Dose-response pathway analysis for gene expression: Graphical
Contents
1. Ranked based Local statistic t Figure 17 DR Pathway Analysis Results window Genes Plot 6 4 Tree The Tree tab utilizes the structured vocabulary whereby genes are annotated in GO from broad to narrow levels of classification in a directed acyclic graph DAG As such many categories are highly related in their gene membership and visualizing results across the ontology can be useful in ascertaining the relationship among multiply significant categories 19 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL By default nodes with unadjusted p values less than 0 1 are drawn in brown less than 0 5 are drawn in green and less than 0 7 are drawn in purple more than 0 7 in grey all other categories are colored in cyan User defined cutoffs for these colors can be specified using the menu Options gt Color scheme By clicking on any tree the node graph will be refreshed and the selected node will become the top of the tree showing child categories You can easily undo that operation by pushing Undo button By pushing the Big tree button a new window will open showing the tree containing all the categories in selected package By checking Show all children DR Pathway Analysis will show all the categories which are not in threshold results Tree color scheme p value lt 0 1 0 1 lt p value lt 0 5 0 5 lt p value lt 0 7 p value 0 7 GO cat not on array JJ Go 002. Loading initial packages Figure 2 Progress window If installation was successful you will see a successful install message displayed Installer will place startup and uninstall link inside R DRGUI directory in case it s unable to create startup menu items and Desktop shortcuts for DR Pathway GUI Message i DR GULR2 15 1 were successfully installed CK Figure 2 Installer success message J DR GUI R2151 L n Run DR GUI R2151 with status pannel fa Uninstall DR GUI R2151 Figure 4 DR GUI folder in Windows Start menu If installation fails with error message below please follow installation instructions for User Access Control below 2 2 1 Installation under Windows Vista As Windows Vista has UAC User Access Control which doesn t allow copy files in Windows you should undertake some additional steps 1 Before installation make sure that you have Full Control over your R installation directory 8 To check that right click on the R folder usually it s here C Program Files R l i THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL 9 Select Properties from menu that will appear 10 Navigate to Security tab 11 Click Edit button 12 Select Users in the list of Group or user names 13 Check Full control box under Allow 14 Click OK 2 Double click on DRGUIInstaller jar file 3 Click Install button to proceed 4 If you will see message Cannot create Start menu Shortcu
3. FDR adjustment can be specified THE UNIVERSITY Nl of NORTH CAROLINA i at CHAPEL HILL CONFIDENCE ANALYSIS OPTIONS Confidence analysis refers to quantifying uncertainty in the pathway dose response curve This is performed only for pathways meeting the Pathway FDR threshold The pathway dose response curve is created by i selecting genes meeting threshold for inclusion For each of these genes a four parameter logistic curve is fit to represent the gene For the included genes bootstrap resampling of the gene expression and dose values is performed In contrast to the testing phase the confidence phase treats the entire expression profile and dose for each sample as a combined unit for bootstrap resampling ii For the observed data and each resample a pathway dose response curve is created either by a summing the individual dose response curves rescaled to the unit interval or b fitting a single logistic curve to all of the genes which have been rescaled to the unit interval P value threshold for inclusion gene specific p value threshold to select genes that are to be included in the confidence analysis Default is 0 1 Number of data re samples An integer value to specify the number of bootstrap resamples performed The entire curve fitting procedure to create the pathway dose response curve is performed for each bootstrap Pathway FDR or Bonferroni Holm threshold The criterion for
4. 9018 GO 009019 Figure 18 DR Pathway Analysis Results window Tree tab 20 THE UNIVERSITY 1 j of NORTH CAROLINA i at CHAPEL HILL 6 5 DR Path Detector Plot tabs Table Graph Tree DR Pathway Plot Change GO 0017085_ response to insecticide p 0 000002 standarized expression o o a o o d 1 0 0 5 0 0 log dose ShowEC5 Smoothed EB Pathway DR profile II 95 cr ShowECi0 Non Smoothed E Fitted curve of significant genes in the pathway W Homes ci J upper 95 cr Show EC50 Hide all Ec Figure 19 DR Pathway Analysis Results window DR Path Change tab DR Path Detector Change Up Down Plot tab contains information and graphical representation of the pathway dose response profiles Users can see the EC5 EC10 and EC50 values on the table and on the plot by selecting one of the radio buttons The EC values and 95 confidence intervals are in red Clicking on the EC buttons will also cause them to appear in the table of all significant pathways The shaded areas indicate extrapolation of results beyond the actual range tested As with any extrapolation these should be interpreted with caution Zooming ability is available and behaves in the same manner as Gene plots 21 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL Tatie Graph Tree DR Patnay Pot Change OR Pathway Pot Up DR Patnay Plot Donn GO 0017085_ _ response to Ins
