2. Some Microbial Trivia
• Microbes rule the planet
• There are 100x more microbial cells in the
human body than human cells
• Microbes are really small
• Human health may be determined more by
microbes than environment or genes
• Most microbes in/on humans are beneficial
- known as commensals
3.
4. Top Known Benefits of Human
Commensals
• Preventing infection by pathogens
• Development of the immune system
• Digestion of food
• Vitamin production
• Toxin degradation
• Appearance and odor
5. Why Study Commensals?
• Diagnostic for health status
• Disturbed by antibiotics
• Colonization disrupted by C-sections
• Imbalances may cause disease (autoimmune, IBS,
obesity)
• Probiotics and prebiotics could improve health
• Likely have many as of yet unknown functions
6. Microbes are Very Small
• Makes them hard to study
• Even when examined in microscopes -
appearance is not a reliable indicator of
microbial type or biology
• Culturing allows biology to be studied in
detail in the lab
• Genome sequencing of cultured species
very informative
11. Guide to the Normal Bacterial Flora of Humans
Clostridia
• Members of the genus
Clostridium
• Found in the the
intestinal tract
Clostridium difficile. Clostridia are
anaerobic endospore-forming bacteria,
found mainly in the large intestine.
19. Limitations of Culturing
• Can’t perfectly mimic niche
• Can’t mimic communities
• Great plate count anomaly
20. The Uncultured Majority
• Vast majority of microbes have never been
cultured (total numbers and phylogenetic
diversity)
• Particularly true for endosymbionts and
extremophiles
• Main questions
– Who is out there?
– What are they doing?
– Connect who and what.
28. Phylotyping Can Be Used to Count
Phylotyping Diversity Indices
Hugenholtz 2002 Bohannan and
Hughes 2003
29. Zoetendal, Erwin G., Vaughan, Elaine E. & de Vos, Willem M.A microbial world within
us.Molecular Microbiology ハ 59 ハ (6), ハ 1639-1650.doi: 10.1111/j.1365-
36. Problems with rRNA PCR
• Doesn’t predict biology of organisms well
• Doesn’t work for viruses
• Not very quantitative
37. Genome-Scale Methods and
Uncultured Species
• High throughput rDNA PCR (e.g., Sogin, Eisen)
• rDNA “phylochips” (e.g., Brown, Anderson)
• Virus chip (e.g., Derisi)
• Metagenomics
– Large inserts (e.g., Delong)
– Environmental shotgun sequencing (Venter, Banfield, everyone
doing because of power of random sampling)
• Single cell genomics
41. taxonomic content per SHOTGUN 16S
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G
S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S-
02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 28 29 30 31 32 33 34 35 36
Station
44. What Next?
• Selected as new NIH Roadmap Initiative
• Current state of knowledge incredibly
limited
• 100s of body locations, and likely variation
across people, places, diets, ages, etc need
to be surveyed
• New molecular and informatics methods
needed
45. Binning in More Complex
Systems?
A T
B U
C V
D W
E X
F Y
G Z
46. Metagenomic Challenges
(Examples)
• Fragmentary data
• Sparse sampling
• Parasitizing methods from standard genome
analysis
• Structure of communities unknown
• Analyses frequently cover multiple levels
and multiple fields of methods
Editor's Notes
Phylogenetic analysis of rRNAs led to the discovery of archaea
Functional prediction using a gene tree is just like predicting the biology of a species using a species tree
Extension of rRNA analysis to uncultured organisms using PCR
Metagenomic analysis led to the discovery of a new form of phototrophy in the ocean
This is a tree of a rRNA gene that was found on a large DNA fragment isolated from the Monterey Bay. This rRNA gene groups in a tree with genes from members of the gamma Proteobacteria a group that includes E. coli as well as many environmental bacteria. This rRNA phylotype has been found to be a dominant species in many ocean ecosystems. clone from the Sargasso Sea. This shows that this