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PathwaysTM 4 Software User's Guide

1. m C But PrOD TS o2 99 don9 53 9 ERR E UQUERRURIEA E a2 0D RR uoa Ed RP 309 es aes ERONMCU SC C m Chapter 12 Profiling 12 1 122 12 3 12 4 12 5 Introduction to Profiling Analysis 1 cece eens Profiling Toolbar 12s qo dciri md dU Rb te ee dh PUES PCS beaten eben SOEUR CR Red n m C m ou Gru sha wees beep meee ee haha de ee beeen eyed eee ens l c fn abu be eke eee hs eh ode a tue E he hae ee eee ae eee 5 oe Chapter 13 Clustering 13 1 15 2 15 3 13 4 15 5 13 6 13 7 13 8 13 9 Introduction to Clustering 2s o9 dox 8 3 ac EHROR EYE shee eda tsan kat pe eee aes Clustering veu go eeviadueseetaeeceadnoeess vei adeweuedaduene es Custer ViSWalIZaUON 2x e scs 8 dod esi Gne nash aoe dae ee ade Sea eee eee Clustering in Path hways uoa em mE dc8 3 8 3 3 9 9 8 ea Dedicado eae dL oe thee KMecans CINSICHING s goeu 2 3 9 9 392 RE IRE mEt T 209 535 EUER GU TE CRCRR a UR Hierarchical Clustering PPT SOM Cluse 2s 239 224 2d 81 E E RRE bee deeded bee 0 9 RE d lau dM ici Trrrrcr wrrr 15 10 CIBSICESTONTI 4 a a 93209 98 73 99 9 229 EXER eee ee Gabe eet eee wee a RU eet Chapter 14 Pathways Data Updates 14 1 Web Links and the Integrated Web Browser elles 14 2 Adding and Editing Web Links lllllllllll res 14 3 14 4 14 5 Introduction
2. Report GF Importer Data Description Batch Data Source Plugin Interactive Data File Pathways Universal allows the importing of microarray data from several different sources and formats In addition to ResGen GeneFilters microarrays the standard Pathways framework now supports other microarray formats through the use of the Array Designer refer to Chapter 4 for details about the Array Designer Pathways Universal also comes bundled with new framework plug in modules which can take data from spreadsheet files Gene Expression Markup Language GEML files and other formats e g databases through a pluggable interface Through the use of the framework plug in modules Pathways can take microarray data from nearly any source and transform that data into a format the program can use for analysis 27 RGMA10011 rev B Affymetrix spreadsheet Spreadsheet Framework Other spreadsheet GF Path Clontech GeneFilter Description athways Framework Plugin Raw Universal Image Description Data Source Create Project Spreadsheet Plugin Importer Batch Interactive E Other Y Other Descriptions Data Fil formats Pluggable iai ai Normalize Data Analyze Affymetrix Genechip GATC file Data Rosetta Rosetta GEML Flexjet DNA GEML F k Microarrays onductor ramewor BioDiscovery AutoGene
3. Control Trials Mori Trias 2 Mant Trigs 3 Mori Trials 141 RGMA10011 rev B 15 8 Example 2 Clustering Analysis As a final step in the time study analysis locate clones that behaved similarly to the cancer genes Clustering the analysis data allows us to group the set of clones into clusters of similarly behaving clones As with the profiling analysis start with the appropriate menu selections for a condition grouping Profile SISNET Tools Help window Micro arrayts Microarray pairis conditionis Condition pairis _ Cluster Analysis of Conditions Fa Control Trials 1 Month Trials 2 Month Trials 3 Month Trials Average by Microarray Address Average by Clone Key cancel 142 RGMA10011 rev B The Clustering dialog box allows us to specify the type of clustering algorithm and the proper ties for the algorithm Choose a KMeans cluster with 50 means clusters Clustering E Clustering Algorithm IMeans Cluster Variable Cluster on Log Properties Distance Correlation Mo af Means 5 hax Iterations f Tolerance 0000000001 To view the clusters for specific cancer genes activate the cluster filter at the bottom of the screen Open the Find Clones window from the cluster window toolbar and click on the clones in this path to display the corresponding clusters Find Clones Find clones by clone key Path Name C Find clones by microarray address f F
4. a T T T a f a T T a f a T T TT E T T T T 5 n a T T T n a T rU d un un m a a T T T n n Sse ste ete ee RJJ rt ee ccd i E Ba ste ate ate ie ie ok ak E le Sd cd ste ote oe EEEIEE E EEEE AEE BEERTA EE EEE E gpr ate ate ote ie Control Points amp Housekeeping Genes Data Points Figure 2 Example of a GeneFilters Mammalian microarray format all releases The control positive or total genomic spots are shown as filled black circles The putative house keeping genes are shown as filled red circles Data spots are shown as open circles GeneFilters Tissue Specific and Named Gene microarray releases have the same overall format but there may be some areas on the membranes without DNA spots depending on the number of cDNAs included on each membrane B GeneFilters yeast microarrays The GeneFilters Yeast microarrays consist of 6 144 gene ORFs spotted on a set of two nylon membranes GF100 I and GF100 II each containing 3 072 ORFs Each membrane is cut in the upper right corner and the DNA is on the labeled side of the membrane Figure 4 illustrates the format of the GeneFilters Yeast microarrays membranes Each membrane is divided into two fields Fields 1 and 2 for GF100 I and Fields 3 and 4 for GF100 II Fields 1 and 3 are at the top Fields 2 and 4 are at the bottom Each field 1s further divided into eight grids per field Grids are laid out right to
5. 157 RGMA10011 rev B portions of this license 9 NO LIABILITY FOR CONSEQUENTIAL DAMAGES IN NO EVENT SHALL RESGEN OR ITS SUPPLIERS BE LIABLE TO YOU FOR ANY CONSEQUENTIAL SPE CIAL INCIDENTAL OR INDIRECT DAMAGES OF ANY KIND ARISING OUT OF THE DELIVERY PERFORMANCE OR USE OF THE SOFTWARE EVEN IF RESGEN HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES IN NO EVENT WILL RESGEN OR ITS SUPPLIERS LIABILITY FOR ANY CLAIMS WHETHER IN CON TRACT TORT OR ANY OTHER THEORY OF LIABILITY EXCEED IN THE AGGRE GATE THE LICENSE FEE PAID BY YOU IF ANY 10 GOVERNING LAW This license will be governed by the internal laws of the State of Alabama without regard to that State s conflicts of law provisions The United Nations Convention on Contracts for the International Sale of Goods is specifically disclaimed 11 ENTIRE AGREEMENT This is the complete and entire agreement between you and ResGen and its suppliers and it supersedes any prior agreement or understanding whether written or oral relating to the subject matter of this license This license may not be modified or altered except by written instrument duly executed by both parties UNITED STATES GOVERNMENT RESTRICTED RIGHTS Any distribution or license of the SOFTWARE and its supporting written materials to the United States Government or its agencies or instrumentalities the Government 1s made only with RESTRICTED RIGHTS Use duplication or disclosure by the Government is
6. ment process This cropping window should not be confused with the template bounding box which is described later in this chapter The Pathways importer only requires the x and y location of the spot centers in a refer ence layout and the choice of autocenter mode in order to execute the auto crop mode for determining centers 4 4 Concepts Template Mode Sometimes noise levels in the image or other characteristics of an experimental image prevent the auto crop mode from successfully aligning the image If this is the case the user will manu ally generate the seed locations for the spots using templates Templates are sketches of the ideal position of the spots which are superimposed upon the experimental image in the Pathways importer to generate the initial seed positions All templates are tied to a global bounding box which is used for initial sizing and positioning of the templates This global bounding box is by default anchored at the minimum and maximum positions on the microar ray but can be set by the user to for example lie on top of control points on the user s array Once the global adjustments are complete each point on each template can be individually fine tuned before generating the seed locations 41 RGMA10011 rev B In a simple microarray geometry such as the one shown below the user may choose to have a single template that lies on top of the global bounding box this is the default configuration for the Arra
