large scale functional data mining: what can we find in the data we have? curtis huttenhower...
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Large scale functional data mining:What can we find in the data we have?
Curtis Huttenhower
08-12-10Harvard School of Public HealthDepartment of Biostatistics
2
Greatest biological discoveries?
Our job is to create computational microscopes:
To ask and answer specific biological questions using
millions of experimental results
3
A computational definition offunctional genomics
Genomic data Prior knowledge
Data↓
Function
Function↓
Function
Gene↓
Gene
Gene↓
Function
4
A framework for functional genomics
HighSimilarity
LowSimilarity
HighCorrelation
LowCorrelation
G1G2
+
G4G9
+
…
G3G6
-
G7G8
-
…
G2G5
?
0.9 0.7 … 0.1 0.2 … 0.8
+ - … - - … +
0.8 0.5 … 0.05 0.1 … 0.6
HighCorrelation
LowCorrelation
Fre
quen
cy
Let.Not let.
Fre
quen
cy
SimilarDissim.
Fre
quen
cy
P(G2-G5|Data) = 0.85
100Ms gene pairs →
← 1
Ks
data
sets
+ =
5
Functional networkprediction and analysis
Global interaction network
Carbon metabolism network Extracellular signaling network Gut community network
Currently includes data from30,000 human experimental results,
15,000 expression conditions +15,000 diverse others, analyzed for
200 biological functions and150 diseases
HEFalMp
6
Functional network prediction from diverse microbial data
486 bacterial expression
experiments
876 raw datasets
310 postprocessed
datasets
304 normalized coexpression networks
in 27 species
Integrated functional interaction networks
in 15 species
307 bacterial interaction
experiments
154796 raw interactions
114786 postprocessed
interactions
E. Coli Integration
← Precision ↑, Recall ↓
7
Cross-species knowledge transferusing functional data
PinakiSarder
)P()|P()|P( sssss FRFRDDFR ),P( ts FRFR
)|P( DFRs
)},{|P( ssts DFRFR
)P()|},P({ sssst FRFRDFR
st
stD
sss FRFRFRDFRs
)|P()|P()P(
TaFTan
8
TaFTan: Cross-species knowledge transfer using functional data
E. coli
B. subtilis
P. aeruginosa
M. tuberculosis
Species-specific data
Species’ data excluded
All species’ data
log(
prec
isio
n/ra
ndom
)
log(recall)
• Important to take advantage of all
available data for any one organism
• Important to take advantage of all
available data for every organism
• Scalable to dozens of organisms with
hundreds of functional datasets
• Currently working on making this
more context-specific
9
Meta-analysis for unsupervisedfunctional data integration
Evangelou 2007
Huttenhower 2006Hibbs 2007
1
1log2
1'
'
''
z
eiey ,
ieeeiey ,,
i
ieiee yw ,*,̂
22,
*, ˆ
1
eie
ies
w
Simple regression:All datasets are equally accurate
Random effects:Variation within and
among datasets and interactions
10
Meta-analysis for unsupervisedfunctional data integration
Evangelou 2007
Huttenhower 2006Hibbs 2007
1
1log2
1'
'
''
z
+ =
11
~2000
AML/ALLTemperature
DNA damage
Geneexpression
Batcheffects
Functionalmodules
So what does all of this have to do with
microbial communities ?
12
2010
Healthy/IBDTemperature
Location
Taxa &Orthologs
???
Niches &Phylogeny Test for
correlatesMultiple
hypothesiscorrection
Featureselection
p >> n
Confounds/stratification/environment
Cross-validate
Biological story?
Independent sample
Intervention/perturbation
13
What features to test?
16S reads
WGS reads
Taxa
Orthologous clusters
Pathways/modules
Functional roles
Pathway activity
Genomic data(Reference genomes)
Functional data(Experimental models)
Binning
Clustering
Microbiome data
14
MetaHIT: Data features
WGS reads
Pathways/modules
KO clusters
KEGG pathways
85 healthy, 15 IBD +
12 healthy, 12 IBD
ReBLASTed against KEGG since published data obfuscates read
counts
10x bootstrap within training cohort, test on
12+12 as validation
Taxa
PhymmBrady 2009
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MetaHIT: Taxonomic CD biomarkersBacteroidetes
Firmicutes
Methanomicrobia
Enterobacteriaceae
Chromatiales
Desulfobacterales
OxalobacteraceaeRhodobacteraceae
Bradyrhizobiaceae
iTOLLetunic 2007
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MetaHIT: Functional CD biomarkers
