Exercise 6: Gene Ontology Analysis

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1 Overview: Intrductin t Systems Bilgy Exercise 6: Gene Ontlgy Analysis Gene Ontlgy (GO) is a useful resurce in biinfrmatics and systems bilgy. GO defines a cntrlled vcabulary f terms in bilgical prcess, mlecular functin, and cellular lcatin, and relates the terms in a smewhat-rganized fashin. This exercise utlines the resurces available under Cytscape t perfrm analyses fr the enrichment f gene anntatin (GO terms) in netwrks r sub-netwrks. In this exercise yu will: Learn hw t navigate the Cytscape Gene Ontlgy wizard t apply GO anntatins t Cytscape ndes Learn hw t lk fr enriched GO categries using the BiNGO plugin. What yu will need: The BiNGO plugin, develped by the Cmputatinal Bilgy Divisin, Dept. f Plant Systems Bilgy, Flanders Interuniversitary Institute fr Bitechnlgy (VIB), described in Maere S, et al. Biinfrmatics Aug 15;21(16): Epub 2005 Jun 21. galfiltered.sif used in the earlier exercises and fund under sampledata. Please dwnlad any missing files frm Dwnlad and install the BiNGO plugin, as fllws: 1. Get BiNGO frm the curse page (see abve) r g t the BiNGO page at (This site als prvides dcumentatin n BiNGO). 2. Unzip the cntents f BiNGO.zip t yur Cytscape plugins directry (make sure the BiNGO.jar file is in the plugins directry and nt in a subdirectry). 3. If yu are currently running Cytscape, exit and restart. First, we will lad the Gene Ontlgy data int Cytscape. 1. Start Cytscape, under Edit, Preferences, make sure yur default species, defaultspeciesname, is set t Saccharmyces cerevisiae. Set it if needed. 2. Re-start Cytscape if yu had t set the defaultspeciesname. Nw lad the netwrk galfiltered.sif. As yu may recall, this file cntains a netwrk f yeast, S. cervisiae, prteins frm the galactse pathway.

2 3. Under the File menu in yur Cytscape Desktp, select Lad and Bi Data Server. G t Cytscape2.1/anntatin directry and lad the manifest file (the anntatin dir is in the same lcatin as the plugins dir). 4. Nw, we will apply Gene Ontlgy anntatins t the ndes in the netwrk, and brwse thrugh them. 5. On the Cytscape Desktp, click n the Anntatin buttn, shwn belw: 6. This will bring up a windw labeled Anntatin, shwn belw: 7. Click n the + sign next t GO, Bilgical Prcess. This shuld bring up ne link per GO level, 1 thrugh 13. These levels crrespnd t the depth f the tree t wrk at: higher levels represent mre general classificatins. 8. Click n 3, and click the buttn labeled Apply Anntatin t All Ndes. 9. On the right-hand panel, an entry shuld appear labeled GO Bilgical Prcess (level 3) with a + sign at the left. Click n the + sign t expand this list. 10. The right-hand panel shuld nw list the Level 3 terms, as shwn belw: 11. On the right-hand panel, click n the term cell cycle. Ntice hw this actin selects several ndes n the Cytscape canvas.

3 12. The level 3 GO Bilgical Prcess anntatin are nw available t the Nde Attribute Brwser, and may be mapped as nde labels. 13. In the Anntatin windw, select sme ther Level 3 GO Bilgical Prcess, and ntice hw ndes selected n the Cytscape canvas. See if yu can find any sectins f the netwrk with a cncentratin f a particular anntatin term. Here we will use the BiNGO plugin t see if GO term enrichment is statisticallysignificant. 1. Select a candidate sub-netwrk f yur chsing r the first neighbrs f RAP1 r GAL4 (e.g. Select -> Ndes -> By Name, enter RAP1 r GAL4 and click Search. Select -> Ndes -> First neighbrs f selected ndes). 2. Select these ndes and all edges int a new netwrk, creating a child netwrk. This will make subsequent steps in this tutrial easier. 3. Select all the ndes in this child netwrk. 4. In the Plugins menu, select BiNGO. This shuld bring up a windw called BiNGO Settings. 5. Fill in yur BiNGO Settings as fllws: 1. Give yur cluster a shrt name such as "test". 2. Leave the Get Cluster frm Netwrk bx checked. 3. Under Select a statistical test, select Hypergemetric. Binmial testing is used when the amunt f data is very large, but hypergemetric testing is apprpriate fr mst Cytscape usage scenaris. 4. Under Select a multiple testing crrectin, chse Benjamini & Hchberg False Discvery Rate (FDR). This is less cnservative than Bnferrni testing, but still sufficient fr mst cases. 5. Under Chse a significance level, enter This threshld cntrls which GO classes are detailed in the utput. This is nt a cnservative threshld, but later, ne can chse GO classes with lwer P values interactively. 6. Under Select the categries t be visualized, select Overrepresented after crrectin. With very few exceptins, this is the setting yu will want. 7. Under Select reference set, select Test cluster versus cmplete anntatin. This will cmpare yur set f ndes t all genes in the yeast genme. 8. Under Select ntlgy, select GO Bilgical Prcess. 9. Click Start BiNGO. 6. After a brief pause, a netwrk will appear n yur canvas such as the ne shwn belw