7. 2 0 images must be reimported into Pathways 4 using the original images These images must be generated by a phosphor imager not by Pathways 2 0 a nec essary step because of the different way that data is handled and stored in Pathways 4 With the batch importing feature reimporting images is faster and the process 1s easier Batch importing automatically imports multiple images using the software s autocentering algorithms The images can then be visualized to ensure proper alignment before continuing with analysis An option to perform interactive importing allows manual image alignment see below 151 RGMA10011 rev B Interactive import The interactive importing of Pathways 4 goes beyond what was available in Pathways 2 0 For instance when an image has been imported in an incorrect orientation Pathways 4 allows it to be rotated A global alignment feature allows a template to be dragged over the image This template includes alignment points that help locate the corresponding reference spots on the image A magnifier with zooming capabilities aids in centering the points for better image alignment Should there be difficulty finding spot centers a pseudo color option is available to help with visualization After aligning the image Pathways 4 detects clones with a centering algorithm and a verification window appears Select spots to check for proper alignment and readjust them if necessary These features were una
8. 28 RGMA10011 rev B Since each framework may provide a tool for importing microarray data into Pathways the File menu may list multiple import options The number of import options in the File menu depends on the frameworks available with the Pathways installation For example when only the standard Pathways framework is present or when other frameworks that do not import other file formats are present the File menu lists a single import option Import Image P Pathways 5ample pwp Edit Comparison Profile Cluster Tools Help Window New project Ctrl M Open project Creo Save project Ctrl 8 Save project as Ctrl A 1 ClRamalPathway Output FilesiSample pwp 2 CaARamalPathway Output FilesiTest Project pwp Exit Ctr o When an additional framework 1s present that supports the importing of other file formats such as a database the Import Image option becomes a cascading menu with entries for all frame works that support importing the database framework is available as a reference implementa tion 1n source code format upon request A Pathways 5ample pwp TGE Edit Comparison Profile Cluster Tools Help window New project Ctrl e M Open project Cil o Save project Ctr 8 Save project as Ctr A PathwaysFramework 1 ClRamalPathway output Filesisample pvp DBF ramnework 2 CaARamalPathway Output FilesiTest Project pwp Other features may depend on the type of framewo
9. 3 image files and projects so current Pathways 3 users should encounter no difficulties when they migrate to Pathways 4 Changes to the user interface have been kept to a minimum in order to provide the user with a familiar experience Pathways 4 contains many new features making the use of Pathways 4 files impossible on older versions of the software Pathways 3 users should carefully read through this manual to learn how to access the new features available to them with Pathways 4 Pathways 3 users need to obtain a new license key to use Pathways 4 from the ResGen Customer Care Unit ccu resgen com Older license keys will not work with Pathways 4 154 RGMA10011 rev B Appendix III Exporting Images from Fuji Software The Pathways 4 Fuji plug in reads the Fuji img inf file combination which is output by Fuji Bas scanners The img image file contains raw image bytes while the inf information file contains fundamental image information such as width height encoding parameter et cetera The following paragraphs describe how to export inf img formats from the Fuji ImageGage software Obtaining Images in MacOS ImageGauge 3 3 Obtain images by selecting File Export Fuji Exchange Format in the menu Doing this outputs the two files the img and the inf which is in the directory read by the converter It is possible to select File Export IRAW but the inf information mu
10. C Average by Clone Key Cancel Isolate the cancer genes by unclicking Show non members in the Path filter list The chart title is entered in the chart customization dialog box which is generated by right clicking Cancer Genes Time Siudy fori Trials t Mom Thak 1 Monit Tizh 3 Mpnihi Trial Y m I maid Clones Bow nan mami ore inten ety ram Paths RITU To learn more about the most highly expressed gene in the control study select this gene from the chart view and activate a web link to Unigene 140 RGMA10011 rev B bii sd Fer mmm mm gu pomii Pole Cue h cO en j Core Ten F nn EI IELT E E moo pom ewe tenpe Narag oe Bead HN NOU WS BH mH wm f mm 1 Se bM Ww M jJ Lao oe p E E Z T rcs Tun Flur Tess joe TR LALALA AAAI Ai 1 i a 1 Fi P i i d n 1 I ul 1E u B a IE pa se I DTE Fem Fur j Dm epoca tu mem rar pem m i pm m rr p Pun EXTEESGEIH Ha CRIM ATKEIR cDNA Bran Eremi Coon ao Poke By ATIS Bade MAWAGERE SECUENTCES 4j f L PE E hi FETA a fella w Baal Finally to view the overall trending of the cancer genes rather than the detailed plot of each gene open the bar chart view The bar chart view presents the average of the cancer genes along with standard deviation bars Prefle Conbline try kari C Lal z m PN a a a
11. ILS Dg o IL 6 Data Management and Updates 0 0 0 0 0 eee II 7 Making the Change s acus o 3 0 oue emm RR Ro nm ei es IL8 Migrating from Pathways 3 2 ee enn Appendix III Exporting Images from Fuji Software Appendix IV License Agreement Appendix V Technical Support Glossary Index RGMA10011 rev B 146 146 146 148 151 151 151 152 153 153 153 154 154 155 156 159 160 167 Book I Introduction and Overview RGMA10011 rev B Chapter 1 Highlights 1 1 Introduction As a comprehensive tool for the analysis of microarray data the Pathways 4 software unleash es microarrays potential for discovery From image importing through data mining data analy sis is rapid accurate and extensible This manual describes the process of data analysis in Pathways 4 Book I offers a product overview including program highlights and a tour of the graphical interface Book II introduces new features exclusive to Pathways Universal Book III describes the flow of data and the image importing process Book IV addresses some of the core concepts in Pathways data analysis including project organization data normalization and filtering tools for large data set reduction Book V describes the three primary types of data analysis comparison profiling and clustering and it offers practical examples of each technique 1 2 Highlights of the Software The Pathways 4 software encompasses a sophisti
12. On gef Files input fie C Program FiesiesOeniPalmeaws 4i3ample gi225 tl jCaPmogram FlleerasGamPaltreas AXarmpla grzz5 s Hr Sample gfz25 1 Sampla gfz75 J Kragram FilgstFresGerP atiam 4 130 RGMA10011 rev B The interactive importing process in this example consists of three steps after the image has been automatically loaded 1 Align Template Click the Template button Instructions on creating resizing and moving the template appear on the screen to the left of the image T n de Ex em 9 md ee raa nh of Har cma Tu mmm pes lo prm De oci ee a ey RII ae pl Uncheck the Adjust Global box and check Use Magnifier to fine tune the alignment using the template hints that appear on the screen to the left of the image In this example 16 alignment points are adjusted for the GeneFilters microarray images 131 RGMA10011 rev B Load image AlignirapiTemplate 11 Sample gr225 1 B e 88 Adjust Global F usel In the magnifier box the alignment points can be moved by left clicking anywhere in the mag nified image Up and Down arrows on the keyboard increase or decrease magnification when the magnifier window is active 2 Compute centers Click the Next button after alignment Centers are computed automatically 3 Verify centers Verify centers by clicking the crosshair on any alignment point in the image A detail viewer in the window to the left shows the detail view of each poin
