1. The Microbiome and Metagenomics
Catherine Lozupone
CPBS 7711
September 25, 2014

2. What is the microbiome?
“The ecological community of commensal, symbiotic, and pathogenic microorganisms that share our body space”
Microbiota: “collection of organisms” Microbiome: “collection of genes”
Bacteria, Archaea, microbial eukaryotes (e.g. fungi or protists) and viruses.
Body Sites
Important roles in health and disease: Gut, Mouth, Vagina, Skin (diverse sites:Nasal epithelial)
Important roles in disease: Lung, blood, liver, urine

3. The big tree Majority of life’s diversity is microbial
Majority of microbial life cannot be grown in pure culture
Vastly different metabolic capabilities, tolerance to temperature, depth, salinity
Pace, N.R.,The Universal
Nature of Biochemistry. PNAS
Vol 98(3) pp

4. The Human Gut Microbiota
100 trillion microbial cells: outnumber human cells 10 to 1!
Most gut microbes are harmless or beneficial.
Protect against enteropathogens
Extract dietary calories and vitamins
Prevent immune disorders
List of diseases associated with dysbiosis ever growing
Inflammatory Diseases: IBD, IBS
Metabolic Diseases: Obesity, Malnutrition
Neurological Disorders
Cancer

6. What do we want to understand?
What does a healthy microbiome look like?
How diverse is it?
What types of bacteria are there?
What is their function?
How variable is the microbiome?
Over time within an individual?
Across individuals?
Functionally?
What are driving factors of variability?
Age, culture, physiological state (pregnancy)
How do changes affect disease?
What properties (taxa, amount of diversity) change with disease?
Cause or affect?
Functional consequences of dysbiosis
Host Interactions
Evolution/adaptation to the host over time.
Immune system

7. Culture-independent studies revolutionized our understanding of gut bacteria
Culture-based studies over-emphasized the importance of easily culturable organisms (e.g. E. coli).
Culture-independent surveys
2.PCR amplify SSU rRNA gene (which species?)
Sequence random fragments (which function?)
Extract DNA from environmental samples.
3. Evaluate
Sequences

8. Gut microbiota has simple composition at the phylum level
Different phyla: Animals
and plants
Data from: Yatsunenko et. al Nature.

9. Diversity of Firmicutes in 2 healthy adults
Each person harbors > 1000 species.
Some species are unique (red and blue)
Some shared (purple)
We know very little about what most of these species do!
1.8 million sequences per person.

10. Sequencing technology renaissance enabled more complex study designs
Sanger Sequencing (thousands)
Pyrosequencing (millions)
Illumina (billions!)
Illumina (100 million)
Pyrosequencing

11. Metagenomics
The study of metagenomes, genetic material recovered directly from environmental samples.
Marker gene
PCR amplify a gene of interest
Tells you what types of organisms are there
Bacteria/Archaea (16S rRNA), Microbial Euks (18S rRNA), Fungi (ITS), Virus (no good marker)
Shotgun
Fragment DNA and sequence randomly.
Tells you what kind of functions are there.

12. Small Subunit Ribosomal RNA
Present in all known life forms
Highly conserved
Resistant to horizontal transfer events
16S rRNA secondary structure

13. Other ‘Omics MetaTranscriptomics (sequence version of microarray)
Isolate all RNA
Deplete rRNA
Sequence all transcripts
Sometimes phenotype only seen in activity of the microbiota
Metabolomics
What metabolites does a community produce?
E.g. in feces or urine
MetaProteomics
What proteins does a community produce?

14. Integrating Data Types
16S rRNA -> shotgun metagenomics
What gene differences cannot be explained by 16S?
Selection by HGT
16S/ genomics -> transcriptomics-> metabolomics
What species or genes (or combination of species or genes), when expressed, are responsible for producing a given metabolite?

17. Sequencing Technologies
Sanger -> 454 Pyrosequencing -> Illumina

18. Short reads (pyrosequencing) can recapture the result.
UW UniFrac clustering with Arb parsimony insertion of 100 bp reads extending from primer R357.
Assignment of short reads to an existing phylogeny (e.g. greengenes coreset) allows for the analysis of very large datasets.
Liu Z, Lozupone C, Hamady M, Bushman FD & Knight R (2007) Short pyrosequencing reads suffice for accurate microbial community analysis. Nucleic Acids Res 35: e120.

19. My presentation is going to cover analysis of data with QIIME
My presentation is going to cover analysis of data with QIIME. This shows many of the steps within QIIME. I am going to discuss certain steps in some detail, and cover the workflow scripts that automate many of the internal steps.