MetScape 2.3.2 User Manual
Contents
1. Entrez Gene ID Human 6880 al ig 12 15 18 21 det Entrez Gene ID Rat 373541 Symbol TAF9 TAF9 RNA polymerase IL TATA box binding protein TBP associated Niomega ADE Hs factor 32kDa ey Like Entrez 6880 cs cis c10 13 4 ANIMATION OF DATA Create an animation of the data to see how it changes over time and across treatments Building an Animation 1 Create a subnetwork for example create a subnetwork for the TCA Cycle See the Concept Selection Data in the Data Panel section for how to do this Note Itis not necessary to create a subnetwork before doing data animation the animation can be done on any network 2 From the Plugins menu choose MetScape gt Animate data Manage Plugins Update Plugins Advanced Network Merge MetScape New Session MiMI Plugin Animate data MIMI Plugin About MetScape Start BINGO 2 42 Register MCODE Shortest path d Agilent Literature Search ClueGo 3 A new window pops up titled Initialize Coupled Animation of Multiple Data Columns 4 Column headings from your data are listed on the right side of the new window Determine how many animations number of rows and data items per animation Page 26 of 35 number of columns you will need Enter this information into the Rows and Cols boxes If you change these numbers from the default click on Reconfigure Layout an Initia2. Option 2 Load an experiment file Use to load experimental data to visualize and explore compound networks over time or in varying experimental conditions The input can be an Excel comma or tab delimited file MetScape allows users to load three types of files compound file gene file and concept file Each type is optional e g you can load only compounds only genes only concepts or any combination of the above Compound File The compound data file must meet the following requirements e The first row must be a heading row in which o The columns in the first row are column headings to label the data e All other rows contain experimental data in which o The first column contains KEGG Compound IDs or names o The remaining columns contain experimental data Multiple experimental values are permissible in the same spreadsheet Below is a portion of an example compound file with Compound IDs significance values and fold change values Page 9 of 35 A B C 1 Fold Change P value 2 0 51 0 0061 74265 3 1 14 0 646801757 0 99 0 970303452 2 12 0 0660528 5 6 0 91 0 1153 7626 T 0 99 0 904658012 8 0 935 0 574835153 9 1 09 0 8091 72269 10 COO 0 56 0 100 724407 11 C0007 1 36 0 189989302 12 000049 0 92 0 755 789133 13 CO0054 0 46 0 186707182 14 C0006535 1 22 0 805151076 15 c00073 0 80 0 356405722 Gene File The gene file must meet the following requirements e The first row must be a heading row tha
3. Reaction pathway Pathway involving reaction Reaction reversible T reaction is reversible F reaction is non reversible Reaction rid Reaction identifier Type Node type zene enzyme reaction or compound canonicalName Node name that is shown on node label Gene locations Subcellular location Please note that the reactions shown in the Reaction equation attribute display all compounds while the networks built from the list of input genes compounds show only main compounds see Ma et al Mol Syst Biol 2007 3 135 for details Pathway specific networks show all compounds Below is a table of available attributes for each edge A B Attribute Description fa Enzyme names Enzyme name Enzyme ecnums Enzyme Comission number Reaction equation Equation for reaction Reaction locations Reaction subcellular location EHMN database Reaction pathway Pathway involving reaction Reaction reversible T reaction is reversible F reaction is non reversible Reaction rid Unique identifier for reaction canonicalName Common name interaction Type of interaction 5 Select compounds and or edges in the graph to view their attributes in the Data Panel Page 19 of 35 82 112 142 iati hA ee jealatraanoyLCoA jeno pea mg Pl nel Re T e F C gt Zy F iT i at tein rir E iii iiis cis TO H doocosapeantaancyHooA eicosaZE AZ 112 2 peaniaanoybooA ie Aikaan cis 15 ta Ee a m CE4812
4. Biosynthesis of unsaturated fatty acids el cyde Citrate cyde TCA cyde 25 ytokine cytokine receptor interaction 132 DNA replication 30 Node Attribute Browser Edge Attribute Browser Network Attribute Browser Concept 4 The Reapply Selection button will re select the last selected concept in the graph If you select a concept and then select off of it clicking the Reapply Selection button will re select that concept The concept information can be saved as a file To save this information 1 Under Concept Filter tab click the Save Concepts button 2 Anew window pops up asking where to save the file Choose the desired location 3 Click Save The file saves as a CSV file that can be opened with most text editing or spreadsheet programs The file format is identical to the input concept file Page 24 of 35 Neth res of Citrate cyde TCA cyde 25 Cytokne cytokine receptor interaction METSCAPE RESULTS PANEL ADDITIONAL NODE INFORMATION Additional information can be obtained for each node by double clicking on it To get additional information about any compound gene reaction or enzyme 1 Double click on the node of interest 2 On the right side of the screen the MetScape Results Panel will show up F 3 Use the scroll bar or click on the undock icon to see all the data Page 25 of 35 ois cis 2 12 154824 Gene N B 1 7 won S 3
