Lecture 5. Functional Analysis with Blast2GO Enriched functions. Kegg Pathway Analysis Functional Similarities B2G-Far. FatiGO Babelomics.

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1 Lecture 5 Functional Analysis with Blast2GO Enriched functions FatiGO Babelomics FatiScan Kegg Pathway Analysis Functional Similarities B2G-Far 1

2 Fisher's Exact Test One Gene List (A) The other list (B) Are this two groups of genes carrying out different biological roles? Biosynthesis 60% Biosynthesis 20% Sporulation Sporulation 20% Biosynthesis No biosynthesis A 6 4 B 2 8 C ont i ng enc y t ab l e Genes in group A have significantly to do with biosynthesis, but not with sporulation. 20% We can do this for each GO, mirna, InterPro,...!!! 2

3 Multiple testing correction Many tests Many false positive = we need correction! FDR control is a statistical method used in multiple hypothesis testing to correct for multiple comparisons. In a list of rejected hypotheses, FDR controls the expected proportion of incorrectly rejected null hypotheses. FWER control: The familywise error rate is the probability of making one or more false discoveries among all the hypotheses when performing multiple pairwise tests. (more conservative) 3

4 Different types of comparisons Compare one condition against another Remove Common Ids Test and Ref-Set are interchangeable Common IDs Set 1 Set 2 Compare a subset against the total Gossip default setting Test and Ref-Set are NOT interchangeable TestSet Common IDs RefSet RefSet Common IDs TestSet 4

5 FatiGO in Blast2GO Two-Tailed test not only identifies over but also under represented functions. If no Ref-Set is chosen all annotations are used as reference 5

6 FatiGO in Blast2GO Result table with link outs to sequence lists 6

7 FatiGO in Blast2GO Retains only the lowest, most specific enriched term per GO branch 7

8 FatiGO in Blast2GO Export enriched terms data as DAG graphs! reduce => To draw all nodes, set filter to 1 8

9 FatiGO in Blast2GO Export enriched terms as chart! => Filter results % of sequences in Test group % of sequences in Ref group If Test > Ref = over-expressed If Ref > Test = under-expressed 9

10 Outline Enriched functions Fatigo Babelomics Export annotations to other tools e.g: FatiScan Kegg Pathway Analysis Functional Similarities B2G-Far C04018C10 GO: C04018C10 GO: C04018C10 GO: C04018C10 EC: C04018E10 GO: C04018E10 GO: C04018A12 GO: C04018C12 GO: C04018C

11 Babelomics WEB tools suite A complete suite of web tools for the functional analysis of groups of genes in high-throughput experiments 11

12 Babelomics Key features Functional profiling by functional enrichment method (FatiGO) and gene set method (Fatiscan) Many functional and regulatory definitions (GO, KEGG, Biocarta, text-mining derived bioentities, Transcription factors, CisRed, mirnas, InterPro, etc.) Wide coverage of model organisms (more than 10 species) Import your own annotations 12

13 Babelomics Tools FatiGO: Finds differential distributions of functional terms between two groups of genes, these terms can be: Gene Ontology, InterPro motifs, SwissProt KW, two lists transcription factors (TF), gene expression in tissues, bioentities from scientific literature, cis-regulatory elements CisRed. Tissues Mining Tool: compares reference values of gene expression in tissues to your results. MARMITE: Finds differential distributions of bioentities extracted from PubMed between two groups of genes. FatiScan: detect significant functions with Gene Ontology, InterPro motifs, ranked list Swissprot KW and KEGG pathways in lists of genes ordered according to differents characteristics. MarmiteScan: Use chemical and disease-related information to detect related blocks of genes in a gene list with associated values. 13

14 Babelomics Databases Interpro Gene Ontology KEGG Babelomics Ensembl SwissProt Transcription Factors Bioentities Literature MicroRNA Integrated Biological DB of Functional Annotation for more than 10 species Cisred 14

15 Babelomics Fatiscan features Interpret a ranked list of genes There is not need for choosing a cut-off (all information is included) One statistical test for each Functional Block of annotation Multiple testing context (hundreds of annotation) Filtering of annotation is convenient (the less tests the best correction) 15

