Motif Enrichment Analysis in Co-Expressed Gene Sets and High-Throughput Sequence Sets

1. Motif Enrichment Analysis in Co-Expressed Gene Sets and High-Throughput Sequence Sets Wyeth Wasserman Jan. 18, 2012 opossum.cisreg.ca/oPOSSUM3

2. 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

3. Webinar Format • Introduction • Walk-Through • Summary • Q&A

4. INTRoduction

5. 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

6. p=0.66 p=0.55 p=0.04 Motif Enrichment Analysis Background Target Finds over-represented TFBS in co-expressed gene sets

7. 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

8. Conserved Region Selection Gene CR1 CR2 CR3 CR4 Threshold phastCons Score Genomic Position

9. TFBS Profiles • JASPAR 2010: Portales-Casamaret 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)

10. 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

11. 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 Foreground Background KS score = -log(KS test p-value) MPC

12. Analysis Methods

13. Walk-Through

14. http://opossum.cisreg.ca/oPOSSUM3

15. Human SSA - Input

18. Human SSA - Results

23. oPOSSUM methods

25. Human aCSA - Input

26. Human aCSA - Input

27. Human aCSA - Input

28. Human aCSA - Results

31. TFBS Cluster Analysis TFBS Profile Cluster

32. TFBS Cluster Analysis (TCA) Gene CR1 CR2 CR3 CR4 TFBSs Merge TFBS Cluster Hits Overrepresentation Analysis based on merged TFBS cluster hits

33. Human TCA – TFBS cluster selection

34. Human TCA - Results

35. TFCluster Info Page

37. Seq SSA - Input

38. Seq SSA - Input

45. Seq SSA - Results

46. KS score

48. Seq TCA - Input

49. SUMMARY

50. 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