5. Cytoscape website at http cytoscape org 2 After Cytoscape is installed start the application 3 To install the MetScape plugin select Plugins gt Manage Plugins from the Cytoscape menu 4 Under Available to Install click the plus sign to expand the Integrated Analysis group Page 5 of 35 Enter key words to search Core 20 Network and Attribute I O 1 Network Inference 1 Available for Iyi 316 ngata import 33 nemer 2 Integrated analysis 32 Data visualization 20 Other 72 Scripting 30 Clustering 29 Network generation 30 J Graph analysis 20 Interaction database 20 Ontology analysis 10 Utility 18 it L Messages 5 Find and click the most recent version of the MetScape plugin 6 Click Install 7 When installation is complete click Close A MetScape option is added to the Cytoscape Plugins menu Registration MetScape is a free program We ask you to register because it helps us to keep track of the number of downloads Your information will be stored in a secure database and we will not share it with anyone We may send you infrequent e mails about future MetScape releases You may also choose to decline the registration If you decide to register later the registration window can be found on the main MetScape menu Note This plugin requires Cytoscape 2 8 to run correctly ENTERING DATA 1 To begin a Cytoscape session with the Me
6. to the appropriate view size zoom the same way you do with any Cytoscape graph 2 On the Animation Controls window click on Realign All This brings all the treatment graphs to the same view Page 28 of 35 4 DataSeries 01 Manipulating the animation bar chart 1 3 A The bar chart in the Animation Controls window shows the range of the data and its frequency The range of measurements are on the x axis while the frequency with which they occur in the experimental data set are on the y axis Click on a bar in the bar chart and a vertical line slider will show up on the bar chart The slider can be moved by clicking on the bar chart and dragging the line When moved the values shown in black are also shown in black in the graph and the set of the color scale is reset The selected portion of the graph determines what the animation will show This allows outliers to be removed if desired The color range can be changed by clicking the dropdown arrow next to Red Blue Select Color Range Redal J Red Blue Page 29 of 35 Playing the animation To play the animation click Play on the Animation Controls window Colors change within the three treatments showing changes over time To stop the animation click Stop on the Animation Controls window o eae DataSeries 11 rat ito the network view ynto the source node then click on d edges specified in S
7. type before the attribute ex Compound name Enzyme name The available attributes will depend on the type of network created Compound data Treatment _t p Compound formula E Compound mw 4 E Compound smile J E Compound synonyms d E Compound name 4 4 F 4 E Enzyme ecnum Enzyme name Ml Gene data we 1 fnild rhanna 4 il 4 When you are done click anywhere outside of the list and the table will be populated with the appropriate data Page 18 of 35 Below is a table of available attributes for each node type Attribute Description kA ID Unique identifier required by Cytoscape Compound approximateMWw Compound Molecular weight integer data source EHMWN database Compound casnum Chemical Abtracts Service compound identifier Compound cid KEGG compound unique identifier Compound file _ name Rows with data specific to your experiments Compound formula Molecular formula Compound mw Molecular weight Compound name Compound name Enzyme ecnum Enzyme Comission number Enzyme name Enzyme name Gene file_name Relative gene data values Gene column_name Gene data p value Gene description Gene description Gene human geneid Human gene identifier Gene symbol Official gene symbol Gene geneid Ortholog gene ID if the input organism is not human Reaction equation Equation for a reaction Reaction locations Reaction subcellular location EHMN database
8. C41 H60N7017P38 CE5114 C41H62N 7017P35 CE5159 C45H 6N 70O17P35 REE Coase ft RE3644 C45HT4N7TO17TP3S RE3104 Node Attrbute Browser Edge Atibute Browser Network Atvbute Browser ConceptSelecion Notes e Some attributes such as Formula Mass and Smile apply only to compound nodes while others such as Enzyme Pathway and Reversibility apply only to reaction nodes e Other plugins used earlier in your Cytoscape session may leave behind attributes that don t apply to MetScape compound or reaction data Rearranging attributes in the data panel To reorder attributes in the Data Panel click on an attribute column heading and drag it to a new location Page 20 of 35 Compound formula Compound name lt 7 om M I aM ae C43H64N7017P3S trans cis cis cis cis 2 10 13 16 19 des a a C29H480 14 Demethyllanosterol Node Attribute Browser Edge Attribute Browser Netwo as gt Compan re RO1218 a CE4852 trans cis cis cis 3 Node Attribute Browser Edge Attribute Browser Network Attribute Browser Concent Se To sort by an attribute click the attribute column heading to sort in the reverse direction click again eee ge 8z memEBASN U O CAH62N7017P35 OO an i NGHINOZ o osphoribosy 5 amino midazolecarbora ogor i OE Edge Attribute Browser twork Attribute Brows Node Attribute Browser Pathway filter in the data pan