13. The Intensity variable is labeled I or II for the first or second set in a paired analysis Paths are discussed in detail below The analysis data is pluggable New data fields that derive from the base types intensity ratios et cetera can be added to the basic set by adding a plug in The outlier plug in for example appears for each paired analysis An outlier describes clones that yield consistently high or low ratios across multiple pairs of microarrays conditions An outlier index near 1 0 indicates consistently high ratios and an outlier index near 1 indicates consistently low ratios The outlier is calculated by first sorting the ratios for each microarray condition pair and then scaling the sort index from 1 to 1 corresponding to the minimum through maximum ratio in the current pair the unscaled sort index would vary from 1 to the number of clones The cor responding scaled sort indices for each clone are averaged across the pairs to yield the outlier index 89 RGMA10011 rev B The Chen test is a statistical analysis plug in that determines whether two sampled intensities are different based on a desired confidence level When a clone is not filtered at a confidence level then the difference in intensity between two samples is statistically significant at the specified confidence level Unlike the t Test this test is applied to paired data microarray pair or condition pair grouping control and experimental data The Chen
14. The last column of the example Affymetrix spreadsheet file contains a text description of the experiment Set the data type for this column to Meta Data to see it during analysis The label for Meta Data may be edited by the user In addition the data type for additional columns may be set to Meta Data to view those columns during analysis Column Identification Data Type Diff Call Ignore Avg Dif Change ignore H A ignore Fold change Ignore sort score Weta Data Sort Score Ignore J intensity Background Primary Key airs Pairs Used Pairs InAvg Pos Fraction AFFXMurlL2_at Secondary Key 20 19 0 25 Data preview AFFX MurlL1 at 8 Coordinates 20 14 0 45 AFFX MurlL4 at Y Coordinates 20 18 0 20 AFFX MurFAS at ESEE 20 19 0 50 A spreadsheet may provide geometry in the form of X and Y coordinates If it does so Pathways will create a synthetic view of the microarray during the importing process Set the appropriate columns to specify X and Y coordinates for a layout 36 RGMA10011 rev B The Data Preview section of the window contains the finished layout of the spreadsheet If the spreadsheet is improperly laid out click the Back button to change its parameters To finish importing the spreadsheet click the Finish button 3 5 The GEML Framework The Extensible Markup Language XML is a syntax format created by the World Wide Web Consortium for creating customized markup languages XML can be used to create
15. Unigene and GenBank Regular updates of GeneFilters data and Plug in features through the ResGen Pathways data server Each of these methods are discussed in detail below 14 1 Web Links and the Integrated Web Browser Pathways 4 has an integrated web browser that can connect a clone and related web sites web Links Unigene Cluster Search Unigene faccession Genecards Purchase Clone Online Month 1 Month 11 4927 lIntorncity In addition a browser window can be accessed through the Tools gt Browser menu The web links that are shown for each clone are implemented through web plug ins These plug ins search meta data for each clone to determine whether key fields like the clone acces sion are present and then dynamically generate web links based on the availability of this data 123 RGMA10011 rev B 14 2 Adding and Editing Web Links Simple web links are managed using the Edit Web Links dialog box Tools gt Edit Web Links di Web Links arian Winigone aoe This dialog box allows web links to be added or modified based on a link name and certain key fields For example access to Unigene for an accession requires a format like http www ncbi nlm nih gov Unigene query cg1 TEXT acc where acc corresponds to the acc accession field in the meta data for each clone An item from this table can generate web links For example if a fictional link required unigene build inform
16. are attached to each target Tools for data analysis and data management are available in Pathways 4 For detailed experimental protocols visit the http www resgen com products GF200_proto col php3 146 RGMA10011 rev B Label with i 33 dCTP by reverse transcription EDNA clones reactions dts thes eroarray dem T m EAE T Pathways analysis Figure 1 An overview of the GeneFilters microarrays system On line resources for GeneFilters microarrays For a current list of GeneFilters microarray products visit http www resgen com products MammGF php3 for GeneFilters Mammalian microarrays and http www resgen com products YeastGF php3 for GeneFilters Yeast microarrays For a list of genes spotted on each membrane visit the ResGen ftp site at ftp ftp resgen com pub genefilters For a query for identifying each spot on a membrane visit http www resgen com resources apps genefilters 147 RGMA10011 rev B I 3 Layout of GeneFilters Microarrays On the GeneFilters microarrays membranes there is a system of controls including total genomic DNA and putative housekeeping genes The controls help to orient and align the mem brane when using Pathways software Familiarity with GeneFilters microarrays membrane layout facilitates image importing A GeneFilters Mammalian microarrays The GeneFilters Mammalian microarrays contain a total of 5 184 genes spotted on a single n
17. comparison of one or more pairs of conditions to determine peak upregulation or downregula tion between the conditions A comparison analysis displays microarray data as synthetic microarrays scatter plots or tables The microarray s and condition s groupings allow determination of minimum and maximum expression levels or analysis of expression levels of a set of clones using Paths and other data filters The microarray pair s and condition pair s allow determination of the following char acteristics Highly upregulated or downregulated clones Analysis of ratios Differences between a set of clones 11 2 Comparison Toolbar The comparison toolbar offers functions for comparison windows The first three icons in the toolbar enable the synthetic microarray scatter plot and table views respectively more about these below The path icon creates a path based on the data remaining after the active filters are applied A dialog box appears and requests the name for the path and whether the path 1s stored by microarray address location in the current microarray type or clone key accession or other unique identifier The path icon is the fourth icon on the toolbar The save image icon camera saves an image of the current data view Images can be saved in either JPEG or PNG formats This icon is disabled for the table view The save image icon is the fifth icon on the toolbar The report icon creates reports or export data bas
18. iterations field sets a limit as to the number of iterations on the grouping process Tolerance the iteration process stops if the difference between the cost average clone to cluster distance at the current iteration and the cost at the previous iteration is less than this number When the properties are set click Ok to proceed with the cluster analysis 13 6 Hierarchical Clustering Hierarchical clustering is a second option for clustering analysis pa Clustering Clustering Algorithm Cluster Variable Cluster an Log Properties Distance Euclidian Complete Linkage As with all clustering algorithms the cluster variable must be chosen before proceeding with cluster analysis 116 RGMA10011 rev B In addition to a cluster variable hierarchical clustering requires the following information Distance plug in calculates the cluster distance Merge Option linkage method see below The merge option linkage dictates where a new cluster created when two clones and or clus ters are combined 1s located relative to existing clones clusters Single linkage dictates that the distance between a new cluster and an existing entity 1s the minimum of the distances between the new cluster s components and the entity in question Complete linkage dictates that the distance between a new cluster and an existing entity is the maximum of the distances between the n
19. E Fea e Click the Browse button to locate the file in the system Once the file has been located click the Next button A window appears and it asks for information about the spreadsheet a Amey langar lgneadchent Ininonalina This page wall kr ama oat Ihe micrearra he fle bapi and athar information for f zpeeadzherd dla mpzot ou ara uzing a piaus sqead sprbadzsaect SH lawl cu can choran il from he drap cdawn lel and click Finistr b eroceed da he amr design siga Propeka File Lzwput PoF 10D aeu In this window enter the microarray File Layout Brand and Type The name entered in the File Layout field is used to signify a set of attributes for the spreadsheet The name entered in the Brand field is the name of the product line and the name entered in the Type field is the kind of microarray from the product line Pathways stores this information and creates a profile for the spreadsheet layout allowing the researcher to quickly import a microarray that uses the same layout If a spreadsheet has been defined for the microarray being imported click the Finish button to proceed to the array design stage 46 RGMA10011 rev B If a microarray layout is being designed for the first time click the Next button to define the attributes of the spreadsheet in the Spreadsheet Import wizard Para Dezira 2 pioanaidizhinet Preven Fi This page will lel you adyuret ihe Gaim 1 staring pore and est qualifier for your le Yo
