1 Species Divergence and the Measurement of Microbial Diversity Cathy Lozupone University of Colorado, Boulder. Washington University, St Louis. Outline • Classes of diversity measures – α vs β diversity – Quantitative vs Qualitative – Divergence/phylogenetic-based diversity vs Taxon/species • Phylogenetic diversity measures that: – Compare the total amount of diversity between samples. • e.g. Is a polluted lake less diverse than pristine? – Test if samples have significantly different membership. • e.g. Do gut samples from HIV positive people have different microbes than those from healthy people? – Identify environmental variables associated with differences between many samples. • e.g. Does pH, organic carbon, soil type, etc correlate with variability across many soils? • These measures are not just for microbes! • Lozupone, C.A. and R. Knight (2008) Species divergence and the measurement of microbial diversity. FEMS Microbiol Rev. 1-22. How do we describe and compare diversity? • α Diversity: – “How many species are in a sample?” • (e.g. 6 colors in A and 6 in B) – e.g.: Are polluted environments less diverse than pristine? • β Diversity: – “How many species are shared between samples?” • (e.g. 2 shared colors between A and B) – e.g.: Does the microbiota differ with different disease states? A B Quantitative versus Qualitative measures • Qualitative: Considers presence absence only – α: How many species are in a sample? • e.g.: 6 colors in both A and B. – β: How many species are shared between samples? • e.g.: A and B are identical because the same colors are present in both. • Quantitative: Also considers relative abundance. – α: Accounts for “evenness”: • e.g. B, where the population is evenly distributed across the 6 species, is more diverse than A, where all species are present but red dominates. – β: Samples will be considered more similar if the same species are numerically dominant versus rare. • e.g. B and A no longer look identical because of differences in abundance. A B
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
Species Divergence and theMeasurement of Microbial
Diversity
Cathy Lozupone
University of Colorado, Boulder.Washington University, St Louis.
Outline• Classes of diversity measures
– α vs β diversity– Quantitative vs Qualitative– Divergence/phylogenetic-based diversity vs Taxon/species
• Phylogenetic diversity measures that:– Compare the total amount of diversity between samples.
• e.g. Is a polluted lake less diverse than pristine?– Test if samples have significantly different membership.
• e.g. Do gut samples from HIV positive people have differentmicrobes than those from healthy people?
– Identify environmental variables associated with differencesbetween many samples.
• e.g. Does pH, organic carbon, soil type, etc correlate withvariability across many soils?
• These measures are not just for microbes!• Lozupone, C.A. and R. Knight (2008) Species divergence and the
measurement of microbial diversity. FEMS Microbiol Rev. 1-22.
How do we describe and comparediversity?
• α Diversity:– “How many species are in a sample?”
• (e.g. 6 colors in A and 6 in B)– e.g.: Are polluted environments less
diverse than pristine?• β Diversity:
– “How many species are sharedbetween samples?”
• (e.g. 2 shared colors between A and B)– e.g.: Does the microbiota differ with
different disease states?
A
B
Quantitative versus Qualitative measures• Qualitative: Considers presence absence only
– α: How many species are in a sample?• e.g.: 6 colors in both A and B.
– β: How many species are shared betweensamples?
• e.g.: A and B are identical because the same colorsare present in both.
• Quantitative: Also considers relative abundance.– α: Accounts for “evenness”:
• e.g. B, where the population is evenly distributedacross the 6 species, is more diverse than A, whereall species are present but red dominates.
– β: Samples will be considered more similar if thesame species are numerically dominant versusrare.
• e.g. B and A no longer look identical because ofdifferences in abundance.
A
B
2
What is a phylogenetic diversitymeasure?
• α Diversity:– Taxon: “How many species are in a
sample?”– Phylogenetic: “How much phylogenetic
divergence is in a sample?”• (e.g. B more individually diverse than A - more
divergent colors)
• β Diversity:– Taxon: “How many species are shared
between samples?”– Phylogenetic: “How much phylogenetic
distance is shared between samples?”• (only related colors from B are in A)
A
B
Advantages of phylogenetictechniques.
• Phylogenetically related organisms are more likelyto have similar roles in a community.
• Taxon-based methods assume a “star phylogeny,”where all relationships between taxa are ignored.
4. Diversity evaluation.
Taxon (Species)-based: Group sequences into
OTUs based on %identity. 97% id forspecies.
Phylogeny-based:
Easily applied to microbial communitysequence data.
1. Extract DNA fromenvironmental
samples.
2. PCR amplify SSUrRNA gene.
3. GenerateSequences:
SangerPyrosequencing
Most (>99%) microbes cannot be cultured.
Adapted from Pace 1997 Science 276:734-740.
Majority of phylogenetic diversity ismicrobial.
3
Phylogenetic Diversity Measures• α Diversity
– Phylogenetic Diversity (PD)– Compare the total amount of diversity between samples.
• β Diversity– Test if samples have significantly different membership.
