User Manual
Contents
1. GIPro User Manual 10 2 4 Heat maps Heat maps allow visual representation of the interactions between genes or complexes There are multiple types of heat maps that can be generated Complex network heat maps Gene heat o Gene heat map select two or more complexes and click the MM Gene heatmap button in the panel to generate a heat map of individual interactions between genes in the complexes The labels beside gene names indicate which complex the genes belong to The gradient of the color indicate the magnitude of genetic interactions o Complex heat map select two or more complexes and click the BA complex heatmap ton in the panel to generate two heat maps one with the average positive score between complex pairs and similarly with the average negative score in the other The gradient of the color indicate the magnitude of enrichment Please go to TreeView Menu to change Settings gt Pixel settings gt Contrast to adjust the contrast in order to display the gradient Expanded view heat maps o Raw interaction data Select one or more genes and click B Display raw interaction data in the panel to generate a heat map of interactions between the selected gene s and all other loaded genes o Sign patterns Select two or more genes and click IM Display sign patterns ip the panel to display two heat maps showing common interactions for the selected genes with same signs all positive2. Display sign patterns or Add correlation edges Allows cutoff to be applied when using both features GIPro User Manual 19
3. gt Export ColorBar to Image Export data matrix To export a generated heat map as a data matrix select Export gt Save data Under Field s to print select YORF for gene identifiers or GID for gene names Choose the save path file name by clicking Browse Finally click Save to export the data matrix Note for more details on using TreeView see hitp jtreeview sourceforge net Technical details 4 1 Cutoff analysis A genetic relation is considered positive if it s score exceeds the positive cutoff value and is considered negative if it is below the negative cutoff The positive and negative cutoffs are specified in the Cutoff Parameters panel and are based on p value percentile or user specified custom cutoffs 1 p value based cutoff The background scores are approximated as a normal distribution then identifying true genetic interactions amounts to finding outliers to the background distribution For each score x in the dataset we determined its probability of belonging to this background distribution by calculating a normalized score z as follows _ xj mM Xi m a GTR IORhnorm where m is the median and IQR is the inter guartile range between the first and third quartile IQRnorm 1 34898 is the inter quartile range of the standard normal distribution with a mean of 0 and a standard deviation of 1 This version of z score is more robust than the conventional z sc
4. or green if a negative correlation and labeled with the Pearson correlation coefficient r If a correlation edge is not added there is insufficient data to generate it ardor ACTA Tip to add correlation edges above a specific threshold hold down Ctrl while clicking and EAF1 specify a positive and negative cutoff 0 204 Correlation tables matrices An alternative way of viewing correlation data is Correlation for ACT1 EAF3 EAF6 EAF7 through a table or a pairwise matrix Select two Correlation P value tables or more genes in the expanded network view Correlation ACT1 EAF3 EAF6 Correlation tables aa Ta Merr i ery EAF3 0 144 0 309 and CEK i TWO EAF6 0 122 0 309 matrices are generated the top one showing EAF7 0 051 0 219 0 588 pairwise Pearson correlation coefficients and he AE Tara Tara i are E ACT1 l 0 044 0 79 the bottom one showing pairwise p values amaan 0 044 4 as representing the significance of the pairwise EAF6 0 79 0 034 l W l EAF7 0 711 0 035 0 correlation A hyphen represents pairs with no data and an asterisk represents pairs with 225 List pairwise genes GA Export tables insufficient data for a correlation coefficient p GIPro User Manual 12 value To export both matrices click _ amp Export tables and select a save directory The matrices are saved in a tab delimited format to the specified directory To see a list of pairwise genes