20. JH Er Ss 1 A I 123 GEE CUE 1 11 LEG 2942 r3 7 58 Kx When a filter setting 1s adjusted a check mark appears beside the filter name in the filter view Refer to Chapter 9 for a detailed discussion of filter use 19 RGMA10011 rev B 2 7 Quick Start The Quick Start palette offers a quick method of accessing core functionality in Pathways 4 including the Array Designer disabled with Pathways 4 GeneFilters E LT M ILERTE lmporia new mucrcaeray wage Open an ensimg project Open the project wand io pade you Der cugth cte eredi of new feaject Analyze relstive iniensiiy values of a Compare bard TIBCTOGETIVE uing mniensiy Palit ans difTer erc ies Corina damir isi of genes buli an i Desig new microarray byos aang the S Amay Deter 19 l Enga qr aaa Quick Start appears automatically at startup unless the option is disabled or it may be launched at any point from the Tools gt Quick Start menu Quick Start reappears after tasks are finished unless the Close button is clicked Quick Start performs the following functions Imports images Opens project files Launches the new project wizard Analyzes one or two microarrays Edits Paths Launches the Array Designer These functions are discussed in detail in this manual The Quick Start menu includes the Array Designer in Pathways 4 Universal Refer to Book II for information about using the Array Designer 20 RGMA10011 rev B 2 8
21. Microarray Brand Microarray type and Sampling type are adjusted appropriately Check the Batch process box to process these imports in a batch mode see the section on batch importing for more details Select the Trim option to automatically trim the image to reduce the size of the saved Pathways image file The Reimport box is active when the selected image format is a Pathways image A Pathways image can be imported as a regular image Otherwise skip the initial centering step and read the computed centers from the Pathways file When reimport is selected the initial centering step is skipped and the import process proceeds directly to data sampling and clone adjustment This feature is useful if the purpose of the reimport is to adjust a single spot or to resample the data using a different sampling technique Normally the importer expects to find black spots on a white background Select the White on 61 RGMA10011 rev B Black option to load the file as a white image on a black background This option should be selected if raw image file consists of white spots on a black background The White on Black option works differently from the Contrast Controller Invert option which takes an image that has already been imported and inverts it turning the black spots into white spots on a black background To add images to the import window click once on the file to highlight the first import file in the file browser and click t
22. Other GEML Sources Other nepon Frameworks Pluggable Frameworks provide a means of loading and saving data but they may serve other functions Frameworks that use raw image files may provide a different way of importing and viewing images The functionality of the framework depends on the type of microarray data being imported Because frameworks are plug in modules new frameworks for previously unsup ported microarray data sources can be developed and integrated into Pathways 3 2 Influence of Frameworks Frameworks influences the File menu where new menu options may be introduced for import ing microarray data Some frameworks obtain their microarray data through a relatively simple process such as reading the data from a spreadsheet file For these frameworks an image import tool is unnecessary For other frameworks where the process of converting the original array image into a useful representation is more complicated the framework must provide an image import tool One example of this kind of framework is the standard Pathways framework which trans forms an image file into a computational description of a microarray For Pathways to identi fy the locations of spots on a microarray and then sample their intensities some user interven tion may be required during the process e g to verify that the auto centering algorithm correct ly identifies the spots centers before it samples their intensities
23. Unigene build New microarrays description files for new GeneFilters microarray products will be added as they become available Plug In enhancements enhancements to existing plug ins are supplied as they become available New plug Ins as they become available new plug ins are added to Pathways automatically Data updates are performed by establishing an internet connection between Pathways and the ResGen data server Pathways sends a message to the data server asking if any updates are available The data server replies with a list of available updates Pathways retrieves each selected update from the data server The updated data become available the next time Pathways is launched Pathways must be shut down and restarted after the update is com plete Additional data can be added to the microarray description files therefore these files must be treated specially during a Pathways update The description file updates are performed as a merge between the old and new files so that auxiliary data added to the description files are not lost 125 RGMA10011 rev B 14 4 Launching the Updater To launch the updater select Tools gt Update Pathways A dialog box appears and indicates that Pathways is contacting the ResGen data server Once a successful connection has occurred Pathways displays a list of available updates Pathways data and or plug ins Awaits Updates The Tod
24. appears automatically alabie pahi R Bess Cancer 43 Excluded Clone The left portion of the Path editor shows the available paths and includes options for adding new paths removing paths and exporting paths to a comma separated text file for use in spreadsheets For the Path icons an envelope is used to depict clone address paths a key 1s used to depict clone key paths and a magnifying glass is used to show automated search paths The right portion of the Path Editor is specific to the type of path being edited Clone key and microarray address paths display a list of keys or a list of entries consisting of Microarray Brand Microarray Type Clone Number respectively Entries can be added and removed using the Add or Remove buttons respectively 96 RGMA10011 rev B The Automated Search editor displays a list of rules for the current path along with a radio but ton to select how these rules are applied Selected path Satisfy any rule all rules Match Method Tet does contain cancer Add iun When the path is set to satisfy any rule a clone is included in this path when any of the rules are satisfied When the path 1s set to satisfy all rules a clone is included in this path only when all the rules are satisfied Each rule requires four inputs Field the meta data field to use for this rule Match field that specifies whether or not clones must
25. as the reference microarray and normaliz ing the ratios of the other microarrays to the first microarray The user can select Normalize using all spots to do this The concept of paths has been added to this algorithm so that the mean ratio can be estimated using only the spots in a path or outside of a path depending on the selected option The user can select Normalize using only the path spots to do this This normalization technique does require that microarrays in a normalization group see below are of the same microarray type 8 3 Normalization Groups Normalization techniques generally fall into two classes auto normalization and dependent normalization An auto normalization technique normalizes intensities based on data that are contained completely in the microarray data set For example the Data Point Normalization technique is performed by dividing sampled intensities in a microarray by the mean sampled intensity value in the same microarray Dependent normalization techniques exhibit a dependency on other microarrays to perform the normalization algorithm The Y C Normalization algorithm for example normalizes a pair of microarrays to the mean ratio of intensities between the arrays The Path Normalization algorithm is structured so that only the elements of the path that are common to all microarrays are used in the normalization algorithm 87 RGMA10011 rev B Pathways normalization groups explicitly assign a nor
