Chris Stoeckert, Ph.D. Dept. of Genetics & Center for Bioinformatics University of Pennsylvania

1. Integrated Data Systems for Genomic AnalysisGenomics and Bioinformatics for the Advancement of Clinical SciencesThomas 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. Using a Genomics Unified Schema (GUS) to ask genomic questions 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.

5. GUS Powers Multiple Genomics Projects AllGenes Allgenes is based on a comprehensive mouse and human gene index. The genes are approximated by transcripts predicted from EST and mRNA clustering PlasmoDB 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 EPConDB is an index of genes expressed in endocrine pancreas. Expression is defined either through microarray experiments or sequence annotation.

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

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

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

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

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

13. PlasmoDB query: "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." This query illustrates several aspects of the GUS database including: 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

14. Choose signal peptide Select Queries from the PlasmoDB homepage

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

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

19. EPConDB query: "Which DOTS assemblies (RNA) represented on the Endocrine Pancreas Consortium’s chip 2.0 are constituents of the insulin initiated signal transduction pathway ?"

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

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

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

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. DoTS RAD TESS SRES Core How GUS Works AllGenes PlasmoDB EPConDB Java Servlets Other sites, Other projects, e.g. GeneDB Oracle RDBMS Object Layer for Data Loading

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. AutomatedAnalysis &Integration Annotator’s Interface WWW queries,browsing, & download Mining Applications Architecture of GUS QTL,POP, SNP, Clinical GenBank, InterPro, GO, etc GenomicSequence microarray& SAGEExperiments GSSs &ESTs MappingData Annotation Object Layer Oracle/SQL DoTS TESS RAD Core SRes Java Servlets &Perl CGI

29. Namespace Domain Highlights Core Data Provenance Evidence SRes (Shared Resources) Shared Resources Ontologies Sequence and annotation DoTS (DB of Transcribed Seqs) Central dogma RAD (RNA Abundance DB) Gene expression MIAME/MAGE TESS (Trans Elem Search Site) Gene regulation Grammars Five domains GUS is divided into 5 domains* (separate name spaces) * Protein Abundance DB domain underway

30. DoTS central dogma schema Gene Gene Instance Gene Feature (isa NA Feature) Genomic Sequence (isa NA Sequence) RNA RNA Instance RNA Feature (isa NA Feature) RNA Sequence (isa NA Sequence) Protein 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 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. BindingSite DoTS.NaFeature Promoter . . . DoTS.NaSequence TESS Schema TESS.Moiety Moiety MoietyHeterodimer MoietyMultimer MoietyComplex TESS.Activity ActivityProteinDnaBinding TESS.FootprintInstance ActivityTissueSpecificity TESS.TrainingSet TESS.Model ModelString TESS.ParameterGroup ModelConsensusString ModelPositionalWeightMatrix TESS.Note ModelGrammar

35. Identify shared TF binding sites Genomic alignment and comparative Sequence analysis RAD DoTS EST clustering and assembly 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. Reln 7q21-22 Sema3a,c,d, e 8 Nos3 12 7q36 Dpp6 Htr5a 15 Hdh, Adra2c 20 4p16.3 Drd5 23 Qdpr 4p15.31 30 Gabrb1 , a2, g1 40 4q12 Pdgfra, Kit, Flk1 43 Clock Areas of Focus on Mouse Chromosome 5 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. Known RefSeq Genes in (72-76Mb) Region as Viewed in UCSC Genome Browser Only 14 RefSeq Genes plus an additional 7 from Ensembl

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

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

43. Putative Genes on Mouse Chromosome 5 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)

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