Growth/replication Motility Transporters Sugar metabolism
Down in CD
Up in CD
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MetaHIT: KO IBD biomarkers
Transporters
Growth/replication
Motility
Sugarmetabolism
Down in IBD
Up in IBD
LEfSe
NicolaSegata
t-tests, ANOVA, MANOVA, Friedman, Kruskal–Wallis…
Metagenomic differential analysis: LEfSe
1. Is there a statistically significant difference?
2. Is the difference biologically significant?
3. How large is the difference? PCA, LDA, mean difference, class or cluster distance…
expert supervision, specific post-hoc tests…
p(ANOVA) < 0.05
pairwise post-hoc Wilcoxon OK
Log(Score(LDA)) = 3.68
LEfSe:
19
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LEfSe: A non-human exampleViromes vs. bacterial metagenomes
Metastats (White 2009): p < 0.001ANOVA: p < 0.05
LEfSE: DIFF!
Hi-level functional category: CarbohydratesHi-level functional category: TransportersHi-level functional category: Nucleosides and Nucleotides
LEfSE: NO DIFF!
Microbial Viral
Dinsdale 2008
21
• Sleipnir C++ library for computational functional genomics
• Data types for biological entities• Microarray data, interaction data, genes and gene sets,
functional catalogs, etc. etc.• Network communication, parallelization
• Efficient machine learning algorithms• Generative (Bayesian) and discriminative (SVM)
• And it’s fully documented!
Sleipnir: Software forscalable functional genomics
Massive datasets require efficientalgorithms and implementations.
It’s also speedy: microbial data integration
computationtakes <3hrs.
22
Recap
TaFTan Meta-analytic integration
LEfSe
• Unsupervised system for
data mining without curated
prior knowledge
• Comparative microbiome
analysis by taxa, orthologs,
and pathways• Sleipnir software for
scalable functional genomics
• Network framework for
scalable data integration
• Cross-species knowledge
transfer from functional data
23
Thanks!
http://huttenhower.sph.harvard.edu/sleipnir
Jacques Izard
Wendy Garrett
Sarah Fortune
Pinaki Sarder Nicola Segata
Levi Waldron LarisaMiropolsky
WillythssaPierre-Louis
25
Predicting Gene Function
Cell cycle genes
Predicted relationships between genes
HighConfidence
LowConfidence
26
Predicting Gene FunctionPredicted relationships
between genes
HighConfidence
LowConfidence
Cell cycle genes
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Cell cycle genes
Predicting Gene FunctionPredicted relationships
between genes
HighConfidence
LowConfidence
These edges provide a measure of how likely a gene is to
specifically participate in the process of
interest.
28
Comprehensive Validation of Computational Predictions
Genomic data
Computational Predictions of Gene Function
MEFITSPELLHibbs et al 2007
bioPIXIEMyers et al 2005
Genes predicted to function in mitochondrion organization
and biogenesis
Laboratory ExperimentsPetite
frequencyGrowthcurves
Confocal microscopy
New known functions for correctly predicted genes
Retraining
With David Hess, Amy Caudy
Prior knowledge
29
Evaluating the Performance of Computational Predictions
106Original GO Annotations
Genes involved in mitochondrion organization and biogenesis
135Under-annotations
82Novel Confirmations,
First Iteration
17Novel Confirmations,
Second Iteration
340 total: >3x previously known genes in ~5 person-months
30
Evaluating the Performance of Computational Predictions
106Original GO Annotations
Genes involved in mitochondrion organization and biogenesis
95Under-annotations
40Confirmed
Under-annotations
80Novel Confirmations
First Iteration
17Novel Confirmations
Second Iteration
340 total: >3x previously known genes in ~5 person-months
Computational predictions from large collections of genomic data can be
accurate despite incomplete or misleading gold standards, and they
continue to improve as additional data are incorporated.
31
Validating Human Predictions
Autophagy
Luciferase(Negative control)
ATG5(Positive control) LAMP2 RAB11A
NotStarved
Starved(Autophagic)
Predicted novel autophagy proteins
5½ of 7 predictions currently confirmed
With Erin Haley, Hilary Coller
32
Functional mapping: mining integrated networks
Predicted relationships between genes
HighConfidence
LowConfidence
The strength of these relationships indicates how
cohesive a process is.
