The Microbiome and Metagenomics

Catherine LozuponeCPBS 7711

October 13, 2015

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

The big tree

Pace, N.R.,The UniversalNature of Biochemistry. PNAS Vol 98(3) pp 805-808.

• Majority of life’s diversity is microbial

• Majority of microbial life cannot be grown in pure culture

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

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

Culture-independent studies revolutionized our understanding of gut bacteria

• Culture-based studies over-emphasized the importance of easily culturable organisms (e.g. E. coli).

1. Extract DNA from environmental samples.

2.PCR amplify SSU rRNA gene (which species?) Sequence random fragments (which function?)

3. EvaluateSequences

Culture-independent surveys

Gut microbiota has simple composition at the phylum level

Data from: Yatsunenko et. al. 2012. Nature.

Different phyla: Animalsand plants

• 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!

Diversity of Firmicutes in 2 healthy adults

Sequencing technology renaissance enabled more complex study designs

• Sanger Sequencing (thousands)• Pyrosequencing (millions)• Illumina (billions!)

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.

Small Subunit Ribosomal RNA

• Present in all known life forms

• Highly conserved• Resistant to horizontal

transfer events

16S rRNA secondary structure

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?

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?

Sequencing Technologies• Sanger -> 454 Pyrosequencing -> Illumina

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.

Preprocessing pyrosequencing datasets

• Quality filtering: Discard sequences that:– Are too short and too long (200-1000 range)– With low quality scores– With long homopolymers– Can trim poor quality regions from the ends

• PyroNoise and Chimeras– Can greatly inflate OTU counts– Pyronoise algorithm uses SFF files to fix noisy

sequences

• Use barcodes to assign sequences to samples

Defining species: OTU picking

• Cluster sequences based on % identity– 97% id typical for species– CD-HIT, UCLUST

• For Phylogenetic diversity measures need to make a tree– Align sequences: NAST, PyNAST– Denovo tree building: FastTree– Assign reads to sequences in a pre-defined

reference tree

Comparing Diversity

• Overview of methods for evaluating/comparing microbial diversity across samples using 16S rRNA– diversity: Measures how much is there?– diversity: How much is shared?

• Phylogenetic verses taxon based diversity. • Quantitative verses Qualitative diversity.• What types of taxa are driving the patterns? Which

species are associated with measured properties?• Tools: UniFrac/QIIME/Topiary Explorer• Lozupone, C.A. and R. Knight (2008) Species divergence and the

measurement of microbial diversity. FEMS Microbiol Rev. 1-22.