Changes occur independent of diet

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Other Gastrointestinal Studies

• Crohns disease (Robert Hettich and Janet
  Jannson)
• Necrotizing enterocolitis (Steven Zeichner)
• Role of the immune system in regulating GI
  microbial composition (HIV patients and patients
  undergoing chemotherapy)

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Human Microbial Metagenomics Grand Challenges

• Do we all share an identifiable core microbiome
  surrounded by a shell of diversity? Is this best
  defined by species, gene content, or functional
  capabilities?
• Should differences in our microbiome be viewed,
  along with our immune and nervous systems, as
  features of our biology that are profoundly
  affected by both our genotypes and by our
  individual environmental exposures?
• How is the human microbiome evolving (within and
  between individuals) over varying time scales as
  a function of age, diets, disease, lifestyle, and
  biosphere?
• Are changes in community composition the cause of
  disease or a read-out of a disease process?

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Human Microbial Metagenomics Technical Issues

• How do we define normal when there appears to
  be a great deal of inter-individual variability?
• What are the appropriate meta-data to collect?
• How often should communities be sampled?
• What is the best approach for sample archiving?
• What are the best surrogates for longitudinal
  monitoring of communities?
• __________________________________________
• Better tools for deep community sampling
• Better tools for measuring function at both the
  single-cell and community level
• Better tools for measuring metabolite and gene
  flow in situ
• Better ways of storing and analyzing disparate
  data sets

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Acknowledgements

• Microbial Genomics Group
• Jonathan Eisen
• Rob Fleischmann
• Steve Gill
• John Heidelberg
• Barbara Methe
• Garry Myers
• Karen Nelson
• Scott Peterson
• David Rasko
• Jacques Ravel
• Herve Tettelin
• Naomi Ward
• Robert DeBoy
• Emmanuel Mongodin

• Bioinformatics Group
• Owen White
• Neil Hall
• Jennifer Wortman
• Collaborators
• Jeffrey Gordon (Washington University)
• David Relman (Stanford University)
• Rob Knight (University of Colorado Boulder)