5. or all negative in one heat map and alternating signs positive negative or negative positive in the other Tip to add thresholds for same and alternating sign interactions hold down Ctrl while clicking Note array genes that are not interacting are not displayed Once the heat map is generated click and drag to select one or more rows to be viewed in more detail View Status 2 genes kalai 16 arrays sel 4 Genes from Cy Azen fenna I gain Usage Hints use arrow keys to move selection click and drag to select genes hold shift to select arrays too Click ang drag to select Detailed view GIPro User Manual 11 To modify the contrast of the pixels navigate to Settings gt Pixel settings and adjust the contrast sidebar Note o Hierarchical clustering is applied to heat maps where needed For more details see the technical details Section 4 1 o All heat maps are generated using TreeView For more information on how to use TreeView visit http jtreeview sourceforge net 2 5 Pearson correlation coefficient Correlation edges In order to see how well a pair of genes is correlated a Pearson correlation coefficient VID30 0 048 EC63 can be generated In the expanded network view select two or more genes and choose oF Add correlation edges to add correlation edges for all possible pairs of selected genes to the current subnetwork These edges are dashed colored red if a positive correlation
6. Positive cutoff 0 0 Begin analysis Cancel 1 3 File input 1 File input Genetic interactions file ummer 2011 Sample data sample genetic_interactions tab Browse Protein complex file chive Summer 2011 Sample data sample complex_file tab Browse Physical interactions file er 2011 Sample data sample physical interactions file tab Browse Name map file optional Archive Summer 2011 Sample data sample name_map tab Browse Loadfiles Clearall Load example data _ Filter interactions with at least one member in a complex There are three required and one optional files used for this plugin The purpose of each file is described below 1 Functional relations file This file is used to specify genetic interaction scores between pairs of genes Optionally a p value can be included for each relation In this case relationships that do not meet the p value cutoff score of 0 05 are ignored GIPro User Manual 4 Delimited by tab Header none Columns 1 Gene A 2 GeneB 3 Score 4 P value optional Sample file without p value YBLO75C YDL133W 0 00087 YDLO32W YBLO22C 0 01625 YERO54C YBLO12C 0 02615 Sample file with p values YBLO75C YDL133W 0 00087 0 012 YDLO32W YBLO22C 0 01625 0 005 YERO54C YBL012C 0 02615 0 072 2 Protein complex file This file defines protein compositions for complexes by listing complex IDs and protein ORF names that belong together Delimited
7. User Manual Oo ANP Wodak Lab SickKids H THE HOSPITAL FOR Research Institute SICK CHILDREN http wodaklab org support wodaklab org Table of contents Introduction 1 Getting started 1 1 Installation 1 2 Initiation 1 3 File input 1 4 Parameters 2 Displaying and visualizing data 2 1 The protein complex network 2 2 Expanding protein complex views 2 3 Search and display 2 4 Heat maps 2 5 Pearson correlation coefficient 2 6 Histograms 3 Export information 3 1 Complex enrichment information 3 2 Pearson correlation matrices tables 3 3 Heat maps 4 Technical details 4 1 Cutoff analysis 4 2 Enrichment analysis 4 3 Hierarchical clustering 4 4 Pearson correlation coefficient 5 Shortcuts and tips GIPro User Manual 2 CO BH OD WW WW U Introduction The advent of high throughput technologies in proteomics and genetic screening has yielded hundreds of protein complexes and millions of genetic interactions respectively in the budding yeast However analysis of large scale datasets continues to be overwhelmingly challenging for biologists despite recent progresses in systems biology For example how can one find out which complexes are enriched with aggravating alleviating genetic interactions How can one use genetic interactions to determine the functional relationships between two complexes How can one visually inspect the distribution of genetic interactions and physical interactions among protei
8. a p value an algorithm is run to determine the Gaussian positive and negative solid cutoff Left ta values 2 Percentile cutoffs specifying a value such as 10 percent gives a Custom score cutofi positive cutoff value at the 90th percentile and a negative cutoff value at the 10th percentile of the relation scores Note that the GIPro User Manual 6 value must be non negative 3 Custom score cutoffs a user can enter their own positive and negative cutoff values which can either be positive or negative values themselves as long as the positive cutoff is larger than the negative As different values are entered the custom positive and negative score cutoffs are updated accordingly These are the actual cutoffs that will be used and have a direct impact on how the program is run Refer to the technical details section for details Note with the use of large data sets the positive and negative cutoffs may take a few seconds to update Enrichment analysis parameters 1 Multiple testing corrections A false discovery rate is entered This value is used to compute the p value cutoff used to filter the outputted data A Fishel smaller FDR value will generally Simulation result in a smaller p value See technical details for more information 3 Enrichment analysis Multiple testing correction Simulation Parameters 2 Number of Trials the number of simulation trials performed for each complex See the technical d
