Agilent Bioinformatics & GeneSpring overview GX 10 Guided & Advanced Data Analysis

1. GeneSpring GX 10 for Gene Expression Analysis • Agilent Bioinformatics & GeneSpring overview • GX 10 Guided & Advanced Data Analysis • Practice & Discussion February 2009 Antoni Wandycz Elise Chang Agilent Technologies GeneSpring

2. GeneSpring Workgroup Data storage & Computation Share & Collaborate RNA DNA CH2OH Protein Agilent Bioinformatics Suite Transcriptome ‘GX 10’ miRNA, QPCR, Exon DNA ‘DNA Analytics’ ChIP, Methyl, CGH Metabolome ‘GX 11’ Proteome ‘GX 11’ GeneSpring

3. 2004 2005 2006 • Agilent acquires Stratagene 2007 2008 2009 • Agilent acquires Silicon Genetics • GX 11 • GX 9 • GX 9 development on avadis platform • GX 10 • GX 7.3 functions • miRNA, Exon, QPCR analysis • Pathway analysis • Support for eArray • GX 7.3.1 Released History and Future of GeneSpring GeneSpring

4. GeneSpring GX: Multiple-Platform Compatibility • Agilent Feature Extraction files (>FE v8.5) • Affymetrix CEL, CHP • llumina BeadStudio (>v 3.1) • ABI SDS, RQ Manager (for QPCR) • Custom Formats (ALL 1 & 2-color microarrays) • .GPR files from AXON Scanners (GenePix software) GeneSpring

5. GeneSpring GX 10 – Key features • Guided Workflows • New Applications - miRNA, QPCR, Exon & more in future • Project-based organization & Translation-on-the-fly • Biological Context - Pathway Analysis, GSEA, GO, IPA, etc. • Customization - Scripting in Jython and R GeneSpring

6. GX 10 Key features: Guided Workflows • Pre-determined steps: • Normalization • QC • Statistics • GO • Pathways GeneSpring

7. Project-based organization GeneSpring

8. GeneSpring GX 10: Translation (Chap 3 in GX 10 manual) • Comparing Platforms • i.e.Affymetrix vs. Agilent vs. Spotted • Comparing Species • i.e. Mouse vs. Human -- Homology Table • (NCBI’s Homologene) • Comparing Applications: • i.e. Gene Expression & QPCR or miRNA GeneSpring

9. Homology table displayed GX 10 Key features: Translation Compare platforms, applications, species GeneSpring

10. Venn Diagram • Compare experiments from different platforms, applications, & species GeneSpring

11. GX 10 Key features: Biological Context • GO Analysis (Fx, Process, Location) • GSEA(Gene Set Enrichment Analysis) • Pathway Analysis GeneSpring

12. GeneSpring GX 10:Gene Ontology (GO) Analysis • Likelihood that your genes of interest fell into a GO category, just by chance HELP always available GeneSpring

13. Pathway Analysis in GX 10:Two types of Pathway Analysis in GX 10:1. ‘Pathway Analysis’ ToolBuilding networks of related entities8 Pathway Interaction Databases and NLP 2. ‘Find Significant Pathways’ ToolEntity-list enrichment with known pathways (Step 8 in Guided Workflow) BioPax format pathways (.owl) GeneSpring

14. Overlay Networks with Expression Data/Conditions GeneSpring

15. Cellular Location Overlay of Network GeneSpring

16. ‘Find Similar Pathways’ Tool Significant enrichment of my genes in particular pathways? • Analysis performed on all pathways imported into GX 10 Significant pathways are added to experiment GeneSpring

17. e-Seminars & Workshops www.genespring.com • Recorded Seminars: • Introduction to GX 10 • Analysis of miRNA & GE data • Analysis of QPCR & GE data • Alternative Splicing • Pathway Analysis GeneSpring

18. Affymetrix Files Getting Started in GeneSpring GX 10 Advanced Workflow: (To Find Differentially Expressed Genes) GeneSpring

19. Cardiogenomics dataset: Affymetirx data • Congestive heart failure (CHF)is a degenerative condition in which the heart no longer functions effectively as a pump. • The most common cause of CHF isdamage to the heart muscle by not enough oxygen.  This is usually due to narrowing of the coronary arteries which take blood to the heart. • Idiopathic cardiomyopathy results in weakened hearts due to an unknown cause. • Ischemic cardiomyopathy is causedby a lack of oxygen to the heart due to coronary artery disease. GeneSpring

20. Cardiogenomics dataset: Affymetirx data • Experimental Goal: • To identify the molecular mechanisms underlying congestive heart failure, gene expression profiles were compared between male and female patients with idiopathic, ischemic or non-failing heart conditions. CEL files generated by Affymetrix GCOS GeneSpring

21. Experimental Setup in GeneSpring Gender Interpretation Condition 1: Female (Samples 1, 2, 5, 6, 9, 10) Condition 2: Male (Samples 3, 4, 7, 8, 11, 12 ) CHF Etiology Interpretation Condition 1: Idiopathic (Samples 1, 2, 3, 4) Condition 2: Ischemic (Samples 5, 6, 7, 8)Condition 3: Non-failing (Samples 9, 10, 11, 12) Gender/CHF Etiology Interpretation Condition 1: Female/Idiopathic (Samples 1, 2) Condition 2: Male/Idiopathic (Samples 3, 4) Condition 3: Female/Ischemic (Samples 5, 6) Condition 4: Male/Ischemic (Samples 7, 8) Condition 5: Female/Non-failing (Samples 9, 10) Condition 6: Male/Non-failing (Samples 11, 12) The selected Interpretation determines how the samples are displayed in the various views and the comparisons that are made in analyses such as statistics. GeneSpring