26. been located click the Next button A window appears and it asks for information about the GEML file T Arap Deng GEMI sl This page wili lei you set the mitr iay brand ype and other information for fhe GENL file import Properties Brani osetia Inpharmiat 5 a Tene furman Finish In this window enter the microarray Brand and Type Pathways stores this information and creates a profile for GEML files of this type Once the microarray Brand and Type have been entered click the Finish button to proceed to the array design stage 4 8 Reading from a Clontech Atlas Array Gene List File To design a microarray layout for a Clontech Gene List file open the Array Designer and select Read layout information from a Clontech Gene List file from the Array Designer Data Source window Then click the Next button 49 RGMA10011 rev B A window appears and it asks for the file location and for the Array Type of the gene list file Array Designer Clontech Information Ea Please enter the full path to the file containing a Clontech Gene Lists file ar click the Browse button to locate this flle using a file browser dialog box Also specify array type corresponding ta that file Filename Browse Array type f 2 Array Clontech 1 203 1 2 Array Clontech 1 2K Trial Array Clontech Demo Small Array Clontech One Large Array Clontech 588 Cancel Select the appropriate Array Type
27. clustering This is the self organizing map clustering algorithm A clustering algorithm that finds clusters in an input data set by mapping the data onto a two dimensional array of nodes and then running these nodes through a series of iterative calculations Synthetic microarray This is the view of the microarray image s currently being analyzed in Pathways software This image is a synthetic or cleaned up version The spots appear to be the same size with differing intensities allowing analysis and comparison between different microarrays t Test This is a statistical analysis plug in that determines whether two sampled intensities are different based on a desired confidence level The t Test is a specific application of ANOVA for two conditions Template A template 1s a computer generated sketch of the critical points in a microarray that can be overlaid on top of an image Templates can be stretched and rotated and template points can be dragged to aid the location of clones in a microarray image during an import process 165 RGMA10011 rev B Thumbnail A thumbnail is an enlarged and enhanced picture of a spot as it appears on the original image stored in the Pathways image format file for the current microarray This pic ture is intended to be a visual reference and it is useful only when numerical intensity values are used simultaneously TIF file or TIFF File This image file format created by phosphor imaging syst
28. data is known about the array layout Using the Array Designer is a simple matter of telling Pathways some information about the type of microar ray to be used Different microarray types use different array layouts To import an image Pathways must know what kind of array layout to expect The Array Designer allows the user to give the program the layout information it needs to import a microarray image for use in analysis During the analysis process the user may want to know more information about a microarray spot with a certain intensity The Array Designer allows the user to associate descriptive information meta data with intensity for each microarray spot in the layout The Array Designer provides customized microarray layouts for GEML files Clontech Atlas Array Gene List files and Corning CMT Map files It can also design layouts for any other type of microarray that 1s described in a spreadsheet format The spreadsheet must include the X and Y coordinates for the array layout and the associated meta data for each spot on the microarray The Array Designer takes this information from the spreadsheet file and cre ates a visualization of the microarray which can then be imported for analysis When a layout is designed using the Array Designer the user will enter the Brand and Type of the microarray A microarray brand is usually the name of the product line e g GeneFilters while a microarray type is the specific kin
29. file can be 5 MB or larger Pathways image files can be read back into Pathways 4 A Pathways image file can be treated as a regular image file with the usual import procedure In addition Pathways 4 files can be reimported into Pathways without locating the clones because this information 1s already in the Pathways image file Therefore it is possible for example to change the sam pling technique for a file without finding clone centers Likewise it is possible to reimport and adjust clone centers if the center is found to be in error without performing global alignments 60 RGMA10011 rev B Chapter 6 Importing 6 1 Image Import Dialog The image import dialog sets the relevant parameters for an import session Open the import dialog File gt Import Image CTRL P mne lapai E ras fol ume mae xw bise J Pate 4 al ej att image firma A tines T Fhupiza utri Microarray beard eser ino J Tana i ninia ala M ma hpr GF 225 ir L3 pia Sarqng ira em IIT ex Bakh peres TH image FF Hemse anpe gnis in Loge A 7 Bama p715 i Sample i5 7 gt LA Peery ee Pe Pathways Universal users may see a completely different import dialog if they are using a Framework that implements importing other than the Pathways Framework Before adding images to the import window adjust the Files of type selection to indicate the appropriate image extension Next assure that the Image Format
30. follow this rule Method conditions for the text as a substring in the field contain at the beginning of the field start with as the end of the field end with or for an exact match equal Text the text for which to search in the clone data field After editing a Path click Ok to accept any changes and close the dialog box Cancel to revert any changes or Apply to apply any changes to the current analysis but leave the dialog box open 9 9 Path Filtering Path filtering allows including or excluding members from paths The Path filter window offers a list of paths and check boxes for showing members or non members of the Path Shoe members 5 3 Exrluded Clones J Prostate Cancer 44 tqDNA 97 RGMA10011 rev B The default behavior is to show both members and non members for all Paths To include only members of a path uncheck the Show non members check box To include only non members of a path uncheck the Show members check box Unchecking both boxes for a path excludes all data points because all points can be categorized as being either members or non members of a path When path filtering is applied to more than one path the results set includes the intersection of the modified filters For example showing only members of the path Breast and non mem bers of the path Cancer yields clones that are in the Breast path but not the Cancer path Showing only members of the Breast and Cancer paths
31. generates a menu that allows adding a microarray to the condition or renaming or removing the condition Right clicking on a microarray generates a menu that allows removing the microarray from the project Adding or removing conditions or microar rays to the project generates a prompt to close any active analysis windows that would be affected by the modification 16 RGMA10011 rev B 2 5 Detail View The detail view area of the GUI shows thumbnail pictures of the analysis window s currently selected clone along with a table of information on the selected clones The detail view area starts with web links then thumbnail s and finally an information table associated meta data The detail view area of the GUI may appear different if a framework is used that does not supply geometry The Web Links button at the top of the detail view opens a web browser that connects to Unigene Genecards and other web sites that display information related to the clone These sites can be expanded by either editing links through the Tools menu or through the pluggable web interface refer to the Data Updating chapter The forms of the thumbnail section depend on the grouping selected for the analysis Microarray s A thumbnail image is shown for the currently selected clone Microarray Pair s A thumbnail image is shown of the current clone from each of the two microarrays The ratio and difference of the normalized intensities are shown in th
