1. EMBRACE Gene Ontology Workshop, 7 th – 9 th November 2007 Bari High throughput functional annotation and analysis with the Blast2GO suite Ana Conesa Bioinformatics Department Centro de Investigaciones Prínicpe Felipe aconesa@cipf.es

2. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Credits Biomedical Informatics UPV, Valencia Juan Miguel Gómez Montserrat Robles Bioinformatics Department CIPF, Valencia Blast2GO Development: Blast2GO special thanks to: ANNEX :Simen Myhre, Henrik Tveit (MTNU)‏ GOSSIP: Nils Blüthgen (MicroDiscovery GmbH)‏ ZVTM: Emmanuel Pietriga (INRIA)‏ goslim.tair.obo: Suparna Mundodi (TAIR )‏ Bioinformatics Department CIPF, Valencia Ana Conesa Stefan Goetz Centro de Genómica IVIA, Valencia Javier Terol Manuel Talón

3. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Motivation Numerous EST/genome projects Large amounts of NEW sequence data Functional Genomics Studies Need of Functional Annotation Which kind of tool? Easy to set up & run Versatil & Universal High-throughput & interactive Combine annotation & function analysis www.blast2go.org

4. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Gene Ontology based annotation more general more specific Molecular Function Biological Process Cellular Component IP2GO GO2EC

5. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Similarity between Sequences Quality of existence annotation Precision vs. “recall” Resolution Level in GO hierarchy Selection of recovered annotation data B2G Annotation Rule Consistency of assigned annotation Concepts of automatic annotation

6. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Annotation Rule Blast2GO Annotation Rule Lowest term satisfying the requirements Quality of source annotation Evidence Codes Possibility of abstraction Similarity requirement Recall vs. Precision

7. EMBRACE Workshop, 7 th – 9 th November 2007, Bari InterProScan GO-Slim GO Second Layer Graph Visualization Enrichment Statistics KEGG maps Validation localB2GDB Pipeline Batch Mode Compare Annotation (GO,IPR,EC)‏ Main functions within Blast2GO costumDB GeneIDs Additional Features: BLASTMAPPINGANNOT.RULE Manual Curation

8. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Blast2GO Schema

9. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Blast2GO use Species Citrus, nicotiana, maize, soybean, tomato, grape… Streptococcus, Trichoderma, Schistosoma, Cyanobacteria… European Flounder,pig, flidder crab, rat, honneybee, human… Metagenome projects…

10. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Where to find Blast2GO More info: Bioinformatics 2005 21: 3674-3676 Blast2GO tutorial: http://www.blast2go.org Web: http://www.blast2go.org http://blast2go.bioinfo.cipf.es http://www.geneontology.org http://groups.google.com/group/Blast2GO

11. EMBRACE Gene Ontology Workshop, 7 th – 9 th November 2007 Bari Blast2GO Guided Tour Ana Conesa Bioinformatics Department Centro de Investigaciones Prínicpe Felipe aconesa@cipf.es

12. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Start Blast2GO www.blast2go.org  Desktop application  Java webstart technology  Internet connection

13. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Load Sequences

14. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Run BLAST search

15. EMBRACE Workshop, 7 th – 9 th November 2007, Bari BLAST results

16. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Blast Distribution Charts

17. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Exercise 1  Launch Blast2GO  Open FASTA file (unizip examples.zip)‏  Browse number of sequences and sequence length  Unselect all sequences  Select 5 sequences  Run Blast against NCBI nr (change parameters if desidered)‏

18. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Exercise 2  Open blast_example.dat  Examine Distribution charts

19. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Mapping

20. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Mapping Resources Hit ACC/GI GO-Terms EC sim % GO mapping resources: Full Gene Ontology DB NCBI Flat Files: gene2accession (4 079 414 entries) gene_info (1 635 614 entries) PIR - Non-Redundant Reference Protein Database: including PSD, UniProt, Swiss-Prot, TrEMBL, RefSeq, GenPept y PDB Resources of mapping

21. Annotation EMBRACE Workshop, 7 th – 9 th November 2007, Bari GO DAG Validation Annex GOSlim EC/KEGG InterPro

22. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Gene Ontology annotation

23. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Annotation Charts

24. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Exercise 3  Select 10 first sequences  Run Mapping and Annotation  Select non annotated sequences and re-annotate with milder parameters  Lo annotation_example.dat file  Visualize Results on Mapping/Annotation Charts

25. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Sequence menu

26. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Modulation of annotation Change annotation manually EC-Codes Seq. Description GO-Term ACC Summarize annotation by “GoSlim” OBO GO-Slim File Extend annotation by the GO “Second Layer” Biological ProcessCellular Component Molecular Function acts inis involved in Myhre et al, Bioinformatics 2006

27. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Exercise 4  Browse BlastResults to (select one sequence and use sequence menu): Draw annotation graph View Annotations Edit/change annotation  Select a few sequences to run GoSlim  Run Annex

28. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Enzyme annotation and Kegg Maps GO  Enzyme Codes  KEGG maps

29. EMBRACE Workshop, 7 th – 9 th November 2007, Bari InterproScan

30. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Exercise 5  Select a few sequences to run InterProScan  Change terms view GO ID/term, IPS/GO  Merge IPS results with Blast Annotations  Load annot_interpro_annex_example.dat  Export GO Annotations  Export IPS Annotations  Save Project and Sequence Table

31. EMBRACE Workshop, 7 th – 9 th November 2007, Bari GO Graph Visualization as tool to explore data Interactive and “zoomable” graphs Color graphs highlighting areas of interest Node Score of Annotation Content 31 2.4 2.5 1 13 Visualization

32. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Level and Multilevel Charts Visualization :Pies

33. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Exercise 6  Select some sequences using select by names function (use test.example.txt)  Create a GO Combined Graph  Create Pies at level 4 and Multilevel Pie at score 3  Play with filters to simplify the graph (set score filter to 3)  Export GO Graph data as table and visualize

34. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Functional analysis with B2G Enrichment Analysis (Fisher)‏

35. EMBRACE Workshop, 7 th – 9 th November 2007, Bari Exercise 7  Run Enrichment Analysis using test and reference set files  Create Bar Chart  Create Enriched Graph and modulate number of nodes  Export results