1. Making Sense Out of Transcriptome Integrative Bioinformatic Approaches Anil Jegga, D.V.M., M.S. Division of Biomedical Informatics Cincinnati Children’s Hospital Medical Center Department of Pediatrics University of Cincinnati College of Medicine Email: anil.jegga@cchmc.org Homepage: http://anil.cchmc.org

2. Acknowledgements Jing Chen* Siva Gowrisankar Vivek Kaimal Amit Sinha* Mrunal Deshmukh Nishanth Vepachedu Divya Sardana Scott Tabar Eric Bardes Bruce Aronow

3. Annotation Databases Gene Ontology, Pathways DNA Repair XRCC1 OGG1 ERCC1 MPG….. DNA Repair XRCC1 OGG1 ERCC1 MPG….. Angiogenesis HIF1A ANGPT1 VEGF KLF5…. Angiogenesis HIF1A ANGPT1 VEGF KLF5…. Gene lists associated with similar function/process/pathway Genome-wide Promoters Putative Regulatory Signatures E2F RB1 MCM4 FOS SIVA….. E2F RB1 MCM4 FOS SIVA….. PDX1 GLUT2 PAX4 PDX1 IAPP…. PDX1 GLUT2 PAX4 PDX1 IAPP…. p53 CDKN1A CTSD CASP DDB2…. p53 CDKN1A CTSD CASP DDB2…. Expression Profile - Gene Lists Enrichment Analysis Observed Expected E2F RB1 MCM4 FOS… E2F RB1 MCM4 FOS… Angiogenesis HIF1A ANGPT1 VEGF….. Angiogenesis HIF1A ANGPT1 VEGF….. DNA Repair XRCC1 OGG1 ERCC1 MPG…. DNA Repair XRCC1 OGG1 ERCC1 MPG…. P53 CTSD CASP DDB2…. P53 CTSD CASP DDB2…. Random Distribution Significant Enrichment

4. Gene Ontology Pathways Phenotype/Disease Association Protein Domains Protein Interactions Expression in other tissues/experiments I have a list of co-expressed mRNAs (Transcriptome)…. Now what? Known transcription factor binding sites (TFBS) Conserved Non-conserved Unknown TFBS or Novel motifs Conserved Non-conserved MicroRNAs 2.Identify the underlying biological theme 1.Identify putative shared regulatory elements Give a man a fish and you feed him for a day. Teach a man to fish and you feed him for a lifetime Give a man a fish and you feed him for a day. Teach a man to fish and you feed him for a lifetime

5. Biological question: Are co-expressed genes co-regulated? Gene Regulatory Networks (GRNs) Do they share cis-elements or TFBSs? Are there any significant common motifs within the promoter regions? Do they share cis-elements or TFBSs? Are there any significant common motifs within the promoter regions?

6. http://genometrafac.cchmc.org

9. 1 1 3 3 2 2 You need to have a login account; contact anil.jegga@cchmc.org

10. 4 4 6 6 5 5

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18. 14 13

19. DiRE: http://dire.dcode.org

21. http://www.cisreg.ca/oPOSSUM/

24. Genome Browser Gateway choices: 1.Select Clade 2.Select genome/species: You can search only one species at a time 3.Assembly: the official backbone DNA sequence 4.Position: location in the genome to examine or search term (gene symbol, accession number, etc.) 5.Image width: how many pixels in display window; 5000 max 6.Configure: make fonts bigger + other options 1 45 6 3 2 Golden Path: http://genome.ucsc.edu

28. 1 1 2 2 3 3 4 4 1.Select “refFlat” under “table” 2.Ensure that “region” is “genome” 3.Click on “paste list” 1.Paste the gene symbols 2.Remember it is case-sensitive: Human: all upper case (e.g. XRCC1) Mouse: lower case (first letter upper case. E.g. Xrcc1) Enter number of bp you want to analyze/download Select the output format as “custom track” Describe your track

29. 5 5 6 6 7 7 8 8 Try GTF output too 1.Select “Variation and Repeats” under “Group” 2.Click on “create” under “intersection” Change the “group” to “Custom Tracks” and select the appropriate “track” and “table”

30. 9 9 10 One drawback with this output is it doesn’t tell you which SNPs are in the upstream region of which gene. However, since the positions of SNPs are included, you can compare them with the gene coordinates and figure it out. Genome Browser view that lists all the SNPs lying within the upstream 1 kb (the region we queried) region of one of the genes analyzed.

33. Biological question: Are co-expressed genes functionally similar? Functional Networks Do they share same GO terms or pathways? Are there any significant enriched terms within a group of gene list? Do they share same GO terms or pathways? Are there any significant enriched terms within a group of gene list?

34. http://david.abcc.ncifcrf.gov/

36. You can compare multiple lists! http://www.pantherdb.org/

38. ToppGene – General Schema http://toppgene.cchmc.org

39. TOPPGene - Data Sources 1.Gene Ontology: GO and NCBI Entrez Gene 2.Mouse Phenotype: MGI (used for the first time for human disease gene prioritization) 3.Pathways: KEGG, BioCarta, BioCyc, Reactome, GenMAPP, MSigDB 4.Domains: UniProt (Pfam, Interpro,etc.) 5.Interactions: NCBI Entrez Gene (Biogrid, Reactome, BIND, HPRD, etc.) 6.Pubmed IDs: NCBI Entrez Gene 7.Expression: GEO 8.Cytoband: MSigDB 9.Cis-Elements: MSigDB 10.miRNA Targets: MSigDB

43. http://www.fatigo.org

46. http://vortex.cs.wayne.edu/projects.htm

49. My NCBI: Can create/store queries; Set mail options to receive new articles on your chosen queries/subjects PubMed Limits: Useful to refine your queries and get more appropriate results Preview/Index: You can customize (intersect, etc.) your previous queries My NCBI: Can create/store queries; Set mail options to receive new articles on your chosen queries/subjects PubMed Limits: Useful to refine your queries and get more appropriate results Preview/Index: You can customize (intersect, etc.) your previous queries