9. by tab Header yes 2 column header Columns 1 Complex ID name 2 ORF in complex Sample file Complex name Gene name TRAPP complex YERO54C TRAPP complex YDLO33C Rpd3l complex YBLO22C 3 Physical interactions file This file contains physical interaction scores for pairs of proteins Delimited by tab Header none Columns 1 ORFA 2 ORF B 3 Score Sample file GIPro User Manual 5 YBLO75C YDL133W 0 00087 YDLO32W YBLO22C 0 01625 YERO54C YBL012C 0 02615 4 Name map file optional This file contains a mapping between ORF names of proteins to gene names Delimited by tab Header yes 2 column header Columns 1 ORF name 2 Gene name Sample file ORF name Gene name YDLO33C SLM3 YERO54C GIP2 This information can also be found via the plugin Roll over the file text fields or Browse button for more information You may also check the Filter interactions with at least on member in a complex check box This is useful if a large relations file is being loaded If this box is checked only those relations where at least one of the genes belongs to a complex will be loaded 1 4 Parameters Cutoff calculation parameters The solid positive and negative cutoffs are used to determine if an interaction is positive 2 Gi score cutoff parameters or negative see technical details section for more information There are three ways to specify them ski 1 P value based cutoffs by specifying
10. complex means the complex is enriched with positive interactions green represents enrichment with negative interactions and blue represents an enrichment of both positive and negative genetic interactions Complexes have a default dark grey color if they are not enriched with either type of interaction Edges edges exist between a complex pair if their between complex interactions are positively or negatively enriched o Color a red edge represents enrichment with positive interactions while green represents enrichment with negative interactions Double edges are created between complex pairs who s between complex interactions are enriched with both positive and negative interactions o Thickness proportional to the significance of the enrichment p value The more significant the thicker are the edges 2 2 Expanding protein complex views By clicking the xpand complexes _ button in the left panel a network of interacting genes contained in the selected complexes is created Genes belong to a complex are laid out in a circle and the relative position of the circle is determined by its location in the original complex network Some of the network properties are listed below GIPro User Manual 8 Nodes o Node color represents the complex that the gene belongs to Nodes with thick border indicate that the proteins are shared by multiple complexes o Node label is in italic font if no genetic interactions exist in
11. etails section for more information The recommended default is 1000 Note This value must be a positive integer 3 Trial for Each Complex This check box should be checked if the user would like simulations to be re run for complexes with the same number of interactions otherwise they share a distribution Usually this box should remain unchecked unless the number of trials parameter is small To begin computations click Begin analysis Computation progress and details will be displayed in progress dialog and when finished a protein complex network will be created in Cytoscape After the complex network is displayed in Cytoscape if the user decides to change the parameters click Adjust parameters on the bottom left of the Cytoscape panel to bring up the update parameters panel to enter new parameters then click Apply changes to restart the analysis The current network will be destroyed and a new complex network will appear GIPro User Manual 7 Displaying and visualizing data 2 1 The protein complex network In the generated complex network the nodes represent complexes and an edge exists between two complexes if and only if there exists a significant enrichment of positive and or negative interactions Nodes o Size scaled according to the number of genes within the complex using a logarithmic scale o Color represents whether the complex is enriched with genetic interactions A magenta
12. ficance and 1 representing negative significance or a hyphen if no significance exists File saved as givenName_ matrix txt Sample output file complex_name complex1 complex2 complex3 complex1 1 1 complex2 1 1 complex3 1 1 3 2 Pearson correlation matrices tables Correlation P value matrices When exporting correlation p value matrices two files are saved GIPro correlation matrix txt GIPro pvalue matrix txt In the following format gene name RPL38 HHF1 MSO1 RPL38 0 823 0 052 HHF1 0 823 i MSO1 0 052 Ranked pairwise correlation p value list When exporting the pairwise pair wise list generated one tab delimited file is saved GIPro rank list txt In the following format GeneA GeneB r p value N RPL38 HHF 1 0 823 0 032 56 HHF1 MSO1 0 997 0 001 89 MSO1 RPL38 0 012 0 572 13 3 3 Heat maps Export to image You can export a generated heat map as an image by selecting Export gt Export to Image in the heat map window Next to Total size adjust the dimension of the heat map being exported choose a save path file name by clicking Browse and choose an image format from the drop down menu Finally click Save to export the heat map as an image GIPro User Manual 15 Note to exclude gene and or array dendogram from the exported file deselect the checkboxes Gene Tree and or Array Tree Similarly the legend can be exported as an image by selecting Export