32. left A through H in all fields The grids are organized into nine Columns and 24 Rows Columns are numbered 1 through 9 right to left in each grid Rows are numbered 1 through 24 from top to bottom in each grid Control positives are in Column 1 every other Row in all grids in both fields The spacing between each spot for GeneFilters Yeast microarrays is 1 000 microns from center to center 149 RGMA10011 rev B CE E ee OR Ee FB Ee Ocho n oly deben aly dee o oy OR Dn e csbBD cconncesanpocesanacs Chien d n ele Did a TAADA dera eera Ser eos i BS 000 Soe oS SSS ood 7 Oo SSO IO ata S 0ta F008 000 eaten SHG S oS Sooo CcCoOcCUOUCU m inch EH i is nr pr tae San aan TEE RM NETS HS ri orm 1 h T ti i nau mE dan ar DO SOG SESS SOS OS Sele OS Dob omimnocoesc ic TET Lo Tu ie E B Figure 3 Example of a GeneFilters Yeast microarray format all releases The control posi tive or total genomic spots are shown as filled black circles Data spots are displayed as open circles 150 RGMA10011 rev B Appendix II Migrating to Pathways 4 Universal II 1 Introduction Compared to Pathways 2 Pathways 4 offers additional capabilities like statistical analysis tools and graphical visualization of data Data storage has been improved in Pathways 4 with data stored in sharable files Other useful features include hyperlinks to online databases and an
33. menu it is possi ble to view the intensities of filters 1n an overlay This means that the intensity data of each fil ter 1s still viewed separately and no ratios are calculated In graphical representations of the data both the intensities of each filter are displayed on the same graph each with a different color coding In the same manner multiple sets of microarrays can be added into the microar ray pair s comparison analysis Pathways 4 also has an option for a new broader level of data analysis termed a condition By grouping data under a condition a researcher can include more than one microarray i e repeated experiments and even microarrays of different types 1 e ResGen GF200 and GF211 together in one group The behavior of that set of data ver sus that of another set of data i e condition A versus condition B can be analyzed by compar ing Condition pair s Other Pathways 4 analysis tools are profiling and clustering 152 RGMA10011 rev B II 4 Normalization and Data Filters Because of the large amount of data generated by microarray experiments normalize and filter data Select the way that Pathways 4 normalizes data from a preset list or even customize the process The software extracts the information to display intensities and calculate ratios from the normalized data In Pathways 2 0 intensity ratios are displayed in a fashion to show the upregulation and downregulation of genes respectiv
34. other centering modes that can be used to recalculate the positions of spot centers Profile Profile autocentering scans the area around each spot and finds a peak intensity both horizontally and vertically The two intensities are cross matched finding the area of highest intensity which is usually the center of the spot This mode is useful when the image is high contrast Centroid Centroid autocentering scans the area immediately around the current position of the spot center The center position is moved towards the nearest area of higher intensity This movement occurs until all the areas around the center position are of lower intensity This mode is useful when the image is low contrast The user may need to experiment with different Autocenter modes to find what works best The Color by field allows the user to set which microarray attribute to use in differentiating color between the spots in the image allowing for more accurate visual comparisons when cre ating a bounding box or template In addition the user may set the field to None to show the location of the spots The Color by field affects only the image during the layout design stage In the upper left section of the window three buttons appear The first button allows the user to create a bounding box P Array designer dE f Trim size 4 0 Select bara opposite comer pomts of the bounding box or press ESC to restart A bounding box is automati
35. tags that focus on a particular type of data XML based tag sets are designed according to their content allowing them to provide specific information about their content and how that content relates to other data They are self describing eliminating the need to include extraneous documenta tion when transmitting data The Gene Expression Markup Language GEML is an XML based tag set that provides a method for exchanging gene expression data and related annotations Rosetta Inpharmatics developed GEML as a means of transmitting data between different gene expression systems databases and tools GEML separates data collection and reporting from the methodology used to collect and report that data enabling the analysis of data derived from differing method ologies through the use of the same syntax Pathways Universal features a GEML compatible framework that takes GEML files and translates them into a format the program can use in the analysis of data Pathways processes GEML files directly with the GEML framework Other gene expression data files can be converted to GEML files through a program like the Rosetta GEML Conductor The GEML framework does not provide an import tool so new microarrays must be added by browsing the file system In this way adding a GEML file is similar to adding a spreadsheet file To add a new microarray to the project from a GEML file select the project condition to a
36. test plug in is extended from a manuscript by Yidong Chen Chen Y et al J Biomedical Optics 2 4 364 374 October 1997 ISBN 1083 3668 This statistical test is based on an assumption that the coefficient of variation is constant across microarray condition data points Before using the plug in care fully review this manuscript including the assumptions involved in derivation of the test The manuscript limits the test to an assumed distribution of the data The Chen test plug in as an option extends the test to a distribution free form The t Test is a statistical analysis plug in that determines whether two sampled intensities are different based on a desired confidence level When a clone is not filtered at a confidence level then the difference in intensity between two samples is statistically significant at the specified confidence level This test 1s applied to paired data with repeats and is limited to condition pairs The t Test plug in is an implementation of the commonly used Unrelated t Test Student s test Unlike the Chen test this test involves a single microarray sampled multi ple times It determines whether the difference between two condition averaged clones is sig nificant compared to the standard deviation of the sampled intensities for each of the condition averaged clones This plug in offers both a Gaussian distributed and a distribution free form Analysis of variance ANOVA is used to test if the differenc
37. the top of the window allows toggling the grid on or off displaying of x s to mark invalid clones exporting the sampled data to a spreadsheet file comma separated text file or annotating the image with the researcher name and comments To bring clones into focus in the detail viewer Click the image at the appropriate location or Use the up down left or right arrow keys The detail window on the left of the screen can be used to zoom in and out of the image to bet ter review the overall alignment of the image up arrow key zooms in and down arrow key zooms out when the cursor 1s in the detail viewer 40096 1 If the overall alignment of the image appears to be inaccurate click the Back button and repeat the initial alignment step When repeating the alignment step add a cropping window or over lay a template for 1mages that are problematic If clone alignment appears to be inaccurate then 71 RGMA10011 rev B this clone can be adjusted individually To adjust clones perform the following steps 1 Select the clone so that the appropriate clone is in the detail window 2 Press the Ctrl key and drag the alignment circle in the detail viewer Click the Done button to complete image import When additional images are specified in the import dialog box the next image loads automatically When no additional images were speci fied the import process stops 6 6 Invalidating a Clone For microarrays origi
