1. Exploratory Gene Association Networks October 2009 Jesse Paquette Helen Diller Family Comprehensive Cancer Center University of California, San Francisco

2. What EGAN is Software that runs on a biologist’s computer –Java 6 and Java WebStart –Utilizes Cytoscape libraries for graph rendering A searchable library of genes and gene annotation –Links out to web resources (Entrez/PubMed/KEGG/Google/etc.) A visualization tool that shows how genes and annotation terms are related –User constructs dynamic hypergraphs using experiment results and enrichment statistics

3. Why EGAN was made To accelerate exploratory assay analysis by providing a pre-compiled knowledge network As an alternative to presentation of exploratory assay results as gene lists To allow researchers to combine multiple analysis results from potentially different platforms

4. Exploratory assays AKA high-throughput experiments –Measure hundreds to millions of entities Empirical assays –Expression microarrays –aCGH –MS/MS proteomics –Yeast two-hybrid interaction assays –QTL/SNP associations –DNA Methylation –ChIP chips –Next-gen sequencing In-silico algorithms –Sequence –Structure –Literature

5. The exploratory assay workflow

6. Post-computational analysis questions Given a set of entities (genes): S –How are the entities in S related to each other? –What annotation terms/pathways are enriched in S? –How are the entities in S and the annotation terms related? –Are there any pertinent literature references? –Are there any entities not in S that have relationships with multiple entities in S? –How does S compare to the set published by Soandso et al.? –What changes when entities are added to or removed from S? (e.g. when the p-value cutoff is changed)

7. EGAN lets the biologist investigate results quickly and independently Point-and-click interface –Buttons –Context-specific pop-up menus –Spreadsheet-like data tables –Graph visualization All network information is pre-collated –No programming/scripting –No data transfer/download steps Automated gene-level integration of multiple experiment results

8. How are computational analysis results commonly presented to the biologist?

9. Gene lists –Show gene annotation (but too much at once) –Do not show gene-gene relationships Enriched annotation lists –Do not identify the genes annotated with each term –Do not show which genes share annotation terms Gene graphs –Show gene-gene relationships –Do not adequately show annotation

10. Gene lists

12. Reducing information by significance cutoff

13. Reducing information by taking away genes Prevents the user from wasting time investigating actual negatives But what about genes that just missed a stringent cutoff? –These genes are likely to have some importance –Biologists are often given the impression that genes that fail to pass the cutoff are negatives Valuable information is lost by only focusing on a “significant” set –See Gene Set Enrichment Analysis (GSEA), Subramanian (2005)

14. Enriched annotation lists

15. What is enrichment? Annotation terms/pathways define sets of genes Enrichment –Overrepresentation Set-based enrichment –Given a significant set, S of genes (or a cluster) –Use hypergeometric distribution to compute overlap between each gene set, T and S Global empirical enrichment –Use generated statistics for each gene in the assay –Summarize the statistics for all genes in each set, T –Test to see if the statistics show a non-random trend –GSEA

16. Enriched annotation lists

17. Gene graphs

18. Canonical pathway maps Start with fixed pathway graph Color the gene nodes by empirical values (only significant genes?) Enriched annotation terms not shown Most useful when –This pathway is expected to be affected in experiment –Little interest in other pathways/unassigned genes –Most genes in pathway graph have significant empirical data values –These conditions are rare in exploratory experiments GenMAPP, Dahlquist (2002)

19. Association enrichment graphs Calculate enrichment of terms Nodes are annotation terms Edges are ontological relationships Color represents enrichment score What about other annotation types? Which genes are implicated? BiNGO, Maere (2005)

20. Custom gene set graphs Start with significant set of genes or cluster Show gene-gene relationships as edges How is gene annotation shown? –Hypergraphs Ingenuity IPA, www.ingenuity.com PubGene, Jensen (2001)

