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www.cmmt.ubc.ca
MOTIF ENRICHMENT ANALYSIS IN CO-EXPRESSED GENE SETS AND HIGH-
THROUGHPUT SEQUENCE SETS
Wyeth WassermanJan. 18, 2012
opossum.cisreg.ca/oPOSSUM3
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Welcome
• If you encounter any technical difficulties during the webinar– Type a report using the chat option
• Slide presentation ~20 min• Compile Questions as they are submitted
and answer them during the final Q&A/discussion period
• During the discussion session, we’ll allow audience speaking
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Webinar Format
• Introduction• Walk-Through• Summary• Q&A
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INTRODUCTION
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Overview
• Given co-expressed gene sets, what are the key mediators of co-expression?– Focus on TFs
• Web-based software system for motif enrichment analysis– Co-expressed genes or sequences– Multiple sets of analysis methods– Available for human, mouse, fly, worm, yeast
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Motif Enrichment Analysis
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Background Target
0
0.2
0.4
0.6
0.8
1
TFBS1 TFBS2 TFBS3
Pro
po
rtio
n o
f g
en
es
co
nta
inin
g
TF
BS
Background
Target
p=0.04 p=0.55 p=0.66
Finds over-represented TFBS in co-expressed gene sets
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What do we need?
• Region selection– Where to look for enriched binding sites– Use conservation filter to restrict search
space• TFBS profiles to search for
– Need a pool of validated profiles• Scoring metrics for enrichment
– How to measure motif over-representation
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GeneCR1 CR2 CR4CR3
Threshold
Genomic Position
phastConsScore
Conserved Region Selection
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TFBS Profiles
• JASPAR 2010: Portales-Casamar et al. Nucleic Acids Research 2009.
• Expanded collection of TFBS profiles– 130 vertebrate profiles– 105 insect profiles– 5 nematode profiles– 177 yeast profiles– PBM (104), PBM_HOMEO (176), PBM_BHLH (19)
• Standardized 2-level TF classification (class, family)
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Scoring Metrics
• Z scores– Based on the number of occurrences of the
TFBS relative to background– Normalized for sequence length– Simple binomial distribution model
• Fisher scores– Fisher exact probability test
• Fisher score = -log(Fisher p-value)– Based on the number of genes containing the
TFBS relative to background
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Additional Metric for Seq-Based
• KS scores– Kolmogorov-Smirnoff
test– Compares the empirical
distribution of the distances of the binding sites from the maximum point of confidence (MPC) to the background
– Expect real binding sites to be centered around the MPC
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MPC
Foreground
Background
KS score = -log(KS test p-value)
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Analysis Methods
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WALK-THROUGH
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http://opossum.cisreg.ca/oPOSSUM3
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Human SSA - Input
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Human SSA - Results
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TF HNF1A
JASPAR ID MA0046.1
Class Helix-Turn-Helix
Family Homeo
Tax Group Vertebrates
IC 15.548
GC Content 0.259
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Target Gene Hits 19
Target Gene Non-Hits 36
Background Gene Hits 1113
Background Gene Non-Hits 3887
Target TFBS Hits 41
Target TFBS Nucleotide Rate 0.0269
Background TFBS Hits 2127
Background TFBS Nucleotide Rate 0.009
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Z-score 15.134
Fisher score 3.646
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oPOSSUM methods
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Human aCSA - Input
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Human aCSA - Input
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Human aCSA - Input
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Human aCSA - Results
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TFBS Cluster Analysis
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TFBS ProfileCluster
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GeneCR1 CR2 CR4CR3
TFBSs
TFBS Cluster Hits
Merge
Overrepresentation Analysisbased on merged TFBS cluster hits
TFBS Cluster Analysis (TCA)
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Human TCA – TFBS cluster selection
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Human TCA - Results
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TFCluster Info Page
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Seq SSA - Input
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Seq SSA - Input
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Seq SSA - Results
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KS score
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Seq TCA - Input
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SUMMARY
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oPOSSUM-3
• Web-based system for motif enrichment analysis in co-expressed gene sets and sequences from high-throughput experiments
• Important functionalities– Gene-based vs. Sequence-based– Single site vs. Anchored combination site– Individual vs. clusters of TFBS profiles– Human, mouse, fly, worm and yeast
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Development Team
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Version 1 CSA Version 2 Version 3
• Ho Sui, SJ• Mortimer, JR• Arenillas, DJ• Brumm, J• Walsh, CJ• Kennedy, BP• Wasserman,
WW
• Huang, S• Fulton, DL• Arenillas, DJ• Perco, P• Ho Sui, SJ• Mortimer, JR• Wasserman,
WW
• Ho Sui, SJ• Fulton, DL• Arenillas, DJ• Kwon, AT• Wasserman,
WW
• Kwon, AT• Arenillas, DJ• Worsely
Hunt, R• Wasserman,
WW
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QUESTIONS & ANSWERS
Please take a moment to type questions/comments into the chat box.
The questions will be answered shortly.
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