4 7. Within this netwrk Each nde represents a GO term, and is labeled accrdingly (zm int the netwrk t see labels). The tplgy depicts the hierarchy f GO bilgical prcesses. Apply the yfiles hierarchical layut. The yellw and range ndes represent terms with significant enrichment, with darker range representing a higher degree f significance, as shwn by the legend n yur screen: White ndes are terms with n significant enrichment, but are included because they have a significant child term. Branches f GO with n significant terms are nt shwn. The size f each nde in a BiNGO graph is prprtinal t the number f ndes in yur query set with that term. 8. G t Visual Styles editr (Visualizatin -> Set Visual Prperties -> Define -> Nde Label), and lk at the available attributes under Map Attribute. Yu shuld see several mre, including: descriptin_test: the name f the GO bilgical prcess adjustedpvalue_test: the p-value fr the nde, adjusted fr multiple hypthesis testing (nte that the un-adjusted p-value is als there, with the name pvalue_test, but this P value is less useful fr mst applicatins). n_test: the number f genes in the yeast genme with this GO term.

5 x_test: the number f ndes that yu have selected which have this GO term. N_test: the ttal number f genes in the yeast genme with GO anntatins. X_test: the ttal number f genes that yu have selected. These last fur quantities are used in the calculatin f the adjusted P value. 9. Select these attributes. Nw, select sme ndes in yur BiNGO graph, and lk at their attributes under the Nde Attribute Brwser 10. Select sme f these terms, and brwse thrugh the ndes in yur BiNGO graph. 11. Here is a gd case fr Cytscape's hiding cntrls. When we zm int this netwrk, we see the fllwing: Ntice hw the regin n the right cntains tw ndes f marginal significance, plus several ndes f n significance included because they are parents f these ndes. 12. Select these ndes. Click n the Hide Selected Regin buttn (shwn) These ndes will disappear frm the canvas. 13. Yu can make these ndes visible again with the Shw All Ndes and Edges buttn (belw) 14. BiNGO will ptinally prduce an utput file listing the p-values f all ndes with significant enrichment, as fllws:

6 . Return t the child netwrk yu created previusly, and make sure that all ndes are still selected. i. Return t yur BiNGO Settings windw. ii. Specify a new cluster name: test2. iii. iv. Near the bttm f the windw, click n Check bx fr saving data. Click n the buttn labeled Save BiNGO Data file in:, and select a directry fr BiNGO's utput file v. Rerun BiNGO. vi. In the specified directry, yu shuld nw have a file called test.2.bg. Yur screen shuld shw a new windw titled test.2 BiNGO Results. Bth f these shuld summarize yur BiNGO parameters, and reprt n the enrichment f all terms meeting yur p-value threshld. 15. Recall that this BiNGO graph reprts n the enrichment f a subnetwrk centered n RAP1, GAL4 r the particular ndes yu selected. Recall als that this entire netwrk cnsists f ndes invlved in ne single pathway: galactse utilizatin. S when we lk at the enriched GO terms in yur BiNGO graph, which terms relate t galactse utilizatin in general, and which relate t yur sub-netwrk specifically? Here, we shall see hw t answer that questin.. i. Return t yur parent netwrk and verify that the sub-netwrk is still selected. ii. Return t yur BiNGO settings windw, and chsing Test cluster versus netwrk under Select reference set. Specify a new name in the Cluster name: bx, "test3". iii. Rerun BiNGO. iv. Cmpare the new BiNGO netwrk against the ld ne. Yu shuld see fewer significant GO terms in the new BiNGO netwrk. Which terms are lst? These are prbably assciated with galactse utilizatin in general. v. G t the Nde Attribute brwser, and click n Select Attributes. Nte that the available attributes include adjustedpvalue_test (reprting enrichment against the cmpleted genme) and adjustedpvalue_test3 (reprting enrichment against the galfiltered netwrk). Select these tw attributes fr a side-by-side cmparisn f the p-values f sme ndes in the BiNGO graphs. Let's say that BiNGO shws enrichment in sme GO categry that yu find especially interesting, and yu want t see the ndes in yur netwrk with that GO categry. The fllwing steps utline hw yu can d that. We will illustrate the prcess with nde 5975, "carbhydrate metablism". 1. G t the parent netwrk n the Cytscape canvas, and un-select any ndes currently selected. 2. G t ne f yur BiNGO graphs and carefully cunt the shrtest path t the term frm the nde 'bilgical prcess'. That indicates the level f the term. Fr instance, 'carbhydrate metablism'' is a distance f fur frm 'bilgical prcess: there are three ndes in between them.

7 3. If yu still have the Anntatin windw pen, return t it. 4. Under the Anntatin windw, and select the Bilgical Prcess anntatin fr that level: Level 4 fr the case f "carbhydrate metablism". Click n the buttn labeled Apply Anntatin t All Ndes 5. On the right side f the windw, expand the list f GO terms fr Level 4. Scrll dwn the list t "carbhydrate metablism" and click n it. 6. Lk at yur parent netwrk n the Cytscape canvas. Yur netwrk shuld appear smething like as shwn belw: 7. Ntice hw many f the Carbhydrate Metablism ndes cme frm tw prtins f the netwrk, and that these tw sub-netwrks cnsist mstly f Carbhydrate Metablism ndes. This suggests that these are the prtins f the graph specific t carbhydrate metablism activity.