1. Integrated Data Systems for Genomic Analysis Genomics and Bioinformatics for the Advancement of Clinical Sciences Thomas Jefferson University, Oct. 14, 2002 Chris Stoeckert, Ph.D. Dept. of Genetics & Center for Bioinformatics University of Pennsylvania

2. Plasmodium genomics: Genomics and proteomics pave the way for controlling malaria Nature, October 3, 2002

3. Thinking Genomically Genome Phenotype Genome structure Genes and function Pathways Expression patterns (Complex) diseases

4. Genomic Unified Schema (GUS) is a relational database that warehouses and integrates biological sequence, sequence annotation, and gene expression data from a number of heterogeneous sources. User-friendly web interfaces present slices of the GUS database and allow researchers to execute structured queries for information concerning gene structure, function, and expression. Using a Genomics Unified Schema (GUS) to ask genomic questions

5. GUS Powers Multiple Genomics Projects AllGenes PlasmoDB EPConDB Allgenes is based on a comprehensive mouse and human gene index. The genes are approximated by transcripts predicted from EST and mRNA clustering PlasmoDB is the official database of the Plasmodium falciparum genome project which provides an integrated view of genome sequence data including expression data from EST, SAGE, and microarray projects EPConDB is an index of genes expressed in endocrine pancreas. Expression is defined either through microarray experiments or sequence annotation.

6. "Is my cDNA similar to any mouse genes that are predicted to encode transcription factors and have been localized to mouse chromosome 5?" http://www.allgenes.org/ Steve Fischer, Debbie Pinney, Brian Brunk, Joan Mazarelli, Jonathan Crabtree, Yongchang Gan, Sharon Diskin Nikolay Kolchanov, Alexey Katohkin Data IntegrationData Analysis Tools RHMap GOFunction Sequence GOFunction assigments Boolean function History function BLAST This query illustrates several aspects of the GUS database including: allgenes.org query

7. Select the allgenes.org boolean query page Click on the "AND" button

8. Choose the RH map and GO function queries Select mouse chromosome 5 and "transcription factor"

9. There are 26 mouse RNAs (assemblies) that meet these criteria: This query result set now appears on the query "history" page:

10. Now use the BLAST page to identify RNAs similar to my cDNA The results of the BLAST search appear in the query history

11. Intersect ("AND") the BLAST search with the previous query: And we have our answer (the third row on the query history page):

12. Predicted GO function(s) (some manually reviewed) Other transcripts from the same gene External links Mapping information Gene trap insertions Protein/motifs, etc. predicted protein CAP4 assembly EST expression profile UCSC BLAT

13. "List all genes whose proteins are predicted to contain a signal peptide and for which there is evidence that they are expressed in Plasmodium falciparum's merozoite stage." http://plasmodb.org/ David Roos, Jessie Kissinger, Bindu Gajria, Martin Fraunholz, Jules Milgram, Phil Labo, Amit Bahl, Dave Pearson, Dinesh Gupta, Hagai Ginsburg Jonathan Crabtree, Jonathan Schug, Brian Brunk, Greg Grant, Trish Whetzel, Matt Mailman, Li Li Data Integration Data Analysis Tools Genome annotation Mass spec Sequence analysisHistory function This query illustrates several aspects of the GUS database including: PlasmoDB query:

14. Select Queries from the PlasmoDB homepage Choose signal peptide

15. Choose chromosome and Gene/prediction type-submit There are 651 genes with predicted signal peptides

16. Choose Gene Expression from the queries page, then Proteomics Then choose chromosome, lifecycle stage, evidence - submit There are 828 gene predictions that satisfy this query

17. Go to the history page and choose which simple queries to combine. Select intersect. We have an answer. There are 86 predicted genes that satisfy our complex query Click on a gene to get a full report

18. There is a variety of information available from the report page including: Predicted protein features and gene models

19. "Which DOTS assemblies (RNA) represented on the Endocrine Pancreas Consortium’s chip 2.0 are constituents of the insulin initiated signal transduction pathway ?" EPConDB query: Data Integrationes3www w Data Analysis Tools Sequence Microarray experiment Transduction pathway BLASTHistory function