21. Hypergraphs A graph is a collection of nodes and edges A hypergraph is a graph with hyperedges A hyperedge is a set of nodes –Annotation terms and pathways are hyperedges Choice of hypergraph visualization method (HVM) is critical as the number of nodes and hyperedges scales upwards

22. Hypergraph visualization methods

23. HVM: Venn diagram Draw a curve around nodes in a set Shows hyperedge overlap effectively Limited to 3 hyperedges No legend required

24. HVM: Clique Use edges to fully connect all nodes in a set Scales poorly –For a hyperedge with n nodes, 0.5n 2 – 0.5n edges must be used Layout algorithms use additional edges Legend required

25. HVM: Node-coloring Give all nodes in a set the same color or shape (Ingenuity uses shapes) Scales poorly –Nodes associated with multiple hyperedges must be divided –Hyperedge count limited to number of distinguishable colors Layout algorithms do not use hyperedges Shows hyperedge overlap poorly Legend required

26. HVM: Association node Hyperedges as association nodes on the graph –Connect each association node to its node members –Incomplete, semi-bipartite graph –Association nodes given different shapes/colors Scales well –For a hyperedge with n nodes, 1 node and n edges must be used Extra association nodes/edges complicate dense graphs –Exploratory assay gene graphs are sparse Layout algorithms use hyperedges No legend required

27. HVM comparison

28. EGAN

29. EGAN features Entire pre-collated hypergraph is available in memory –Mostly defined by NCBI Entrez Gene –Allows dynamic selection of genes and genes sets Useful interface tools for finding genes and terms/pathways of interest –Advanced queries using mouse clicks –Spreadsheet-like tables –Selective addition and removal of information Association node HVM –Thought-provoking display of genes and annotation Node and Edge references –Nodes link to NCBI/UCSC/AmiGO/KEGG/etc. –Edges can link to PubMed

30. Mockup from 12/2007

31. EGAN as of 10/2009

32. Data in the default human gene association network as of 06/08/2009 Node TypeSource# Nodes# EdgesNode LinksEdge Links GeneNCBI Entrez Gene405560Entrez Gene, UCSCN/A MeSHNCBI PubMed162041113983MeSHPubMed ID Conserved Domain NCBI Conserved Domain Database17168295287CDDNone Gene Ontology ProcessNCBI Entrez Gene6779211391AmiGOPubMed ID MIMNCBI Entrez Gene39515082OMIMNone Gene Ontology FunctionNCBI Entrez Gene311468240AmiGOPubMed ID CytobandNCBI Entrez Gene98767422None Gene Ontology ComponentNCBI Entrez Gene93741040AmiGOPubMed ID KEGGNCBI Entrez Gene1958017KEGGNone NHGRI GWA CatalogNCBI Entrez Gene2141271PubMedNone ReactomeNCBI Entrez Gene493594ReactomeNone PubMed Co-occurrenceNCBI Entrez Gene0118596N/APubMed ID Chromosomal SequenceNCBI Entrez Gene042468N/APubMed ID BioGRIDNCBI Entrez Gene024401N/APubMed ID IntActEBI IntAct022229N/ANone HPRDNCBI Entrez Gene017380N/APubMed ID MINT 011903N/ANone BINDNCBI Entrez Gene03879N/APubMed ID Total901542056183

33. The data is fully customizable The pre-collated network –Stored as flat, tab delimited text –Users can specify alternative/supplemental data files Updates are easily pushed to the end users –Using Java WebStart –Compressed in.jar files (.zip) Additional gene sets are already available at MSigDB –Broad Institute, non redistributable –EGAN loads gene sets in.gmt and.gmx file formats

34. Using EGAN: The simple case

35. Three EGAN use cases 1) Characterize a gene using protein interaction neighbors 2) Characterize an pre-collated gene set 3) Characterize gene set defined by experiment results

36. Characterize a gene using protein interaction neighbors Find gene PPARG in the Entrez Gene Node Table Show PPARG and all gene neighbors Hide protein-protein interaction edges Calculate enrichment for all gene sets Use enrichment statistics to selectively show association nodes on the graph