22. GeneSpring GX 10 Vocabulary • Project – collection of experiments • Entity– gene, probe, probeset, exon, etc. • Interpretation – samples that are grouped together based on conditions. • Technology– A file containing information on array design and biological information (annotation) for all the entities on the array • Biological Genome–a collection of all major annotations (NCBI) for any organism; essential for Generic/Custom arrays lacking annotations GeneSpring

23. Getting Started in GeneSpring Cardiogenomics Experiment:Transcriptional profiling to learn more about molecular mechanisms underlying Congestive Heart Failure (CHF)Sample Data: Myocardial samples from patients with normal hearts and Ischemic & Idiopathic cardiomyopathies (3 Etiologies) Variables: Gender (2) and Etiology (3)Technology:Affymetrix U133Plus2 array GeneSpring

24. Getting Started: Create New Project From Startup screen OR from File/New Project GeneSpring

25. Getting Started with Advanced Analysis Experiment Type:Affymetrix Expression (3 Affy choices!) Workflow Type:Advanced Analysis GeneSpring

26. Select Data for Experiment • Select ‘Choose Files’ to load data files found on your computer. Note: ‘Choose Samples’ option is used when creating experiments with samples already loaded into GX 10 GeneSpring

27. Sample Upload GeneSpring

28. Summarization Algorithms in GX 10 for CEL Files • Summarization of Affymetrix probesand baseline transformation of probeset values. GeneSpring

29. Summarization algorithms in GX 10 • In addition to different calculations, the algorithms differ in the order in which Normalization and Summarization are performed. GeneSpring

30. Preprocessing of Affymetrix Arrays • CEL files are the raw data files that contain signal values for individual probes. • CEL files are preprocessed to generate one value per probeset. • Preprocessing steps are: • 1. Background subtraction • 2. Normalization • 3. Summarization of probeset values • Different preprocessing algorithms are available. Array Hybridization& Scanning DAT File ImageAnalysis GCOSAGCC CHP CEL File + CDF File GeneSpring

31. BoxWhisker plot: Summary of Normalized Intensities GeneSpring

32. Advanced Workflow Experiment Setup • Experiment Grouping • Specify parameters/conditions GeneSpring

33. Experiment Grouping • The experimental parameters are added in this window. • For each array, the particular parameter value (condition) is also specified. • Values can be added manually or loaded from a saved file (circled in Red). GeneSpring

34. Advanced Workflow Experiment Setup Create Interpretation • In the Guided Workflow, only one interpretation is automatically provided. • Here, users can create multiple interpretations GeneSpring

35. Grouping and Interpretation • 2 experimental variables: CHF Etiology and Gender • For this experiment, 3 interpretations could be created: • Gender • CHF Etiology (Ischemic, Idiopathic, non-failing) • CHF Etiology and Gender: This interpretation is automatically created in the Guided Workflow. Example: Gender Only GeneSpring

36. Creating Interpretations: step 2 of 3 GeneSpring

37. Creating Interpretations: step 3 of 3 GeneSpring

38. Advanced Analysis Workflow: Quality Control • QC on Samples and Probes automatically performed in Guided Workflow • Users can specify settings beyond those available in Guided Workflow GeneSpring

39. Quality Control on Samples GeneSpring

40. Filter by Expression GeneSpring

41. Advanced Analysis Workflow: Analysis • Statistical Analysis • Filter on Volcano Plot(both Stats and Fold Change) • Fold Change Clustering Find Similar Entities Filter on Parameters PCA GeneSpring

42. Getting Started with Guided Workflow Experiment Type:Agilent Single-color Workflow Type:Guided Workflow GeneSpring

43. Sample Upload GeneSpring

44. BoxWhisker plot: Summary of Normalized Intensities GeneSpring

45. GeneSpring GX 10: Important Menu options: Project: Import/Export project zip Tools: Script Editor/ R Editor Import BioPAX pathways GS7 data migration Options… Annotations: Update Technology Annotations Create Biological Genome Update Pathway Interactions Help: License Manager Update Product GeneSpring

46. Pathway Analysis To use ‘Find Significant Pathways’ Tool:1. Download BioPax format (.owl) pathways www.biopax.org to your computer2. Import .owl pathways into GX 10 from Tools and ‘Import BioPax pathways’ option3. From Workflows menu (in the right margin of GX 10) select ‘Find Similar Pathways’ and choose your Entity List of interest GeneSpring

47. Performing Pathway Analysis in GX 10:1. In the Annotations Menu, select ‘Update Pathway Interactions’ from Agilent Server 2. Before choosing an organism, GX 10 must first create a Pathway Database Infrastructure. May take >10 min3. Once the Infrastructure database is complete, go back to Annotations/Update Pathway Interactions and choose your preferred organism. May take >20 minutes4. From Workflows menu (in the right margin of GX 10) select ‘Pathway Analysis’ to begin building networks GeneSpring

48. Option 3 is new in GX10: Update directly from NCBI from GX (Biological Genome) Option 2: Update from file Updating Annotations: Chap 3 in GX 10 pdf manual, pg. 51 Option 1: Update from Agilent Server GeneSpring

49. GeneSpring GX 10: Reference pages in Manual • Creating/Updating Technologies & Annotations: • Chapter 3 in GX 10 pdf manual, pg. 51 • From 1) Agilent server; 2) file; 3) NCBI (Biological Genome) • GS7 to GS10 Data Migration: • Chapter 4 in GX 10 manual, pg. 71 and in Quick Start Guide • Translation: • Chapter 3.3 in pdf manual (pg 63) GeneSpring

50. Thank youwww.genespring.comTechnical Support 24 hours/5 days per weekinformatics_support@agilent.com1-800-227-9770 (option 6, 2)elise_chang@agilent.comantoni.wandycz@agilent.comchris_gates@agilent.com (Genomics) GeneSpring