38. upregulation or downregulation of clones over two experiments Profiling analysis views data as a function of experimental conditions e g determination of the general trending of a subset of genes in a time course study Clustering analysis generates associations between clones in a data set automatically 14 RGMA10011 rev B Each analysis menu has four submenus that represent how microarray data could be grouped for the analysis Microarray s Microarray Pair s Condition s Condition Pair s Profile Cluster Microarraytsy k Microarray pairis Conditions Condition pairis These analysis modes and the analysis groupings are discussed in detail in later chapters The Tools menu offers access to the Quick Start palette Path editor an internet Browser win dow and a Web link editor Updating of Pathways data and plug ins is also launched from the Tools menu The Array Designer is only available in Pathways 4 Universal refer to Chapter 4 for more information on the Array Designer ES Help Window show Guick Start Path editor Browser Ctrl B Edit Web Links Ctr eA Update Pathways Array Designer The Help menu offers access to help on the Pathways program including general information about the program and a searchable version of the Pathways manual The Windows menu presents several options for managing the active analysis windows in the workspace For details refer
39. 1 GF211 Project GF200 GF200 GF200 GF211 76 RGMA10011 rev B 7 2 Conditions Conditions can represent any experimental grouping The most common uses of conditions are for state comparison e g normal versus diseased or for time series comparison each condi tion represents a time in the study Each project can contain as many conditions as are neces sary to represent the study Microarrays of a single condition need not be of the same type Instead of analyzing data sets one microarray type at a time which could for example limit the analysis to approximately 5 000 clones for GeneFilters the data set can comprise multiple microarray types enabling analysis of an unlimited number of clones Repeated clones in a condition are detected automatically during analysis If a condition contains repeat clones then the analysis uses the mean of the normalized intensity for each set of repeated clones In addition the existence of repeat elements enables the calculation of experimental statistics for the sampled data sets Pathways searches for repeat elements based on one of two entered methods microarray address or clone key A Compare Conditions Control Manth 1 Manth 2 Manth 3 C Average by Clone Key The microarray address method identifies repeats based on common microarray types physi cal location For ex
40. 5 unz mnhrctmrg pin 7 D AALIS rura irre erret notar baia 5 E 15 5 Example 2 Complex Time Study The following example describes the use of Pathways for a more complex project a time study with repeat data The example offers an overview of the analysis capabilities of Pathways and there is a brief description on the creation of the project and the analysis win dows First an Empty Project is created from the new project wizard Conditions and microarrays are added using menus accessed by right clicking in the project tree 4 Control Trials Control Trial 1 F200 Control Trial 2 F200 Control Trial 3 3F 200 Control Trial 1 F202 Control Trial 4 F202 Co Condition Add micraarrayre 92592529 LS A Rename condition Remove condition 1_ 1_ 1_ 1 Ier 1 Month Trial GFZ 2 1 Month Trial 3 GFZUZ 3 2 Month Trials 2 Month Trial 1 GF200 2 Month Trial 2 GFZ U 2 Month Trial 3 GFZ U 2 Month Trial 1 GFZ Z 2 Month Trial 2 GFZ 2 2 Month Trial 3 GF202 Month Trials 3 Month Trial 1 GFZ 3 Manth Trial 2 GFZ l 3 Manth Trial 3 GFZ U 3 Month Trial 1 GFZ Z 3 Manth Trial 2 GFZ 2 3 Manth Trial 3 GFZ 2 a e OE E E E Sui E E EEE ThE E EEEE This project shows four conditions in a time study Control and 1 2 and 3 Month trials Each condition has two microarray types GF200 and GF202 Each microarray is repeated in tripli cate for each condition Repeated elemen
41. 61 Importing Process 57 130 163 Index column 108 112 120 Intensity 18 89 163 Interactive importing 57 64 Invert 92 Key in 92 KMeans clustering 113 115 143 License Agreement 156 Log 93 106 111 Look and feel 24 Magnifier 69 131 Menus 13 Meta data 18 163 RGMA10011 rev B Index Microarray address 77 163 Microarray address paths 94 Microarray brand 58 61 Microarray grouping 78 89 Microarray name 58 61 Microarray pair grouping 78 89 Normalization 81 84 85 153 164 Normalization Groups 87 164 Normalized Intensity 18 Outlier 89 Path Filtering 97 98 Path Normalization 85 Paths 94 164 Pathways 2 10 Pathways image 58 61 PDF File 99 Plot 106 110 Plug ins Pluggable 10 57 58 59 62 85 89 114 164 ratio 17 93 164 Printing 99 Profile cluster 119 Profiling 109 139 Progress Bars 23 Project Tree 16 Projects 76 79 164 Proxy 24 Quick Start 20 129 164 Refresh 84 Reimport 60 61 165 Repeated clones 77 Reports 99 135 136 144 165 Requirements Hardware and Software 10 Reviewing alignments 71 Sampling Data 61 165 Settings 24 165 Single Microarray Projects 79 SOM 113 116 117 Strict 91 Synthetic Microarray 103 165 Table 108 112 120 Template 59 69 131 165 Thumbnail 60 166 Tiff 58 166 Trim 61 Two Microarray Comparison Projects 81 Unigene 17 123 124 140 Updates 123 125 153 Web Browser 123 Web Links 17 123 140 Workspace 15 Y C Normal
42. 655813 295 053TT 41813345 28122487 11705837 TEER IL 3 00709845 D f5O0B1TS 0 26841683 0 0691 7474 0 22027412 22853753 D 1 38301 3 rh keid TT Wiens i Emir E 14 ir 2i 31 5132728 4 1600185 8047364 3 8524403 3 4765 14553783 4 2719BB07 1 037 1228 0 340388 0 07320428 0 31 site 3 204 5B7 4 0 1551352 hoa a are s by il iz 563353 5 54247 33 E 192466 55406304 54058705 023708938 53172008 151778547 058574224 n13739115 0 48912028 34876008 1805554 lel 1517900 MENE 62788 030252 1 00224 0 82588 0 42800 0 05833 0 01902 002641 D OGOBT DOOT14 When multiple microarrays or multiple conditions are presented microarrays appear in tabbed panels as shown above Each column can be sorted by clicking the label at the top of the col umn Clicking the label again toggles between descending and ascending ordering The index column labels on left side of the table can be modified using the buttons at the top of the table Options for indexing include the clone key microarray address or a meta data item RGMA10011 rev B 108 Chapter 12 Profiling 12 1 Introduction to Profiling Analysis Profiling analysis determines the data trends from one experiment to the next One example of profiling analysis is plotting the intensity of genes associated with cancer at multiple times in a study The microarray s or condition s group
43. 854 rias AALIT TET 1358218 IB 1192247 D TEQ4SJBR Brings ue Faia 8 228 000 D z78Bb3B ID 2424217 o 288161 D 44BBTTBE 0 054100224 B iz 370403 B8 LS IBIBE 4 DU3273810 DOT oe COSI LIZ UARN LIGETI LoL SBS D 1202747 b rejas nd n17311058 TEDZSDSE Laguna rr LL ba berry a 151 GRIT oe piii 0 n and 8 45284 QE 4 09213818 E arioso TALEE 003667110 n 042128854 D1302747 b 163453 isis D TES aps be D2290 D Impr UEST D 2265151 D 44 GB1T81 M SET Items of analysis data e g intensity are displayed in a tab panel as shown above Each column can be sorted by clicking the label at the top of the column Clicking the label again toggles between descending and ascending sort ordering The index column labels on left side of the table can be modified using the radio buttons at the top of the table Options for indexing include the clone key microarray address or any meta data item 112 RGMA10011 rev B Chapter 13 Clustering 13 1 Introduction to Clustering Clustering analysis automatically generates associations between clones in an analysis set For example clustering could find clones that respond similarly upregulation or downregulation over the course of a study Mathematically clustering algorithms locate points that are close together in a multi dimensional clustering data space Clustering is complex and a complete explanation of the clustering pro