13. find significantly enriched complex interaction edges 4 3 Hierarchical clustering When generating complex or query heat maps hierarchical clustering is used to group genes or complexes into groups or clusters such that those within a cluster are closely related to one another The metric used in the clustering is the Euclidean distance and an average linkage criterion For more information see the homepage of the algorithm at http function princeton edu WCluster 4 4 Pearson correlation coefficient When generating a Pearson correlation coefficient for a pair of genes genetic interaction data of both genes is used Only common interactions between both genes are considered during the calculations For example if geneA interacts with genex geneY genez and geneB interacts with geneX geneY only the pairwise scores of geneX and geneY are used GIPro User Manual 18 Shortcuts Shortcut Description Ctrl 1 Generate subnetwork using custom list of one or more complex Ctrl 2 Generate gene heat map using custom list of two complexes Ctrl 3 Generate complex heat map using custom list of two or more complexes Ctrl 4 sort complexes in the tree by the number members in the complex in descending order Ctrl 5 Sort complexes in the tree by the number members in the complex in ascending order Ctrl 6 sort complexes in the tree by the number members in the complex in alphabetical order Ctrl down click When clicking
14. information on the distribution and p values generated for positive and negative genetic relations for every possible complex pair in a spreadsheet format Only complex pairs with significance are displayed File saved as givenName_ between txt Columns 1 2 3 4 5 Complex1 first complex in the complex pair Complex2 second complex in the complex pair Number of pairs the total number of possible pairs between the complexes e g if Complex1 has 3 genes and Complex2 has 4 genes the total number of pairs is 12 Note If the two complexes share a gene the gene self loop is not counted For example if Complex1 contains genes 1 2 3 and Complex2 contains genes 3 4 The total number of pairs is 3 x 2 1 5 an edge is subtracted since gene 3 belongs to both complexes Actual Number of Pairs number of pairs that have a score in the functional relations file This is the number used to perform analysis Pos Neg Zero Relation the number of positive negative and neutral relations in the complex pair GIPro User Manual 14 6 Pos Neg pvalue is calculated during the statistical analysis for the positive or negative interactions See technical details for more information 7 Significance Denotes the significance of the complex pair 3 Complex enrichment matrix this file contains the significance of interactions between complexes These are displayed in matrix form with a value of 1 representing positive signi
15. ive interaction and negative relations given at least one positive interaction in each complex is generated Based on the type of enrichment selected one of the following is performed 1 2 Fisher Exact Test This statistical method compares the number of positive interactions within a complex with the number of positive background interactions to generate a right tailed p value for the complex s positive interactions Similarly a right tailed p value for the complex s negative interactions is generated Note p values for positive and negative interactions in a complex are only calculated when there are at least one type of that of interaction Simulations The simulation creates distributions for the number of positive and negative J relations within each complex or between a complex pair The number of possible pairs n for a given complex is calculated Given the number specified in the number of trials text field m the algorithm makes n draws from the Functional relations file scores m times The number of times the positive relations drawn are greater than or equal to iis recorded and divided by m to generate an empirical p value signifying the likelinood of observing positive interactions by chance A p value is generated similarly for negative interactions Note If the Run trials for each complex check box is unchecked the algorithm will re use distributions for complexes with the same number of relatio