9. IF format to create a node jin an edge and specify the source on the target node to finish the MANIPULATING NETWORKS Expanding a network From a compound node you can expand the current network to include additional reactions and related compounds genes and enzymes Option 1 Expanding a network in the current network window Example using Compound Reaction Network 1 Right click on the compound node you want to expand this node is known as the expansion seed node A menu of options will pop up 2 Goto MetScape gt Expand gt Expand in Existing Network Page 30 of 35 5 10 Methyle C00097 Visual Mapping Bypass Nested Network Use Web Services Hide Node LinkOut MIMI Plugin MIMI Plugig i Evide E from Literature soy Expand Expand in Existing Network Collapse Expand in Subnetwork Restore Original Network 3 Additional compounds and reactions are added to the network As a result the network is often redrawn 4 The edges between the original node and the expanded nodes are now blue R0423 oazat Mor Aion RE281 POOBSE 2 Ox0l Page 31 of 35 Option 2 Expanding a network in a new window Example using Compound Network from Selected Genes 1 Right click on the compound node you want to expand this node is known as the expansion seed node A menu of options will pop up 2 Select MetScape gt Expand gt Expand in Su
10. PIESPONCIBI National Center for Integrative Biomedical Informatics MetScape 2 3 2 User Manual A Plugin for Cytoscape National Center for Integrative Biomedical Informatics July 2012 2011 University of Michigan This work is supported by the National Center for Integrative Biomedical Informatics through NIH Grant 1U54DA021519 01A1 Contents OVER VIEN eee eter een tt ee ern ee ene te et ee tt tr ee eee re 4 Abo t data SOUS ssiri rinna E sees ses ecg es eget gst cesar st E ener tees 4 Workflow OV SU Ny ans ses yes eo acai vances noes gona oesa ead epe cane paavatesieconne tnraceveceoutnera 4 INSTALLING CYTOSCAPE AND THE METSCAPE PLUGIN ssssssseeeeececcececeeeseaaaeeessssseseeeeeeeeeeeeees 5 Launching Cytoscape and the MetScape plugin from the We D ccccccceseccceessececeeseceeeeneeeeeeees 5 Installing Cytoscape and the MetScape plugin on your local computer cccccceeeceeeeeseneneeens 5 ENTERING DATA arts teccresesocttanatdounusad ue vetnaeasenepertontanatseuaaiten E 6 Option 1 Enter a list Of COMPOUNAS cccccccssccccceseccccesececeesececeueecceeeesecessuaecessunceeseueeessenecetsgges 7 Option 2 Load an experiment file ccc ccescccceeseccceescceeeesececceeeceeeeeseceeseaeceseeaeceeseueeeesegecetsgeas 9 VISUALIZATION osere ecco stn cnet gone cece EEEE EOE EA AEEA AEE A ANETA RENEE EENET E aS 14 METSCAPE TAB oron E EEE E A E A E E EEE ER 15
11. RULES USED TO BUILD DIFFERENT NETWORK TYPEG ssssssseeeeececceeecceeceesaaeeesssseeseeeeeeeeeeeeess 16 DATA PANELDISPLAY ceccar EE A 17 Choosing attributes provided through MetScape ccccccsssccccssececcesececeenscceeeeseceseuecesseneceesenes 17 Rearranging attributes in the data panel ccc ccecccccesscccceseceeeeeceeseeecceseeseceeeeeceseugeceesenaeeeeas 20 Pathway filter in the Gata panel isvisicccdsesdceacsnrdcccseradeanssadeesessandsaerinddaaarvsepannavaddesdaceudeenteedicetosidesssuadics 21 Concept filter in the data panel ccccccccssseccceesececeesecceceesececeeececsueceeeeaeceeseneeessuaecetseneceesenes 22 METSCAPE RESULTS PANEL ADDITIONAL NODE INFORMATION ccccccccsssssssssssssseeeseeeeeeeeeees 25 ANIMATION OF DATA recs secnc etsrsraate vcd sins oi ptes sng ig gd ote icrgnne in vd alnge ig csiegn ves need tani eceaersten dss le eisiegn daeseasdototesaorint 26 Building an LUNN UN IIMs Sects pc ec Secs se Ses cs se Sn ce seas srs ceo ee secs Shee eset oes 26 Manipulating the animation ZOOMING cccccccceseccccesseccceesececeesececceeecceteuseceseunecessugeceeseneeeeeas 28 Manipulating the animation bar chart ccccccccsseccccsssecccesececeeseceeceeecceseuseceseuecessueceesenseeenas 29 Playing the animation occsceseces egrets cats nets ren wns ences gat aes scott erase scsaeat Er Er UENEN 30 MANIPULATING NETWORKS 000 cccccccccssss