20. http://www.cbil.upenn.edu/EPConDB Klaus Kaestner, Marie Scearce, John Brestelli, Phillip Le Elisabetta Manduchi, Angel Pizarro, Debbie Pinney, Greg Grant, Joan Mazzarelli, Jonathan Crabtree, Hongxian He,Shannon Mcweeney, Matt Mailman

21. Go to the gene information query page and click on “DOTS assemblies involved in a pathway”

22. Choose the insulin pathway, a p-value, pancreas, the species, and whether an assembly must include an mRNA - submit There are 59 dots assemblies that are constituents of the insulin pathway

23. Return to the gene information query page and select clones sets. Choose chip 2.0 - submit There are 3242 assemblies represented on chip 2

24. Go to the history page, select the queries to combine and select intersect – view the results There are 8 assemblies that satisfy the complex query. Clicking on an RNA retrieves an allgenes report.

25. Using Databases to Think Genomically Draw attention to these resources Show how different data sources and approaches can be used to ask powerful questions This can be done for different organisms, different systems

26. How GUS Works AllGenes PlasmoDB EPConDB CoreSRESTESSRADDoTS Oracle RDBMS Object Layer for Data Loading Java Servlets Other sites, Other projects, e.g. GeneDB Other sites, Other projects, e.g. GeneDB

27. Goals of GUS Generic platform for model organism or disease specific databases Integration of genome, transcript and protein data, including: –Sequence –Function –Expression –Interaction –Regulation –Orthologs and paralogs Support for: –automated annotation and integration –manual curation –data mining/analysis and sophisticated queries –web access http://www.gusdb.org Jonathan Crabtree, Jonathan Schug, Steve Fischer, Elisabetta Manduchi, Angel Pizarro, Junmin Liu, Debbie Pinney, Greg Grant, Trish Whetzel, Li Li, Sharon Diskin, Hongxian He

28. Automated Analysis & Integration WWW queries, browsing, & download Java Servlets & Perl CGI Mining Applications DoTS Oracle/SQL Genomic Sequence microarray & SAGE Experiments Mapping Data GenBank, InterPro, GO, etc GSSs & ESTs Annotation QTL,POP, SNP, Clinical RADCoreSRes Object Layer TESS Annotator’s Interface Architecture of GUS

29. Five domains OntologiesShared Resources SRes (Shared Resources) EvidenceData ProvenanceCore GrammarsGene regulation TESS (Trans Elem Search Site) MIAME/MAGEGene expression RAD (RNA Abundance DB) Central dogma Sequence and annotation DoTS (DB of Transcribed Seqs) HighlightsDomainNamespace * Protein Abundance DB domain underway GUS is divided into 5 domains* (separate name spaces)

30. DoTS central dogma schema Gene Instance Gene Feature (isa NA Feature) Genomic Sequence (isa NA Sequence) RNA Instance RNA Feature (isa NA Feature) RNA Sequence (isa NA Sequence) Protein Instance Protein Feature (isa NA Feature) Protein Sequence (isa AA Sequence)

31. RAD schema uses MAGE/MIAME MAGE Experiment Array BioMaterial BioAssay BioAssayData Protocol, Descr. HigherLevelAnalysis MAGE Experiment Array BioMaterial BioAssay BioAssayData Protocol, Descr. HigherLevelAnalysis MIAME Experimental Design Array design Samples Hybridization, Measure Normalization. MIAME Experimental Design Array design Samples Hybridization, Measure Normalization.