16. l datasets are being used See http cytoscape wodaklab org wiki How to increase memory for Cytoscape for more information on how to increase Cytoscape memory 1 2 Initiation After Cytoscape starts click Plugins gt GIPro and a Wizard will prompt the user for input files and other parameters Fig 1 Input screen used for specifying data files and parameters used by the plugin GIPro User Manual 3 BAD GIPro GIPro About T Need help Hover over items for more information or get the User Manual 1 File input Genetic interactions file ummer 2011 Sample data sample genetic_interactions tab Browse Protein complex file chive Summer 2011 Sample data sample complex_file tab Browse Physical interactions file er 2011 Sample data sample physical_interactions_file tab Browse Name map file optional Archive Summer 2011 Sample data sample name_map tab Browse Load files Clearall Load example data _ Filter interactions with at least one member in a complex 2 Gi score cutoff parameters 3 Enrichment analysis P value based cutoff recommended Multiple testing correction Left tail cutoff 0 025 False discovery rate 0 05 Right tail cutoff 0 025 Fisher exact test recommended i Simulations Percentile cutoff lals OOO Left tail percentile 5 Number of trials 1 Run trials for each complex Right tail percentile 5 No enrichment Custom score cutoff Negative cutoff 0 0
17. ns to expedite the algorithm For example if a complex has 9 proteins and 6 positive interactions and the number of trials specified is 1000 the program randomly draws 36 9x8 2 divided by 2 to get rid of duplicate edges relations from the functional relations scores 1000 times and counts the number of positive interactions each time to generate distributions for the number of positive and negative relations The number of times that 6 or more positive interactions are observed within the 36 draws is recorded and an empirical p value is generated by dividing this number into 1000 The same is done for negative interactions GIPro User Manual 17 Once the p values are generated a within complex p value cutoff is calculated which complexes must meet in order to be considered statistically enriched This is done using the false discovery rate specified in the enrichment parameters Ihe within complex p values are arranged in increasing order and the maximum index i is found such that for all indices smaller than i q Pvalue lt F DR va UE Vi where M is the total number of p values and FDR is the specified false discovery rate The corresponding p value is then the within complex p value cutoff If the positive and or negative p value for a complex is below the p value cutoff the complex is considered significantly enriched with that type of interaction The procedure above is repeated for between complex interactions to
18. ns within one or more complexes simultaneously and make sense out of it The GIPro Plugin for Cytoscape is developed to analyze large scale quantitative genetic interaction data statistically to identify functional relationships between genes and between protein complexes and displays results in Cytoscape Data tables heat maps and histograms are optionally generated for further analysis As an example of application of the GIPro plugin yeast COG complex Conserved Oligomeric Golgi complex is found to be enriched with both aggravating and alleviating interactions in the enrichment analysis by integrating genome wide genetic interaction data with protein complexes A detailed inspection of the genetic interactions within this complex indicate that it may consist two modules consisting of COG1 to COG4 and COG5 to COG8 respectively as aggravating interactions exist between these two modules while alleviating interactions occur within modules This modular decomposition of COG is in good agreement with existing morphological and biochemical evidence This example demonstrates that a detailed analysis of Gls can reveal fine functional differences between modules of the same complex Getting started 1 1 Installation Place the GIProPlugin jar into the Cytoscape plugins folder to install The plug in is automatically initialized when Cytoscape starts Java s default memory should be increased with vm arguments when using this plug in unless smal