5. DR GUI user need to have R and the latest Java installed on your PC NOTE R software should not be older than version 2 15 1 R software could be downloaded from many mirrors around the world You could find these mirrors here http www r project org Please select Windows operation system and then base as a subdirectory One can find Java software here http www java com en After you have successfully installed R and Java you need to download DR Pathway Analysis installation package The most recent version of DR Pathway Analysis is located here http comptox unc edu resources html Before installation make sure that you have Full Control over your R installation directory To check that right click on the R folder usually it s here C Program Files R Select Properties from menu that will appear Navigate to Security tab Click Edit button Select Users in the list of Group or user names UM Br AE Check Full control box under Allow 3 THE UNIVERSITY Nl of NORTH CAROLINA i at CHAPEL HILL 7 Click OK 2 2 Installation process Please unzip DR GUI archive to any place on your PC after you have downloaded DRGUI zip file Then open DRGUI folder and double click on DRGUlInstaller jar file The Installation window should appear DR Pathway installer will look for installed versions of R inside default locations and if you have administrative rights on your PC Java and R properly installed into defau
6. E Ee SERE EG SERE Ge ee REG Ge Ee GREG Gee WEE Ee Ge NE dee 13 6 Results RE ER ER OE OG RO ER OE GE ae 15 6 1 Categories pathways table iese ee ee Ee Re Ee Re ee AE Ge ee AA Ee ee ee ee ee ee 16 sel el N RE EE EE EE EE EE EE 17 Ete Mel es EO OE EE EE OE EE DE OE OE eee 18 E TNE TEE EE AE EE EE EE NE EE tate 19 6 5 DR Path Detector Plot tabs Eis Ese EERS RR casa ese nue lace Re de Rg Ge ek Ge ees vk aN vee ge Rg ee 21 2 THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL 5 1 About DR Pathway Analysis Dose response DR modeling is a key component of quantitative risk assessments When performing microarray studies of transcriptional dose response it is of interest to test the coordinated involvement of transcripts genes from known biological pathways or functional categories However existing gene set testing methods are not well suited for DR pathway analysis which requires careful control of false positive rates and fast DR curve fitting DR Pathway Analysis GUI was made for DR analysis with fast curve fitting procedures and the introduction of a pathway dose response profile A bootstrap procedure is used to obtain confidence envelopes for the pathway DR profile DR GUI is written in Java and for the testing phase uses ideas from the Significance Analysis of Function and Expression SAFE package Barry et al 2005 written in R R Development Core Team 2006 2 Installation 2 1 Requirements In order to use
7. The University of North Carolina at Chapel Hill Gillings School of Global Public Health Departments of Biostatistics and Environmental Sciences and Engineering Dose response pathway analysis for gene expression Graphical User Interface User Manual THE UNIVERSITY mm Il of NORTH CAROLINA i at CHAPEL HILL Contents 1 About DR Pathway Analysis cccssccccsssscecssssececsesseeecsesaececssaeeeesesaeeecessaeeecseauececseaueeseseaueeeseeaaeesenaas 3 2 oi Che a ss EA EE RE terre RE ee terrier GE Ede RE Ee N Ee ee EA Rai 3 2 1 Requirements EE NE EE N sbasceedesaccuecbivene saschecbescdeee 3 2 2 WistallatlOm dels ie OE AE IE N RE RE N ERE OaE Ea 4 2 2 1 Installation under Windows Vista esse esse se ee Ge AA Ge ee Re AA Ge AA Ge ee RR AA ee ee ee ge ee 5 Be MALAI WINGOW EE RE OE N RE EE EE 7 3 1 DR Pathway Analysis Setting Si sake Se SEER ENE ge EE ede BENE Ge GEE Gee GESE Ee a Gn Ge EES Re GR Ee de Resep ge GE ek dee 7 32 MAN EE RE EE RE EE ET EE EE ES 10 ERNS LE EE EE EE EO EE OE EE N OE aed 10 3 2 2 S tting AI TE EE EE degustaicentease Gegussaiendeeesd degassaecde 10 3 2 2 1 Advanced ie dii EE Ee Oe EE Oe N ED EE EE 11 3 2 2 2 Color Setting S corrr N EA NE OR EE EA ER N 12 EER Help TEEN OG EE RE OE ON NE GO ON EE OK 12 4 Input Data Formaat ESE EERS SES BE KS ER KEES Ee KEER EES BE ASe EE ARE Ee Se EE PES EG ee EE Ke Ee Ke 13 5 Running DR Pathway Analysis ie iss Ee EES Ee SAGE Ee VEE Ee SEGE Ee ES EE Ee Ee SER