12. a Note If you select Save on the Select Compound Mappings window you will get a csv file containing mapping information for all compounds that MetScape successfully mapped Note The Select Experimental Data window can also be accessed from the MetScape tab Note Multiple networks can be built within the same user session After a network is built pull up the Select Experimental Data window and change the data files Then build the new network Both networks will remain accessible If you change species however all existing data will be lost The below warning window will pop up in this situation Missing Data Window After the experimental data has been loaded if any of the genes compounds that you Submitted were not mapped to the database objects a Missing Data window will appear Genes compounds and concepts may appear on this missing elements list because e Genes and compounds that you supply may not be in the database If they are not found in the database then they are reported as missing Page 13 of 35 e If your input genes are not human Rat for example then they are mapped to human genes using homologs from NCBI s HomoloGene If this mapping fails then those genes are reported as missing e MetScape will display only the genes that encode metabolic enzymes If an input gene does not encode metabolic enzymes it will appear on the missing elements list e A concept pathway will appear on the missing l
13. bnetwork C00041 Visual Mapping Bypass Nested Network Use Web Services Hide Node LinkOut MIMI Plugin MIMI Plugin Evidence from MetScape Create Sul Expand Collapse Expand in Subnetweork Restore Original Network S Mejiylmalonate semigidenyde 3 Anew network is created in a new window This network includes only the expansion seed compound and its related compounds reactions enzymes and genes depending on the Network Type The original color designations are used when the graph is created in a new window R 3 Amino ened vbropanoat J Methyl opanoate L alanine arate Page 32 of 35 Collapsing a network To collapse a network that is expanded in a current network window 1 2 Right click on the expansion seed compound node Select MetScape gt Collapse and then e To collapse only the branch expanded from that expansion seed compound select Collapse e To collapse all expanded branches select Restore Original Network Creating a subnetwork A subnetwork of a current network can be created and will appear in a separate window A subnetwork will include all highlighted nodes and edges selected nodes should be yellow and selected edges should be red For example 1 2 Select the compound IMP Go to the Select menu in Cytoscape Choose Nodes gt First Neighbors of Selected Nodes Now all the first neighbors of IMP should be y
14. cle TCAKEGG Pathway 32 0 44858373 16 2447876 _1 23E 08 1 67E 06lup 24368 24299 24401 25179 25721 3 Fattyacidmetab KEGG Pathway 28 0 40269739 12 2142885 _1 10E 06 _7 48E 05 up 24158 25363 25618 25757 140547 4 Alanine andaspqKEGG Pathway 18 0 47651019 19 323541 1 93E 06 8 75E 05 up 24379 24401 25721 81670 81829 5 Reductive carbo KEGG Pathway 11 0 55839769 32 1439027 6 68E 06 2 27E 04 up 24368 24399 24401 25721 79250 6 Oxidative phosp KEGG Pathway 44 0 27758123 5 61284966 3 64E 05 9 89E 04lup _ 116550 291103 295923 301011 311 7 Urea cycle and up 24368 24379 24399 24401 24600 8 PPAR signaling p KEGG Pathway 32 0 28585824 5 90912002 _2 33E 04 0 00441518 up 24158 24450 25045 25757 29171 9 Carbon fixation up 24401 25721 1670 81829 114508 10 Arginine and prol KEGG Pathway 20 0 33385131 7 96259603 5 14E 04 0 00699086 up 24368 24379 24399 24401 24600 11 Butanoate meta KEGG Pathway 30 0 26301141 5 12695967 0 00101503 0 01254945 up 24379 24399 24401 24450 25721 12 Valine leucine al KEGG Pathway 24 0 27898384 5 66198894 0 00167291 0 01625109 up 24158 24450 29711 140547 17046 13 Glutathione met KEGG Pathway 24 0 27372392 5 4799003 0 00205204 0 01860519 up 24379 24399 24401 24422 25721 14 Propanoatemet KEGG Pathway 16 0 32105291 7 35380592 0 00265316 0 02122524 up _ 24158 140547 170465 171155 298 15 Nitrog
15. d using the method of Benjamini and Hochberg When results are sorted by p value a FDR lt 0 05 is interpreted to mean that approximately 5 of the concepts with lesser or equal p value are false positives TO map a concept on the graph click on the concept name in the Data Panel The concept will then be highlighted in the graph associated nodes turn yellow and associated edges turn red E Number of Enriched Driving Genes False Discovery Rate 1845 7 Number of Mapped Genes hesis W terpenoids and steroids SMa aturated fatty acids wooo G ee a lt lt APRES se Citrate cyde TCA cyde Cytokine cytkine receptor interaction SS Se DNA een ET cS hh 5011 Gyce serine and threonine metabolism CD a T u T O T T r a a a ey Pa as Node Attribute Browser Edge Attribute Browser Network Attribute Browser Pathway Filter Concept Filter A subgraph can be created for a given concept The subgraph consists of genes considered to be significant to the concept those genes that drive the concept not all genes in the concept To create a subgraph 4 Select a concept in the Concept Filter tab 5 Click the Create Subnetwork button at the top of the Data Panel make sure you are on the Concept Filter tab 6 A new graph appears in the graph window This is the subnetwork for the selected concept Page 23 of 35 eaction Enzym 55 0 61 0