37. PPARG and all protein interaction neighbors

38. Characterize an pre-collated gene set Find the conserved domain DDHD in the Conserved Domain Node Table Show DDHD and all gene neighbors Hide DDHD association node Calculate enrichment for all gene sets Use enrichment statistics to selectively show association nodes on the graph

39. Genes with the DDHD domain

40. Characterize gene set from empirical data Genes reported by Beier et al. (2007) Format custom gene sets Format empirical data (after computational analysis) Load custom gene set file and empirical file in EGAN Find custom gene sets in Custom Node Node Table Show custom sets and all gene neighbors –Border color shows statistic –Border width shows p-value Hide custom set association nodes Calculate enrichment for all gene sets Use enrichment statistics to selectively show association nodes on the graph

41. Gene sets from Beier et al. (2007)

42. Additional functionality in EGAN Comparison of multiple experiments/gene sets –Different normalization methods –Different analysis parameters –Different platforms –Published experiments/gene sets Discovery of third-party genes not present in S Characterization of sequence-derived gene sets –Transcription regulation motifs –Translation regulation motifs –Clusters Scripting for automatic network generation

43. Future plans More diverse, more complete, higher quality data –Species beyond H. sapiens –Activation/inhibition/modification relationships Examples with non-microarray empirical data –SNP, aCGH, MS/MS Quantitative analysis of the hypergraph Mapping of samples into gene set space Restriction of edges by quality parameters Cytoscape 3.0 plug-in? Improved graph layout algorithms

44. Where to get EGAN http://akt.ucsf.edu/EGAN/ –Downloads http://groups.google.com/group/ucsf-egan/ –Documentation –Discussion forum The EGAN manuscript is currently under review at Bioinformatics

45. Acknowledgements UCSF HDFCCC BCB –Taku Tokuyasu –Adam Olshen –Ajay Jain Use of Cytoscape libraries –David Quigley –Scooter Morris –Alex Pico –Alan Kuchinsky Testing –Donna Albertson –Antoine Snijders –Ingrid Revet –Stephan Gysin –Ritu Roydasgupta –Sook Wah Yee –Scot Federman –Mike Baldwin Interpretation of GBM stem cell experiments –Joachim Silber Figure editing –Ben Kopman

46. Methods

47. Example custom gene set file format

48. Example empirical file format

49. Mapping empirical data to genes Exploratory assays don’t directly measure genes Entities may map to multiple genes –EGAN adds the entity statistic/p-value to all genes Multiple entities may map to a single gene –EGAN generates summary statistics/p-values Statistic median (default) P-value median Maximum/minimum |statistic| Minimum/maximum p-value Statistic/p-value mean Entity-to-gene mapping is customizable –Tab-based text format

50. Set-based enrichment Given a set of genes made visible on graph

51. Global empirical enrichment Set Enrichment by Empirical Data (SEED) ParaSEED –Take statistic for each gene in a set S –Calculate summary statistics (s-mean, standard deviation, n) –Two-tailed t-test probability that S is drawn randomly from a normal distribution centered on 0

52. Global empirical enrichment PermuSEED –Take statistic for each gene in a set S –Calculate summary statistics (s-mean, n) –Randomly sample n genes from background p times –Score is fraction of sample means were lower than s-mean Score of 0.001 (p = 1000) means 1 of the 1000 random sample means was lower than s-mean Score of 0.999 (p = 1000) means 999 of the 1000 random sample means were lower than s-mean PermuSEED absolute –Use |statistic| for each gene in S –Pathway gene sets are likely to have activators and inhibitors –PermuSEED absolute finds gene sets that are strongly affected –Parametric version might use variance

53. Multiple testing adjustment Set-based enrichment –Can’t use q-value due to non-uniform distribution of p-values –Optional permutation-based minP method Westfall & Young (1993) When specifically requested by user Global empirical enrichment (SEED) –q-value Automatically generated