16 FatiScan...testing along an ordered list Annotation label A Index ranking genes according to some biological aspect under study. List of genes + Database that stores gene class membership information. A B C Annotation label B Annotation label C Block of genes enriched in the annotation A Annotation C is homogeneously distributed along the list FatiScan searches over the whole ordered list, trying to find runs of functionally related genes. - Block of genes enriched in the annotation B 16

17 FatiScan Web Tool List of genes C04018C12 C04018C13 C04018C14 C04018C16 C04018E18 C04018E19 C04018A21 C04018C33 C04018C List of annotations C04018C13 C04018C14 C04018C15 C04018C16 C04018E17 C04018E18 C04018A19 C04018C22 C04018C32... GO: GO: GO: EC: GO: GO: GO: GO:

18 gene set enrichment analysis (fold-change case-control) + GO2... GO10 GO11 GO1 A: Functional labels (GO, KEGG, etc.) over-represented among overexpressed genes A B D: Functional labels under-represented among underexpressed genes... (Ranked gene list (e.g. fold-chang FatiScan C D B: Functional labels under-represented among overexpressed genes C: Functional labels over-represented among underexpressed genes 18

19 FatiScan Results Significantly enriched GO-Terms Percentages (Test/Ref) p-values Adjusted p-values 19

20 Functional Analysis - Outline Enriched functions FatiGO Babelomics FatiScan Kegg Pathway Analysis Functional Similarities B2G-Far 20

21 KEGG: Kyoto Encyclopedia of Genes and Genomes GO Term Enzyme Code KEGG PATHWAY 21

22 Obtain Enzyme Codes 22

23 KEGG Pathways First, choose a folder to save the KEGG maps Maps are retrieved online Export as text file (tab-sep) 23

24 KEGG Pathways Or d er ed L i st --> Each enzyme in a different color --> Pathways are ordered for abundance 24

25 Functional Analysis - Outline Enriched functions FatiGO Babelomics FatiScan Kegg Pathway Analysis Functional Similarities B2G-Far 25

26 Compare annotation-sets by graphs We have several conditions, tissues, statistical methods different sets of annotations Question: How similar are they? Approach: Superpose its GO-graphs B2G Solution: Generate a.annot file with the different conditions as Seq-Name 26

27 Compare annotations by graphs Make a combined graph: 27

28 How to compare two sets of annotations? How to quantify the similarity of to sets of annotations Sequence X - original annotations: GO: > process -> regulation of transcription GO: > process -> response to salt stress GO: > component -> nucleus GO: > function -> DNA binding?? si di?? mil ss?? ar im?? ity ila?? rit? y Sequence Y - transferred/new annotations: parent (dist2) GO: > process -> regulation of cellular meta... child (dist1) GO: > process -> response to cation stress exact (dist0) GO: > component -> nucleus brother (dist2) GO: > function -> RNA binding 28

29 Measures (dis)similarity 5 groups of matches Identical if the B2G annotation is present among the original annotations of the sequence. General if the B2G annotated GO term is a parent term of one of the original annotations of the sequence. Specific if the B2G annotated GO term is a children term of one of the original annotations of the sequence "Other branch" if no original annotation terms lay in the same DAG branch of the considered B2G annotated GO term "Other Type" if the term can not be compared to any other term because the GO category is not present in the original gene product annotation. 29

30 Use-Case: Compare annotations DBs Blast2GO function: Search one set of annotations in another Example: Annotation of 1100 sequences against NR (873 seqs, 3152 annots) and SwissProt (639 seqs, 4547 annots) Results: Search SwissProt vs. NR annotation: Compared sequences: 610 Compared annotations: 4402 Swissprot has: Exact GOs: % More specific GOs: % More general GOs: 206-5% Other branch GOs: % Other Type GOs: % Compare NR vs. SwissProt annotation: Compared sequences: 610 Compared annotations: 2488 NR has: Exact GOs: % More epecific GOs: 224-9% More general GOs: % Other branch GOs: % Other Type GOs: 159-6% 30

31 Functional Analysis - Outline Enriched functions FatiGO Babelomics FatiScan Kegg Pathway Analysis Functional Similarities B2G-Far 31

32 B2G-FAR 32

33 Hundreds of species now with Blast2GO protein annotations 33

34 Search your sequences in B2G-FAR Download annotations of your species Load annotations into B2G Deselect all sequences 34

35 B2G-FAR - GeneChips 35