16. dropdown menu o Compound Reaction Enzyme Gene o Compound Reaction o Compound Gene o Compound Note When selecting Compound as the Network Type a dropdown menu appears under Query providing the option of choosing between compounds or genes When any other Network Type is selected the only option is to use compounds genes unless using a selected pathway e Add or Remove data e Select a pathway o Use to view all the compounds and reactions associated with a metabolic pathway Select a specific pathway from the drop down list Saturated fatty acids beta oxidation Selenoamino acid metabolism Squalene and cholesterol biosynthesis rihydroxycoprostanoyl CoA beta oxidation Tryptophan metabolism yrosine metabolism Urea cyde and metabolism of arginine proline glutamate a T ell Ss e Save the Output as a File e Build Network graph based on the data Page 15 of 35 Control Panel Ho nork vizmapper Editor Fiters F Build Network Input Organism Human Experimental Data Compounds human_metabolites4metscape 2 _NAMES Genes none Concepts none Compounds Network Type Compound Reaction Enzyme Gene Query Use compounds genes 0 Use selected pathway TCA cyde Buld Network Note When the organism is human the Genes section will show Input ID and Input Symbol However when the organism is not human ex rat the Genes section will Show In
17. ds and their neighboring compounds e Compound C networks built from gene input If a list of genes is provided the resulting network will include the compounds that are related to the query genes via the reactions in which they participate and the enzymes that catalyze these reactions The edges will be drawn between all compounds e Pathway networks Pathway specific C R E G C R and C G networks are built from a set of genes enzymes reactions and compounds defined in the EHMN database Pathway specific Compound networks are built similarly to the Compound networks built from a set of input genes DATA PANEL DISPLAY Attributes of compounds reactions pathways and concepts that you have selected in the network appear in the Cytoscape Data Panel Choosing attributes provided through MetScape To choose which attributes to view 1 To choose attributes for compounds click the Node Attribute Browser tab in the Data Panel You can choose to view node edge or network attributes by clicking on the respective tabs at the bottom of the Data Panel Pathway data can be viewed under the Pathway Filter tab and concept data can be viewed under the Concept Filter tab in the Data Panel 2 Click the Select Attributes icon in the Data Panel toolbar A list of attributes with check boxes will appear Page 17 of 35 Node Attribute Browser 3 Select the attributes you want displayed in the Data Panel Attributes are listed with item
18. el MetScape provides two ways to access pathway information The Pathway Filter tab lists all pathways represented in the network Selecting one or more pathways will highlight in the network all nodes in the pathway s Pathways are also displayed as attributes in the data panel Page 21 of 35 Data Panel fat Create Subnetwork_ reapoiy Secon Pathways 3 0x0 10R octadecatrienoate beta oxidation ars metabolism drogen J estrogen biosynthesis and metabolism De novo fatty acid biosynthesis Di unsaturated fatty acid beta oidalon A subgraph can be created for a given pathway The subgraph consists of a subset of nodes from an active network that belong to the selected pathway To create a subgraph 1 Select a pathway in the Pathway Filter tab 2 Click the Create Subnetwork button at the top of the Data Panel make sure you are on the Pathway Filter tab 3 A new graph appears in the graph window This is the subnetwork for the selected pathway Concept filter in the data panel The content of the created or loaded concept file is put into the Concept Filter tab 1 Concept Name official name of the concept 2 Number of Genes the number of input genes that belong to the concept 3 Direction direction of the change 4 P value the p value for enrichment depletion Page 22 of 35 5 False Discovery Rate the FDR estimate
19. ellow Go to the Select menu in Ctyoscape Choose Edges gt Select Adjacent Edges Now all the adjacent edges should be red Right click on IMP A menu of options will pop up Page 33 of 35 poise 7 oe hase ie oN C00130 Visual Mapping Bypass Nested Network Use Web Services Hide Node LinkOut MIMI Plugin Create Subnetwork Expand Collapse Restore Original Network Se Sieg Beane 6 7 Choose MetScape gt Create Subnetwork Note When choosing options from the menu be careful not to move the cursor outside the menu panels Doing so will remove the highlighting of the nodes and edges resulting in an empty subnetwork 8 A new subnetwork is created in the graph window HE12 deinen beth AMP Page 34 of 35 Destroying a network To destroy a network no longer needed 1 Make sure you really do want to destroy the network e Cytoscape will not ask you to confirm the deletion e Destroying the network is irreversible e Note If you want to destroy a network view without destroying the network itself use Destroy View instead of Destroy Network 2 On the Network tab in the Cytoscape Control Panel right click on the network you want to destroy and select Destroy Network Note If you destroy a network that has subnetworks the subnetworks are NOT destroyed They are promoted up one network level SAVING AND REOPENING A SESSION Saving a session To sa