• UniFrac Significance• P test• LibShuff
– Identify environmental variables associated with differencesbetween many samples.
• Unweighted and Weighted UniFrac• DPCoA
– Compare local and regional diversity• Gain in PD (G)• NRI-NTI
• Sum of branches leading to sequences in a sample.• Qualitative α diversity.• Sample with taxa spanning the most branch length in this
tree represents the most phylogenetically and perhapsfunctionally divergent community.
• Plot the amount of branch length against the # of observations.• Shape of curve allows for estimating how far we are from sampling all of
the phylogenetic diversity.• Allows for comparison of phylogenetic diversity between samples.
Eckburg, P.B., et al. (2005) Diversity of the human intestinal microbial flora. Science308, 1635-1638.
• Do two samples contain significantlydifferent microbial populations?
• Can we see broad trends that relatemany samples and explain them interms of environmental factors?
Phylogenetic β diversity: How isdiversity partitioned among
samples?
4
Unique Fraction (UniFrac) metric• Qualitative phylogenetic β diversity.• Distance = fraction of the total branch length that is
unique to any particular environment.
Lozupone Lozupone and Knight, 2005, and Knight, 2005, Appl Appl Environ Environ Microbiol Microbiol 71:822871:8228
Phylogenetic (P) Test
• The number of changesbetween states(samples) required toexplain the distributionof sequences on thetree (Fitch parsimony).
• Sensitive to treetopology but not tobranch lengths.
Martin, A.P. (2002) Phylogenetic approaches for describing and comparingthe diversity of microbial communities. Appl Environ Microbiol 68, 3673-3682.
“Is the phylogenetic diversity significantlydifferent between samples?”
• Monte Carlo simulations: randomly permute the data (environmentassignments) and determine how often the random data has a moreextreme value than the real data.
• P-values:– P-test: fraction of random trees that have less parsimony changes than the
real tree.– UniFrac: fraction of random trees that have more Unique branch length
than the real tree.UniFrac UniFrac Website: http:Website: http://bmf//bmf..coloradocolorado..edu/unifrac/edu/unifrac/
LibShuff• CX: fraction of
sequences in X that arenot singletons aftergrouping through rangeof sequence distances.
heterozygous formutation in Leptingene interbreed.
• 16S genesequenced forbacteria in gut ofmothers andoffspring.
Ley et al., (2005)Obesity Alters Gut Microbiota, PNAS Vol 102: pp 11070-11075
Clustering of Mouse Data
• Mice cluster perfectly by mother• No obvious effects of obesity• Robust to sampling effort
• Obese micemostly clustertogether
• Not robust tosampling effort
Weighted UniFracUnweighted UniFrac
7
Unweighted UniFrac
Weighted UniFrac
Eckburg, P.B., et al. (2005) Diversity of the human intestinal microbial flora. Science308, 1635-1638.
• Unweighted: allsamples cluster byindividual.
• Weighted: stool looksdifferent.
Comparison of humanstool and mucosal
microbes
Measures in the same classcluster the data similarly
• Double principal coordinatesanalysis (DPCoA)– Another quantitative β diversity
measure.– A matrix of species distances is first
used to ordinate the species usingPCoA.
– The position of the communities incoordinate space is the averageposition of the species that theycontain, weighted by relativeabundances.
• Produces same results as weightedUniFrac.
Short reads (pyrosequencing)can recapture the result.
• UW UniFracclustering with Arbparsimony insertionof 100 bp readsextending fromprimer R357.
• Assignment ofshort reads to anexisting phylogeny(e.g. greengenescoreset) allows forthe analysis of verylarge datasets.
Liu Z, Lozupone C, Hamady M, Bushman FD & Knight R (2007) Shortpyrosequencing reads suffice for accurate microbial community analysis. NucleicAcids Res 35: e120.
Comparison of Local Diversityto Regional Diversity
• β-diversity measures can also relate diversityin a single community to the total diversity ina habitat type or globally.
• Net Relatedness Index (NRI) and NearestTaxa Index (NTI)– Webb CO (2000) Exploring the phylogenetic structure
of ecological communities. Am Nat 156: 145-155.– Overdispersion of sequences in the tree:
Competition important.– Underdispersion of sequences: Habitat Filtering
important.• Gain in PD (G)
8
• Which communities contain the most unseen diversity?• Branches leading only to sequences in a sample.
Lozupone CA & Knight R (2007) Global patterns in bacterialdiversity. Proc Natl Acad Sci U S A 104: 11436-11440.
Summary• Phylogenetic diversity measures can be more powerful
than taxon based measures because they useinformation on how closely related taxa are to eachother.
• Phylogenetic measures are available for both αdiversity and β diversity.
• Quantitative and Qualitative beta diversity measuresproduce complementary insights into how communitiesare related.
• Although several different methods may exist for aparticular class of diversity measure - these are likelyto give similar results (e.g. DPCoA and WeightedUniFrac).
Acknowledgments
• Rob Knight• Micah Hamady• Knight and Gordon Labs