19. ore defined on mean and standard deviation in the sense that median and inter quartile range are less susceptible to the impact of true interactions which always lie at the tails of the distribution thus provide a more accurate definition of the background distribution Each z score corresponds to a p value in standard normal distribution Right tail and left tail probabilities can be specified separately Based on the p value entered a z score is calculated for each tail and the genetic interaction score cutoff that corresponding to the z score is determined 2 Percentile cutoff The scores are sorted in rising order Left tail and right tail percentiles specify the bottom and top x percent of the entire data respectively The value for x can be different for each tail The percentiles are translated into scores internally by the program 3 Custom score cutoff GIPro User Manual 16 Negative cutoff and positive cutoff will be applied to the scores directly Note O lf a fourth column containing the interaction p value is included in the Functional relations file the p value must be greater than 0 05 to meet the filtration requirements A complex is only considered if more than half its genes partake in genetic interactions 4 2 Enrichment analysis In order to determine whether complexes are significantly enriched with positive negative or both types of interactions a p value for the number of positive given at least one posit
20. the provided data for that gene or in normal font otherwise Edges o Color the type of interaction occurring between genes Red edges represent positive genetic interactions green edges represent negative genetic interactions and blue edges represent physical interactions Thickness of red and green edges is proportional to the score of genetic interactions Note with more than 12 complexes expanded you may start to see duplicated colors GIPro User Manual 9 2 3 Search and display lt 4 Editor Filters GIPro Complex network actions 4 Expand complex es Action buttons used to perform actions on the current network IM Gene heatmap Ba Complex heatmap il Histogram 7 Export results Information CEN PETA Complex 7 Nodes and edges information pane displays information of any node or edge upon its selection 4 positive relations p value 0 00673388 15 negative relations p value 0 10 non interacting pairs Complex Gene Tree Tree pane allows navigation of complexes and their genes will be highlighted to reflected gene or complex selections Gene Search Gene search pane used to search for a gene in a complex or expanded network The corresponding complex and gene node if available will be Adjust Parameters UserManual selected when searched l Adjust parameters Modify your cutoff and enrichment parameters here
21. their correlation p value and number of interactions used in the List pairwise genes adi calculations N click ee This list Can BE Display sign patterns Z Export list Sort by r HH be sorted by selecting a field from the drop down GeneA GeneB Correlati P value Size N menu or filtered using the filter text field at the tn gt an a EAF3 EAF7 0 219 0 035 69 bottom of the window To filter enter one or more ACTI EAF3 0 144 0 044 141 A ACT1 EAF7 0 051 0 711 121 gene name separated by a whitespace to show ACT1 EAF6 0 122 0 79 46 pairs containing the entered gene s To generate a heat map of sign patterns between both genes click Mil Display sign patterns See section 2 4 Clicking TZ Export list allows you to export the current list as is to a tab delimited text file to the specified directory Filter genes For more details on how the Pearson correlation coefficient is calculated see technical details 2 6 Histograms Click the LilliH stes am_ button in the panel to generate a histogram of all between and within complex interaction scores Click and drag your mouse to zoom into the histogram ar hinog aen An Genetic interaction scores within complexes Genetic interaction scores ra complexes Zoomed in Click and drag i l _ rar m sama hal ad Lon Score To export them as an image click the Export to image button and specify the save direc
22. tory GIPro User Manual 13 Export information 3 1 Complex enrichment Click the 9 Export results button in the panel to generate files containing information about the enrichment select the files you wish to output and the Export selected complexes only checkbox to export results on the selected complexes in the protein complex network The three files that can be outputted are 1 Within complex enrichment file this file contains information on the p values generated for positive and negative genetic relations for every complex in a spreadsheet format File saved as givenName_ within txt Columns 1 2 N Rd Bias N 8 N Name indicates the name of the complex in question Number of Genes indicates the number of genes in the complex as stated by the complex file Actual Number of Genes indicates the number of genes in the complex that are also in the functional relations file Full List gives a total list of genes Interacting List gives a list of the genes also in the genetic relations file Interactions total number of positive negative and neutral interactions within the complex Pos Neg Zero Relation indicates the number of positive negative and neutral relations in the complex Pos Neg pvalue is calculated during the statistical analysis for the positive or negative interactions See technical details for more information 2 Between complex enrichment file this file contains
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