20. en metabqKEGG Pathway 15 0 30373897 6 60362465 0 00596513 0 04056287 up 24379 24399 24401 25721 29242 16 C5 Branched dibjKEGG Pathway 19 0 26819205 5 29471153 0 00686636 0 04226683 up 24153 24379 24399 24401 25721 17 Pyruvate metabd KEGG Pathway 19 0 26689922 5 25234187 0 00714807 0 04226683 up 81829 298942 306198 307858 361 18 Phenylalanine mKEGG Pathway 21 0 25034932 4 73898475 0 00822755 0 0447139 up 24368 24401 25721 81683 19 Antigen processi KEGG Pathway 21 0 2496329 4 71793226 0 00841626 0 0447139 up 24223 24812 25217 25599 Instead of loading a concept file you can run LRpath directly from MetScape Running LRpath from MetScape If you choose to run LRpath directly from MetScape the directional test against EHMN metabolic pathways with a minimum number of genes in the concept 5 will be performed Only the concepts with p values lt 0 05 will be reported For more options go to the LRpath page 1 Select Plugins gt MetScape gt Select Experimental Data from the Cytoscape menu Select a species Choices are human rat mouse 2 Using the Import File buttons select the compound and or gene files to load 3 Select the appropriate column headings using the dropdown menus for fold change and p value Choose the desired threshold 4 Choose to select an input file with concepts or choose to generate concepts using LRpath generating the concepts with LRpath will take lo
21. ide variety of network layouts provided by Cytoscape e Use color size and other effects to visually reflect a set of attribute values Save your session and reopen it later Page 4 of 35 INSTALLING CYTOSCAPE AND THE METSCAPE PLUGIN You can use the MetScape plugin for Cytoscape in one of two ways e By launching Cytoscape and the MetScape plugin from the MetScape Webpage With this method you launch a temporary copy of Cytoscape and the MetScape plugin Use this method when you don t have the administrative privileges to install software on your computer You will not have access to other plugins when you use this method e By installing Cytoscape and the MetScape plugin on your local computer With this method you can use other plugins that work with MetScape data such as Shortest Path and MCODE Launching Cytoscape and the MetScape plugin from the Web To launch Cytoscape and the MetScape plugin from the MetScape Webpage 1 Go to http metscape ncibi org metscape2 2 On the Webpage click the Launch Cytoscape w MetScape 2 via Java Web Start link After launching the Web version you can do anything described in this User Guide You can save your Cytoscape session on your local computer and reopen it at a later time after launching the application from the MetScape Webpage again Installing Cytoscape and the MetScape plugin on your local computer 1 Install Cytoscape on your computer For more information go to the
22. ist if all of its significant genes are missing The list of significant genes for a concept comes from the input file or from LRpath After viewing the Missing Data information click SAVE to save the data for later viewing or click OK to close the window without making it available for future viewing Missing Data i Total Glucose Total Glutathione VISUALIZATION Access the MetScape Legend from Plugins gt MetScape gt Show Legend Legend i n Legend Compound Reaction Enzyme C Gene C Input C O Significant Up regulated C Down regulated O Expansion gt Page 14 of 35 Additional information about the network is expressed through visualization e A compound is red in the original network and subsequent subnetworks if it was in the original data loaded into MetScape e A green border surrounding a node represents significant gene compound e Node size represents the direction of the change Larger nodes represent an increase and smaller nodes represent a decrease in gene metabolite The actual amount of the change is not represented visually e When a node is expanded the edges between the original node and the expanded nodes become blue METSCAPE TAB This tab has options for choosing Network Types the data you entered and more From this tab you can e Select the type of network that you want to build Select Network Type by selecting one of the following from the
23. lize Coupled Animation of Multiple Data Columns Drag and drop labels from the ist on the left into the table of data labels for each animation Set the number of animations number of rows and the number of data items per animation number of columns in the text fields and Column 1 Column 2 Se treme 0 DataSeries_0 Control_to reatment1_t0 re DataSeries 1 Control_ti reatmenti_ti f A ii DataSeries_2 Control t2 fTreatment1 t2 Urde S DataSeries_1 Undetied Undefnedj Undefined Treatmenti_ti Treatmenti_t2 Treatment2_t0 Treatment2_t1 Treatment _t Drag and drop labels from the ist on the left into the table of data labels for each animation Set the number of animations number of rows and the number of data items per animation number of columns in the text fields and econfigure Layout I Rows 3 Reconfigure Layout Column 1 reatmenti_t0 reatmenti_ti reatmenti_t2 6 After grid is completely filled in click on Build Animation Note If you want to undo what you filled out in the grid click on Restart and the grid will be cleared 7 Three new windows appear in the graph panel each representing a different treatment In addition an Animation Controls window appears Page 27 of 35 l DataSeries 11 DataSeries_0 Manipulating the animation zooming 1 Zoom in on one graph