8. bution function CDF for the ranked local statistics from a given category solid line A significant category will have more extreme associations to the response of interest than its complement resulting in a rightward leftward or bidirectional shift in the CDF away from the unit line dashed line The shaded regions of the plot correspond to the genes that pass a nominal level of significance empirical p values lt 0 05 by default A user can select SAFE plot representation by switching from ranked local statistic to simple local statistic To zoom in out the plot a user can use buttons or by rotating mouse scroll To move zoomed plot push and hold left mouse button and drag the plot right or left If you click on the gene symbol DR Pathway Analysis will open extended gene information in your default browser Gene symbol 4 Be OBS e Jea 2 p 8 GE Gene name on BEE 1368535 1376048 mouse over 1367 713733 71370332 1376075 71376140 71376093 1375250 71376145 21375188 1376139 1367880 1376023 21370058 213 13 1376023_at Rarg Category name Push and hold left description and mouse button Then category empirical p value drag the plot Ranked local statistics 1 250 1 500 Ranked Local Statistic 1 750 2 500 1 000 2 000 2 250
9. deciding which pathways should be included in the confidence analysis Setting this value too high will result in performing the confidence analysis step for a large number of pathways and thus will take a long time for computations to complete By default value is 0 05 BUTTONS Results button will show the results window on click Note that this button became enabled after you run DR Pathway Analysis Show input data button will show your input data file in a table format RUN button runs DR Pathway Analysis method with selected parameters THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL 3 2 Main menu BBB DR Pathway Analysis GUI EI gt File Settings Help Figure 6 DR Pathway Analysis menu On the main DR Pathway GUI window you will see a top positioned menu with three main items File Settings and Help 3 2 1 File Menu Figure 7 DR Pathway Analysis File menu e Print input data table prints the data in table format from selected data file e Save Project saves current project e Open Project allow user to load previously saved project e Open Recent Project gives user a list of recent projects to load e Exit closes DR Pathway Analysis 3 2 2 Settings Menu File Settings Help Advanced settings ee EE Figure 8 DR Pathway Analysis Settings menu e Advanced settings will open Advanced Settings window e Colo
10. ecticide p 0 000002 o a 9 ES 5 ir a co 0 6 N T oO gel D log dose I Fitted curve of significant genes in the pathway how EC10 Non Smoothed HB amp c E tower 95 Cr J upper 95 ci w EC50 Hide all EC Figure 20 DR Pathway Analysis Results window DR Path Up tab 22 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL GO 0017085 _ response to insecticide p 0 000002 50 Ue a 9 N 1 0 0 5 0 0 0 5 1 0 15 2 0 log dose ShowEC5 Smoothed EB Pathway DR profile I 95 cr gt Show EC10 Non Smoothed Le I Fitted curve of significant genes in the pathway W Moves ci J upper 95 cr Show ECS0 Figure 20 DR Pathway Analysis Results window DR Path Down tab Results can be printed or saved from the Results window 23 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL
11. f NORTH CAROLINA i at CHAPEL HILL 6 Results Results window will appear after DR Pathway GUI finishes with all permutations File 176 0007 0 015 ery long chain fatty acid me nos o a 0 0006 0 013 sponse to oxygen radical C R ct nab process E te maturation pateng ofe of blood vessels 64 o ooo o 0025 b nmo auo 034 EE Sa EE gee 2610 0008 0 016 EA EER 88 0 002 7 029 Jothelial cell proliferation on 23 0 00007 7 0028 itive regulation of cell mat tS 42 0008 O oos positive reauiaton of mesenc positive regulation of recepto Local statistic Score Error rate FDR BH Global statistic D Method express Threshold results by FDR 0 05 Figure 13 DR Pathway Analysis Results window Result window is divided by two logical parts Categories part and Specific category part Categories part consists of the categories table which we will describe more precisely below and Analysis parameters such as e Local statistic used for analysis e Global statistic used for analysis e Error rate e Method Express is the only option e Threshold results by FDR User could change this value by dragging the slider 15 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL 6 1 Categories pathways table The Categories table consists of categories that attain a certain level of significance Figure 14 shows significant results for categories that have empirical p values l
12. lt directories you ll be able to use the Automatic installation function If you don t have administrative rights on your PC you will need to install R into any folder under your user account where you have rights to read write modify execute files In order to install DR GUI you will need to use the Manual installation option One will need to click Select file button and select the folder where R was installed The next step is click Manual installation button S Instalation of DR GUI Automatic instalation You can use this option if you have read wright execute user rights over the default R installation folder G Program Files R R 2 15 1 Please dick the button to proceed ia Manual instalation Please use manual instalation if you don t have Administrator rights to read write execute on system folders or you have R installed into specific folder and you have rights to read write execute on the files in that folder Plese select the folder with your R version and then click Manual Installation button Select file Figure 1 Installer main screen DR Pathway GUI will try to download and install all necessary R libraries from the Internet so Internet connection is required at this stage During the installation you will see the progress window and occasionally the R progress window 4 mm THE UNIVERSITY of NORTH CAROLINA J at CHAPEL HILL Installing DR Pathway Analysis GUI