32. http://www.mged.org

33. Journals are Adopting the MGED Standards Use of Minimal Information About Microarray Experiment (MIAME)

34. TESS Schema ModelString ModelConsensusString ModelPositionalWeightMatrix ModelGrammar TESS.Model ActivityProteinDnaBinding ActivityTissueSpecificity TESS.Activity Moiety TESS.Moiety MoietyMultimer MoietyHeterodimer MoietyComplex TESS.FootprintInstance DoTS.NaFeature BindingSite Promoter... DoTS.NaSequence TESS.TrainingSet TESS.ParameterGroup TESS.Note

35. RAD EST clustering and assembly DoTS Genomic alignment and comparative Sequence analysis Identify shared TF binding sites TESS

36. Using GUS for Genomic Research Annotating mouse chromosome 5 –Maja Bucan Identifying novel genes expressed in the endocrine pancreas –Klaus Kaestner, Alan Permutt, Doug Melton Identifying genes regulated by CREB –Allan Pack, Mirek Mackiewicz

37. Annotation of Mouse Chromosome 5 What are all the genes? What is their structure and function? Where are they expressed and how is this regulated? Maja Bucan, Otto Valladeres, Kyle Gaulton Jonathan Crabtree, Yongchang Gan, Joan Mazzarelli, Jonathan Shug

38. Areas of Focus on Mouse Chromosome 5 Dpp6 Adra2c 7q36 4p16.3 4q12 4p15.31 12 20 40 43 Reln Htr5a Drd5 Gabrb1, a2, g1 Clock Nos3 23 8 15 Qdpr 30 7q21-22 Sema3a,c,d,e Pdgfra, Kit, Flk1 Hdh, Rw as a balancer

39. Approach to Annotating Mouse Chromosome 5 Genomic sequence Public release: chromosome 5 has many gaps –Celera –Combine to eliminate gaps where possible Gene models –ENSMBL prediction –Celera predictions BLAT alignment of DoTS –Comparison to human regions

40. Only 14 RefSeq Genes plus an additional 7 from Ensembl Known RefSeq Genes in (72-76Mb) Region as Viewed in UCSC Genome Browser

41. 5033405K12Rik 6030432N09Rik 1810027I20Rik AI836376 Sgcb 1700067I02Rik C78283 2700023E23Rik 1190017B18Rik 6720475M21Rik 1300019H17Rik Lnx1 Chic2 Gsh2 Pdgfra Kit Kdr Gabarapl2 (homolog) Srd5a2l Tparl Clock Pdcl2 Nmu MGI approved symbols Gene symbol synonyms KIAA1458 KIAA0826 LOC231293 KIAA0276 FLJ12552 Identified 28 known genes ~76Mb ~72Mb 15 genes have assigned GO Functions 5 enzyme 4 signal transducer 4 ligand binding or carrier 3 nucleic acid binding 2 transporter Known Genes on Mouse Chromosome 5

42. *Alignment reveals exon differences between RNAs belonging to gene (Alternative forms) * Example of Known Mouse Chromosome 5 Gene - Chic2

43. putative gene mouse chr5 Note:multi-exon alignment; single image clone 583253; polyA signal suggests 3’ end of gene putative gene mouse chr5 Note:Singleton ESTs from IMAGE clone 551428 align putative gene mouse chr5 Note:multi-exon alignment; ESTs from single image clone 515319; possible polyA signal in 3'sequence putative gene mouse chr5 Note:multiple span alignment; 9/02- RNAs also aligning to another region of mouse chr5 putative gene mouse chr5 Note: 3 ESTs in assembly from embryo ……. Total 21 (some putative genes may later be merged) Putative Genes on Mouse Chromosome 5

44. Example of a Putative Mouse Gene Example DT.40155293 image clone sequences (5’ and 3’ in same assembly)

45. Genes on Mouse Chromosome 5 72-76 Mb region –65 genes from automated DoTS analysis –49 manual evaluation –21 Ensembl genes –14 RefSeq genes Whole chromosome 5 (151 Mb) –2157 genes from automated DoTS analysis –1275 Ensembl genes

46. Summary To make links between genotype and phenotype, the output of technologies such as genomic sequencing, microarrays, mass spec, etc., must be integrated Our solution is GUS, Genomics Unified Schema, used for multiple systems: AllGenes, PlasmoDB, EPConDB –GUS is freely available as a system for use and development –RAD as part of GUS and uses microarray standards now available Using GUS for genomic research such as annotating mouse chromosome 5. –Possibly doubling the number of genes in annotated regions! http://www.cbil.upenn.edu