24. match If the compound is not found in the system it will say Not Found Page 7 of 35 Select aay compounds One or more of the compounds in your query Aad multiple matohes in the database Alease select the maich fiat best or none iF you do not wish fhe compound fo aonear in fhe results Input Name Potential Matches Lipoyl Ltysine CE7102 jerome DL Lipoyl L4ysine 7 Click OK 8 After the experimental data has been loaded if any of the genes compounds that you submitted were not mapped to the database objects a Missing Data window will appear After viewing the Missing Data information click SAVE to save the data for later viewing or click OK to close the window without making it available for future viewing See the Missing Data Window section below under Option2 Load an experiment file for more information about missing data Trimethylamine 9 Select a Network Type by selecting one of the following from the dropdown menu Page 8 of 35 e Compound Reaction Enzyme Gene 1 e Compound Reaction 2 e Compound Gene 3 e Compound 4 Note When selecting Compound as the Network Type a dropdown menu appears under Query providing the option of choosing between compounds or genes When any other Network Type is selected the only option is to use compounds genes unless using a selected pathway 10 Click Build Network to query the database and create the network
25. mics and gene expression data in the context of human metabolic networks MetScape uses a metabolite database developed by extracting and integrating information from the following sources 1 Edinburgh Human Metabolic Network EHMN http www ehmn bioinformatics ed ac uk 2 KEGG COMPOUND Database http www genome jp ke compound MetScape allows users to load a list of metabolites with experimentally determined concentrations a list of genes with experimentally determined expression values and a list of concepts or pathways and display them in the context of relevant metabolic networks Workflow overview With MetScape you can Trace the connections between metabolites and genes Integrate multidimensional data Visualize compound reaction enzyme and gene networks and display compound structures as well as information for reactions enzymes genes and pathways Visually animate changes in compound concentrations over time and across experimental conditions The basic steps in the workflow include 1 Enter data You can type or paste a list of compounds and or genes load a file containing experimental data or start from a biological pathway Select compound and reaction attributes Choose which attributes to display ina table as you work with your visual network graph Explore the Visual network and table of attributes e Expand and collapse a network e Create a subnetwork e Visualize your data in a w
26. nger than loading a file with the information Note LRpath performs gene set enrichment testing an approach used to test for predefined biologically relevant gene sets that contain more significant genes from an experimental dataset than expected by chance Sartor et al 2009 To run LRpath you Page 11 of 35 need a Gene Expression file with fold change or log fold change values and p values The Gene Expression file needs to contain all gene records not just those for significant genes LRpath will determine the significant genes from the input 5 Click OK Example of selecting experimental data including importing files and designating fold change p values and thresholds Select Experimental Data i i reshold 1 f Threshold J j F Meee z Threshold Page 12 of 35 Example of ID mapping window from a loaded compound experiment file Select Compounc One or more of he compounds in your query had multioe matohes in the database Please select fhe matoh thats best or none if you do not wish ihe compound fo appear in the results KEGG ID coogi9 Potential Matches L Glutamine Protein glutamine i Frotein L glutamine Poks N5 alkylglutamine none potround trond Td potround tT wra S EN NotFound Sphingomyelin Sphingomyelin potround Retford potround Rotround
27. put ID Input Symbol and Human Symbol RULES USED TO BUILD DIFFERENT NETWORK TYPES e Compound Reaction Enzyme Gene C R E G Compound Reaction C R and Compound Gene C G networks The C R E G C R and C G networks are all built from the same underlying data That data is derived in each case by finding compounds that participate in reactions that are catalyzed by enzymes that are encoded by genes If only genes are input then all the enzymes reactions and compounds that match those genes are used If only compounds are input then all the reactions enzymes and genes that match those compounds are used If both genes and compounds are input then only those C R E G couplings that match both a compound from the compound input and a gene from the gene input are used If a concept file is provided genes from that file will be used as input If a gene file is also provided it will be used only as input for LRpath if user chooses that option Page 16 of 35 If only a gene file is provided all genes from that file are used as input In this case we recommend that you load a smaller set of genes e g most significant differentially expressed genes e Compound C networks built from compound input If a list of compounds is provided the resulting network will include the query compounds shown in red plus any compounds that participate in the same reactions as query compounds The edges will be drawn between seed compoun