13. mXn matrix where appropriate normalization and other pre processing steps have been taken It should be noted that in the current version of DR Pathway Analysis missing values are not allowed in the expression data and must be imputed prior to analysis Example of the input data file Dose Response pathway Analysis GUI input data file format gene names gene symbols dose amount 0 0 001 0 01 di gene names gene symbol control477428063 cel control477428082 cel control477428465 cel 1367452 at Sumo2 10 93432083 10 91483514 10 90085231 1367453 at Cdca7 10 75105423 10 72111071 10 60042972 1367454 at Copb2 10 13433653 10 10070084 10 08669 dose id s expression matrix NOTE Raw data in the input file should be comma separated File extention should be csv 5 Running DR Pathway Analysis After you click RUN button main window will disappear and Status window will appear 13 THE UNIVERSITY mm Il of NORTH CAROLINA i at CHAPEL HILL Platform rae230a db version is 2 8 1 built with R version 2 15 1 Building GO BP categories from rae230a dbGO2ALLPROBES have C list finished safe express Categories completed 20 Figure 12 DR Pathway Analysis Progress window You can see how Status window looks like on Figure 6 In this window DR Pathway Analysis displays status of the current process You can halt execution by clicking Stop button You can clear status window by clicking Clear 14 THE UNIVERSITY o
14. np oasy 1 028 0 1373304 at 0 A Sys 0 676 0 1370332 at homolog D E segs presenilin 1 Ai cassette subfa asso o o be o nema a295 0 135 os o mmea 0 1375250 at P 0 1376145 at eukaryotic translation initiati Figure 15 DR Pathway Analysis Results window Genes table The Genes table is located under the Table tab and could consist of one or two tables On Figure 15 you can see Up regulated genes table versus Down regulated genes table Up regulated are genes with local statistic values more than 0 Down regulated are genes with local statistic value under 0 17 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL Each genes table contains the following information e Gene name e Gene local statistic e Empirical p value e Gene symbol e Gene description 6 3 Graph AO te eE 3 En a a worm 71370332 1376075 1376140 Eik 1375250 1376145 51375188 71376139 1367880 1376023 71370058 1368535 1376048 oom dd what 1 000 1 250 1 500 1 750 2 000 2 250 2 500 Ranked Local Statistic Ranked based Local statistic 18 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL Figure 16 DR Pathway Analysis Results window Genes plot The graph tab contains a plot for a single pathway We have proposed that the differential expression of genes be plotted as a SAFE plot Barry et al 2005 SAFE plots show the cumulative distri
15. ore accurate p values The default of 1000 should suffice unless the sample size is large gt 200 11 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL 3 2 2 2 Color settings p lt 0 1 lt p lt E 0 15 lt p lt H p gt 0 7 Step line color Plot background color Plot shaded area color left Plot shaded area color right Plot shaded area one sided Figure 10 DR Pathway Analysis Color Settings window 3 2 3 Help menu Figure 11 DR Pathway Analysis Color Settings window e About contains information about the DR GUI software e Input data format shows examples of the input data file structure 12 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL e Load example dataset loads example dataset of Rat expression data on the RAE230 platform for demonstration purposes 4 Input Data Format Data in the input data file should be separated using comma delimiter File extension usually should be csv Input data file should be in the following format First row sample names Second row through n row expression data Dose values should be on an original meaningful scale but will be analyzed on the log scale To avoid difficulties with log 0 zero values will be transformed on the log scale to be an increment x lower than log lowest positive dose where x is the average increment between successive log positive dose values The expression data should be in the form of an