Chemotaxis
33
Functional mapping: mining integrated networks
Predicted relationships between genes
HighConfidence
LowConfidence
Chemotaxis
34
Functional mapping: mining integrated networks
Flagellar assembly
The strength of these relationships indicates how
associated two processes are.
Predicted relationships between genes
HighConfidence
LowConfidence
Chemotaxis
35
Functional Mapping:Scoring Functional Associations
How can we formalizethese relationships?
Any sets of genes G1 and G2 in a network can be compared
using four measures:
• Edges between their genes
• Edges within each set• The background edges
incident to each set• The baseline of all edges
in the network
),(),(
),(
2121
21, 21 GGwithin
baseline
GGbackground
GGbetweenFA GG
Stronger connections between the sets increase association.
Stronger within self-connections or nonspecific background connections decrease association.
36
Functional Mapping:Bootstrap p-values
• Scoring functional associations is great……how do you interpret an association score?– For gene sets of arbitrary sizes?– In arbitrary graphs?– Each with its own bizarre distribution of edges?
Empirically!# Genes 1 5 10 50
1
5
10
50
Histograms of FAs for random sets
For any graph, compute FA scores for many randomly chosen gene sets of different sizes. Null distribution is
approximately normal with mean 1.
Standard deviation is asymptotic in the sizes
of both gene sets.
Maps FA scores to p-values for any gene sets and
underlying graph.
100
102
104
100
101
102
103
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
|G1|
|G2|
Null distribution σs for one graph
|)(|||
|||)(|),(ˆ
1),(ˆ
ji
jijiFA
jiFA
GCG
BGGAGG
GG
)(1)( ),(ˆ),,(ˆ, 212121xxFAP GGGGGG
37
Functional Mapping:Functional Associations Between Processes
EdgesAssociations between processes
VeryStrong
ModeratelyStrong
NodesCohesiveness of processes
BelowBaseline
Baseline(genomic
background)
VeryCohesive
BordersData coverage of processes
WellCovered
SparselyCovered
Hydrogen Transport
Electron Transport
Cellular Respiration
Protein ProcessingPeptide
Metabolism
Cell Redox Homeostasis
Aldehyde Metabolism
Energy Reserve
Metabolism
Vacuolar Protein
Catabolism
Negative Regulation of Protein Metabolism
Organelle Fusion
Protein Depolymerization
Organelle Inheritance
38
Functional Mapping:Functional Associations Between Processes
EdgesAssociations between processes
VeryStrong
ModeratelyStrong
NodesCohesiveness of processes
BelowBaseline
Baseline(genomic
background)
VeryCohesive
BordersData coverage of processes
WellCovered
SparselyCovered
39
Functional maps for cross-speciesknowledge transfer
G17
G16G15
G10
G6
G9
G8
G5
G11
G7
G12
G13
G14
G2
G1
G4
G3
O8
O4O5
O7
O9
O6
O2
O3
O1
O1: G1, G2, G3O2: G4O3: G6…
ECG1, ECG2BSG1ECG3, BSG2…
40
Functional maps for functional metagenomics
GOS 4441599.3Hypersaline Lagoon, Ecuador
KEGG Pathways
Org
anis
ms
Pathog ens
Env.
Mapping genes into pathways
Mapping pathways into
organisms
+ Integrated functional interaction networks
in 27 species
Mapping organisms into phyla
=
41
Functional Maps:Focused Data Summarization
ACGGTGAACGTACAGTACAGATTACTAGGACATTAGGCCGTATCCGATACCCGATA
Data integration summarizes an impossibly huge amount of experimental data into an
impossibly huge number of predictions; what next?
42
Functional Maps:Focused Data Summarization
ACGGTGAACGTACAGTACAGATTACTAGGACATTAGGCCGTATCCGATACCCGATA
How can a biologist take advantage of all this data to study
his/her favorite gene/pathway/disease without
losing information?
Functional mapping• Very large collections of genomic data• Specific predicted molecular interactions• Pathway, process, or disease
associations• Underlying experimental results and
functional activities in data
43
Functional maps for cross-speciesknowledge transfer
← Precision ↑, Recall ↓
Following up with unsupervised and partially anchored network alignment
44
LEfSe: A non-human exampleViromes vs. bacterial metagenomes
Metastats (White 2009): p < 0.001ANOVA: p < 0.05
LEfSE: DIFF!
Hi-level functional category: CarbohydratesHi-level functional category: Membrane TransportHi-level functional category: Nitrogen MetabolismHi-level functional category: Nucleosides and Nucleotides
LEfSE: NO DIFF!
Microbial Viral