28. sssssssseeeeecccececeesessssaseeasssseeeeeeeceeeeeeeeessessaaaaeeaseseeeeeeseeeeeeess 30 Expanding a NGTIV ONC orioicaniccesatacdecedsrcsaasacnogtaceneitasabintad edeeninataanaditecaeiedsaevamiseaiesnisssaaneeseacaxtecsaeioanaadess 30 Option 1 Expanding a network in the current network window Example using Compound Reaction NetWork sspears eat oe scr sess seein tess ered races rac a ened yeast cease eo ace avant 30 Option 2 Expanding a network in a new window Example using Compound Network from Selected GENES eee nn eee een ene oni eee eee 32 Collapsing a CW OI secs ann ete scence cscs ate a sateen anes aaanin gunn aeiegadesoaaanaapensiecsesiasan oieeatens 33 Creating a subnetwork Sars scrscrc cs carcasses ace cases acute octane ss oeace sons eaten see eos oon 33 Destroying a NETWOFK cc cc cecccccessecccceeccccesececauececeusececcenseceseueceesenecesseneceesenecessunecessugeceesaneeeetas 35 Page 2 of 35 SAVING AND REOPENING A SESSION oie Ait a i AE Savine a SESSION esac eee ais ees ics dsc eae a Bec us oe ve ac a ws Sag aie on ese aise ese RECORDING a SESSION eres craic tata esate tae iat ne ae eet aati ee Please note that due to continuous software upgrades the images in this handout may not exactly mimic what you see on the screen Page 3 of 35 OVERVIEW About data sources MetScape is a plug in for Cytoscape the bioinformatics network visualization tool The plug in can be used to visualize and interpret metabolo
29. t includes column headings to label the data e The first column contains Entrez Gene IDs or Official Gene Symbols e The remaining columns contain experimental data Multiple experimental values are permissible in the same spreadsheet Below is a portion of an example gene file with Gene IDs significance values and fold change values A B C 1 ENTREZ GENE ID Pvalue Log fold change 2 1 0 002335904 0 02 3 2 2 07E 08 0 06 4 2 2 03E 08 0 04 a 9 0 003919472 0 04 5 10 0 00026716 0 08 T 12 0 000237925 0 05 8 13 0 0024893604 0 05 9 14 0 506396205 0 00 15 6 29E 06 0 04 16 0 022222108 0 01 18 1 47E 08 0 07 18 5 24E 06 0 09 18 2 13E 05 0 07 15 19 0 820594937 0 00 16 19 1 76E 08 0 06 17 19 9 46E 09 0 04 18 19 0 660631201 0 00 19 20 1 16E 07 0 05 Concept File The concept file can be generated by a gene set enrichment analysis tool such as LRpath or GSEA from gene expression data Page 10 of 35 Note Gene set enrichment testing is an approach used to test for predefined biologically relevant gene sets that contain more significant genes from an experimental dataset than expected by chance e GSEA Subramanian at al Proc Natl Acad Sci USA 2005 102 15545 15550 e LRpath Sartor et al Bioinformatics 2009 25 2 211 7 Below is a portion of an example concept file A B c D E F G H I 1 Concept name ConceptType n genes coett odds ratio p value FDR Direction sig genes 2 Citrate cy
30. tScape plugin first start Cytoscape 2 Choose one of the following methods to get started Page 6 of 35 Option 1 Enter a list of compounds 1 Select Plugins gt MetScape gt Build Network from the Cytoscape menu 2 A MetScape tab now appears on the left of the Cytoscape screen 3 Select a species Choices are human rat mouse 4 Manually enter or copy and paste compound ID s or name s and or Entrez gene ID s or symbol s Click Add and enter the appropriate ID type in the popup box Lists of IDs should be separated by spaces cyorone Dep New 6 a File Edit View Select Layout Plugins Help Snliaaga S Hy a Control Panel an Aale Buld Network Organism Human Choose species Experimental Data Compounds none Genes none Concepts none ompounds may be entered all on one line separated by commas or spaces or one per line Compound names should be entered one per line Input ID Input Name Compound Reaction Enzyme Gene Query Use compounds genes Use selected pathway TCA cyde Output as File Node Attribute Browser Edge Attribute Browser Network Att Welcome to Cytoscape 2 8 1 Right click drag to ZOOM Middle click drag to PAN O 5 Click OK 6 If you enter a compound name it will map to its KEGG id a popup window will appear If there is more than one potential match use the dropdown arrow to choose the best
31. ve a Cytoscape session containing one or more MetScape plugin networks i 2 3 4 Select File gt Save or Save As from the Cytoscape menu Browse to a location for saving the file Name the file Click Save Reopening a session To reopen a saved session containing MetScape plugin data 1 2 Select File gt Open from the Cytoscape menu Navigate to the saved file location Select the file Click Open After Cytoscape reports that the session file was successfully loaded click Close Page 35 of 35
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