16. r settings will open Color Settings window THE UNIVERSITY j of NORTH CAROLINA i at CHAPEL HILL 10 3 2 2 1 Advanced settings Min Category Size Max Category Size Use random begining seed Use constant begining seed Begining seed Number of resamplings Genelist cutoff value for Trend Fisher statistic Method Mead X Gradient Max iter Delimiter for data output Double check Comma TAB t Space Pipe I eg 1000 Figure 9 DR Pathway Analysis Advanced Settings window e Category size options allows user to set minimum and maximum category pathway size for the pathways to be included in the results e Randomization options allows user to set random seed or constant seed for the bootstrap resmapling With constant seed option selected you ll need to input sthe eed value and expect that you will obtain reproducible results e Permutation options these options are intended for future versions of DR Pathway e MLE Optimization options allows user to set options for MLE optimization for logistic curve fitting e Delimiter options allows users to set delimiter for the tables which are produced when user tries to save the result data table Usually delimiter is tab space comma or pipe e Safe express options allows user to set grid length to be used for approximating permutation p values Higher grid values produce m
17. sting pathways for a significant dose response relationship among numerous genes in the pathway 7 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL Method Currently express is the only option This is a mathematical approximation to permutation described in Zhou et al 2013 Other options may be added in future releases Global statistic Specifies the global statistic for a pathway gene set or category The statistic D is currently the only option It is a competitive statistic that compares the mean squared score statistic within the pathway to the mean squared score statistic among the remaining genes Local statistic Specifies the gene specific statistic Score is the current default Pathway Database GO MF GO BP GO CC specify the three Gene Ontologies GO CC will form categories from Cellular Compartment ontology while GO BP and GO MF will work with Biological Processes and Molecular Function respectively It is important to note that in the hierarchical structure of the GO vocabularies a gene category is generally thought of as containing the set of genes directly annotated to a term and also to any terms beneath it in the ontology Correction method Specifies the method for computing false positive error rates accounting for multiple comparisons across numerous pathways A Bonferroni Bonferroni FWER Holm s step up Holm FWER and Benjamini Hochberg step down Benjamini amp Hochberg
18. t 0 25 For each category the category name and number of annotated genes in the dataset is displayed along with the p value FDR value and Category description Specific category information including genes contained in selected category will appear when user clicks on any category Processing times for specific results depend on category size Results for smaller categories will appear immediately while results for bigger categories will take some time but only the first time a user click on the category W Results Name Local statistics Empirical p value Symbol Description Si farsa oooem pry sz pia DEER sk padwredes Local statistic Score Error rate FDR BH eier ass nama senate Global statistic D Method express 0 025 Threshold results by FDR 0 05 Figure 14 DR Pathway Analysis Results window 16 THE UNIVERSITY of NORTH CAROLINA i at CHAPEL HILL 6 2 Genes table rif 0 026 0 1374784 at S tan S ker E 3 T S A Rag o ba o bs o isreas at beinocacdreceptor gamma Nel bees oy o ossa heurofiameng ight polypep hea bay O bs O Jiggssanet OO hepsnAasperepepbdese RS os E E E 1376048_at 1370081_a_at TE vascular endothelial growth so te carrier family 11 prot d 3 2 212 0 027 1368387 at Shydroxybutyrate a So ass SS OCOMSSCS i TARTS C iad acyltransferase 1 cd se ba OO hersstet kapegs
19. ts will be created in R directory the program will not create Desktop shortcut and Start menu items But you ll find the shortcuts inside your R directory usually it s here C Program Files R SAFEGUI You can avoid changing user rights by switching off UAC before installation But we do not recommend you to do this THE UNIVERSITY Il of NORTH CAROLINA i at CHAPEL HILL 3 Main window 3 1 DR Pathway Analysis Settings EA DR Pathway Analysis GUI Testing Method Global statistic THE UNIVERSITY of NORTH CAROLINA Local statistic Pathway Database i rillings Sc Correction method jamini Gillings School of Global Public Health Department of Confidence analysis Biostatistics P value threshold for inclusion 01 ooo O Number of data re samples Pathway FDR threshold oos Figure 3 DR Pathway Analysis main screen On Figure 3 you can see the DR Pathway Analysis main screen which allows you to set input parameters for your data analysis First of all you need to set a path to the input data file To do that click on the Select file button File browsing dialog should appear Browse to your data file Click Open Select following parameters when you have selected input data file e Microarray platform name of a Bioconductor annotation package to build gene pathways that corresponds to the array platform that was used to collect the data TESTING OPTIONS Testing refers to te
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