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Computational Metagenomics: Algorithms for Understanding the "Unculturable" Microbial Majority
Sourav ChatterjiUC Davis Genome [email protected]
Background
The Microbial World
Exploring the Microbial World
• Culturing– Majority of microbes currently unculturable.– No ecological context.
• Molecular Surveys (e.g. 16S rRNA)– “who is out there?”– “what are they doing?”
Environmental Shotgun Sequencing
Interpreting Metagenomic Data
• Nature of Metagenomic Data– Mosaic– Fragmentary
• New Sequencing Technologies– Enormous amount of data– Short Reads
Overview of Talk
• Metagenomic Binning• PhyloMetagenomics• The Big Picture/ Future Work
Overview of Talk
• Metagenomic Binning– Background– CompostBin [to appear in RECOMB 2008]
• PhyloMetagenomics• The Big Picture
Metagenomic Binning
Classification of sequences by taxa
Current Binning Methods
• Assembly • Align with Reference Genome• Database Search [MEGAN, BLAST]• Phylogenetic Analysis• DNA Composition [TETRA,Phylopythia]
Current Binning Methods
• Need closely related reference genomes.• Poor performance on short fragments.
– Sanger sequence reads 500-1000 bp long.– Current assembly methods unreliable
• Complex Communities Hard to Bin.
Genome Signatures
• Does genomic sequence from an organism have a unique “signature” that distinguishes it from genomic sequence of other organisms?– Yes [Karlin et al. 1990s]
• What is the minimum length sequence that is required to distinguish genomic sequence of one organism from the genomic sequence of another organism?
DNA-composition metrics
The K-mer Frequency MetricCompostBin uses hexamers
• Working with K-mers for Binning.– Curse of Dimensionality : O(4K) independent
dimensions.– Statistical noise increases with decreasing
fragment lengths.• Project data into a lower dimensional space to
decrease noise.– Principal Component Analysis.
DNA-composition metrics
PCA separates species
Gluconobacter oxydans[65% GC] and Rhodospirillum rubrum[61% GC]
Effect of Skewed Relative Abundance
B. anthracis and L. monogocytes
Abundance 1:1 Abundance 20:1
A Weighting Scheme
For each read, find overlap with other sequences
A Weighting Scheme
Calculate the redundancy of each position.
4 5 5 3
Weight is inverse of average redundancy.
Weighted PCA
• Calculate weighted mean µw :
• Calculates weighted co-variance matrix Mw
• Principal Components are eigenvectors of Mw.– Use first three PCs for further analysis.
å=
=N
1iiiXwwμ
Twi
N
1iwiiw )μ(X)μ(XwM --=å
=
Weighted PCA separates species
B. anthracis and L. monogocytes : 20:1
PCA Weighted PCA
Un-supervised Classification
Semi-Supervised Classification
• 31 Marker Genes [courtesy Martin Wu]– Omni-present– Relatively Immune to Lateral Gene Transfer
• Reads containing these marker genes can be classified with high reliability.
Semi-supervised Classification
Use a semi-supervised version of the normalized cut algorithm
The Semi-supervised Normalized Cut Algorithm
1. Calculate the K-nearest neighbor graph (KNN-graph) from the point set.
2. Update the KNN-graph with information from marker genes.
3. Bisect the graph using the normalized-cut algorithm.
Generalization to multiple bins
Gluconobacter oxydans [0.61], Granulobacter bethesdensis[0.59] and Nitrobacter hamburgensis
[0.62]
Apply algorithm
recursively
Generalization to multiple bins
Gluconobacter oxydans [0.61], Granulobacter bethesdensis[0.59] and Nitrobacter hamburgensis
[0.62]
Testing
• Simulate Metagenomic Sequencing– Variables
• Number of species• Relative abundance• GC content• Phylogenetic Diversity
• Test on a “real” dataset where answer is well-established.
Future Directions
• Holy Grail : Complex Communities• Semi-supervised methods
– More marker genes– Semi-supervised projection?
• Hybrid Methods– Assembly Information– Population Genetic Information
Overview of Talk
• Metagenomic Binning• Phylo-Metagenomics
– Applications– Incorporating Alignment Accuracy
• The Big Picture/ Future Work
Garcia Martin et al., Nat. Biotechnology (2006)
Population Structure of Communities
Yooseph et al., PLoS Biology (2007)
Gene Family Characterization
Manual Masking
• Require skilled and tedious manual intervention
• Subjective and non-reproducible• Impractical for high throughput data
– Frequently ignored. “Garbage-in-and-garbage-out”
Gblocks
Probabilistic Masking using pair-HMMs
• Probabilistic formulation of alignment problem.
• Can answer additional questions– Alignment Reliability– Sub-optimal Alignments
Durbin et al., Cambridge University Press (1998)
Probabilistic Masking
• What is the probability residues xi and yj are homologous?
• Posterior Probability the residues xi and yj are homologous
• Can be calculated efficiently for all pairs (and gaps) in quadratic time.
y]Pr[x,y]x,,yPr[x
]yPr[x jiji
à=à
Scoring Multiple Alignments
• Calculate the “posterior probability matrix” and distances dij between every pair of sequences.
• Weighted “sum of pairs” score for column r :
å
å à
ji,ij
jiji,
ij
d
]rPr[rd
Testing
The Balibase 3.0 Benchmark Database
Testing
• Realign sequences using MSA programs like Clustalw.
• Sensitivity: for all correctly aligned columns, the fraction that has been masked as good
• Specificity: for all incorrectly aligned columns, the fraction that has been masked as bad
Performance
Gblocks
Prob Mask
Sensitivity Specificity
97% 93%
53% 94%
The Final Result
A Phylogenetic Database/Pipeline (with Martin Wu)
Overview of Talk
• Metagenomic Binning • Phylo-Metagenomics• The Big Picture/ Future Work
Population Structure
Venter et al. , Science (2004)
How to integrate information from multiple markers?
Species-species Interactions
Interactions in Microbial Communities
Time Series Data
Ruan et al., Bioinformatics (2006)
Interaction Networks in Microbial Communities
Ruan et al., Bioinformatics (2006)
Functional Profiling
Prediction of Gene Function Prediction of Metabolic Pathway
Functional Profiling (with Binning)
McCutcheon and Moran PNAS.(2007)
Single Cell Genomics
Hutchinson and Venter, Nature Biotechnology (2006)
Single Cell Genomics
Reads From Single Cell “Simulated” Contamination
The Big PictureMicrobial Community
Metagenomic Sampling Single Cell Genomics
Population Structure Functional Profiling
Species Interaction Network
Time Series Data
Acknowledgements
UC Davis• Jonathan Eisen • Martin Wu• Dongying Wu• Ichitaro Yamazaki• Amber Hartman• Marcel Huntemann
UC Berkeley• Lior Pachter• Richard Karp• Ambuj Tewari• Narayanan Manikandan
Princeton University• Simon Levin• Josh Weitz• Jonathan Dushoff