44. Adding microarrays with the Library tab rIT sr Byndabis Coreiiian A Frwreswrk Faire vy D Bang pra V Litres mieis Sara Grrr Oris Thre tf 1 Tere E 2 Heni 1 1 Mrri i dorm 7 d Minh 2 1 Ted L9 16 5 COT 28001 Mrrin F a Ferree nui 7 Criginad 3 C yop Firin Pete imple 05 1 nF i E a D Hii i i a d L r 83 RGMA10011 rev B Adding microarrays with the Browse tab Ag eres Aree ern ab s Corian A Frera Frane x D bara Jg525 Lima Derena Lam jPluireana mj m a em i Gaughan i Tir iih Pa IFTA L tTagls rtieredialie Y omm piaba Fiba para Bansi gI i ps Fiar rd kar Tahara Camps Fiia prorvi Clicking on a microarray in either section of the dialog box activates the Add button in the dia log box To add a microarray to the condition click the Add button or double click on the microarray To remove a microarray from the Condition select the microarray in the condition field and click Remove Clicking the Refresh button in the library section of the dialog box verifies that each file in the library still exists and that the library reflects each file s correct microarray type In addition to the basic project creation options discussed above the Edit menu allows the fol lowing modifications Changing project properties researcher name project title Editing normalization options refer to the Normalization chapter Renaming or removing cond
45. Contrast Controller A contrast controller is available when an experimental image is displayed in Pathways The contrast controller is visible in the left pane of the interactive importing windows Otherwise the contrast controller can be accessed through right clicking on any experimental image such as on the thumbnail in the detail view vial Links Tene O T 1 FILE NC onginal image Save hhumbrailimage Update image lacstion walks cina Add cns t bey pain F Ahd cine 1o address pat P Acid ciana 1o naw naf The contrast controller allows enhancement of experimental images Contrast slider generates minimum contrast when the slider is to the left maximum contrast when the slider is to the right Auto automatically adjusts the brightness levels of the displayed image to the minimum and maximum intensities for the experimental image Invert inverts the image intensities for example black on white image is displayed as white on black Color indicates whether the image is displayed in grayscale or color In addition to the standard settings the contrast controller for the thumbnail has an option to display the entire original image from which the thumbnail is being displayed to save the thumbnail image and to update the location of the saved image Changing the contrast for the thumbnail image changes the contrast for thumbnails that are dis played for the same microarray However this change does not affect
46. DU IHI i E taar 507175 9 0i84b 3785475 CEST AT i 58 C J og reed 5 11207386 8 37 73 5 9 302681 4 1 50 2B 24 LST aor hrTT83B t amp 45D061 8259 22707301 t 7318708 t 1857407 Fey m r iin I 8H2560 2b t fear 2 2589834 LFF EAR 1 36 A The rows of the table include the cluster number and data values for contributing microarrays or conditions Each column can be sorted by clicking the label at the top of the column Clicking the label again toggles between descending and ascending sort ordering The index column labels on left side of the table can be modified using the buttons at the top of the table Options include the clone key microarray address or a meta data item 120 RGMA10011 rev B 13 10 Clustergram The clustergram plug in generates the same information as the cluster table above but colors are used instead of numbers The vertical axis of the clustergram represents a clone number and the colors across the clustergram represent a variable for each contributing microarray condition or microarray condition pair Cheri E del ans Custer Asutis gt exl cl Winualzation Matos 13 11 Hyperbolic tree The hyperbolic tree plug in displays the cluster as a tree of interconnected clones and subclus ters Chevint Hear Chica Ce ing Fursuies L P x 5 e e visualization Method Tree Gusar pakaian ir Lebel Key T Adres o Mebedaa S
47. El ResGen Pathways 4 Software User s Guide For research purposes only International customers refer to www invitrogen com for technical support contact information RGMA10011 rev B Table of Contents Book I Introduction and Overview Chapter 1 Highlights 1 1 1 2 1 3 loci d vr Highlights of the Software 1uucsca aurem dh o Ege m Rm notie a Pew woo dox deg d oes Hardware and Software Requirements 0 0c cece ee eee eens 1 4 Architecture of the Program gen ura eke ee ee aoe a he ee o8 acd 153 1 6 1 7 Pluggable IMriaceS PTT Compatibility with Previous Versions of Pathways 0 000 cee eae Comparison of Pathways 4 Universal to Pathways 4 GeneFilters Chapter 2 Overview of the Graphical User Interface oa 22 2 3 2 4 2 3 2 6 2 2 8 2 9 Layout of the Graphical User Interface 22s ee oh kd die ee le es dnm ees hun Aa P P Or m bacon peren erann ea dee coe eee even eh oa ee eee ee yee ee eo lucc poker sn bees eeu pate ees eu tea dae ee ee ee eee eee EEE oy Detall VieW SeCPT we Piet ViCW esau baw ewes oa ae Soe ew he ee ee ew eee ee we ar Be ee UIC DIAG C ontpasE C OBBPOIIC E uuu egy rao bed ou Od Re deh ee ee ee eee ee ae oe Be oe PrOctess Das diay caeces chan eee eee be eae eee ee ee ee ee eee 210 GenehalSCUINOS ao ee ee ee a Se o
48. IGeneFilter Micraarray type IGF225 Sampling type ResGenmean Ke Batch process GEBE Trim image Reimport E White on black Click the Edit plug in properties button located next to the Sampling type field Sampling type Basic Batch process Edit plugin properties Trim image i 63 RGMA10011 rev B The Sampler plug in Settings window appears The spot sampling percentage may be adjusted by moving the slider bar SamplerPlugln Settings Spot Sampling Percentage The Basic sampling type is a more generalized version of the ResGen mean sampling type that allows the user to adjust the spot sampling percentage Instead of determining the back ground level by sampling intensities in the center of the image the Basic sampling type samples a strip around the edge of the image The spot sampling percentage is set to 75 but the user should experiment with different sampling percentages for different microarray types 6 2 Introduction to Interactive Importing Interactive importing is launched after clicking the Ok button in the Import dialog box without the Batch option checked Interactive importing consists of five steps for each image 1 Load image 2 Align Crop Template 3 Compute centers 4 Verify centers 5 Write output Interaction with the import process is required for only Steps 2 and 4 Pathways performs other steps automatically The import window shows the current step highl
49. M UN HN a A Pathe 1 25 4 94 eee 16 03 19 72 23 42 27 11 30 81 3 The window above shows a simple data filter applied to the Ratio of normalized intensity The histogram is being used to filter data from the current analysis window To use the histogram to filter data drag the edges of the histogram until the hashed data regions encompass only the desired range of data To key in limits check the Key in box and type the min max limits in the text fields The standard mode for the histogram is to include only data between the min max values spec ified Min less than or equal to Data less than or equal to Max The Invert check box selects only data that are outside the min max bounds Data less than or equal to Min or Data less than or equal to Max This option affects both the visual histogram filter and the Key in of min or max values 92 RGMA10011 rev B To generate a menu with options for enhanced viewing right click on the histogram The Ratio menu item appears for only the Ratio filter This option reformats the histogram by the upregulation or downregulation format for ratios if intensity B gt intensity A ratio B A oth erwise ratio A B icu SSSNNNNNNSNNNNSNS e E L A1 aH N aN S E Jm b Ti gt ce 37 9 ET 521 80 6 39 6 99 7 58 8 1 The Log option creates bins based on a logarithmic rather than a linear scheme This option works well with leveling data in a histogram that ha
50. Macintosh OSX or any platform supporting the Java 1 3 runtime environment Video SVGA with 1024 x 768 resolution or better 256 color palette or better 1 4 Architecture of the Program The Pathways 4 software is written in Java 1 3 allowing significant flexibility in the choice of operating systems extensibility of the program and ease of internet data access 1 5 Pluggable Interfaces Pathways is designed with multiple pluggable components that allow the program to be extended at runtime by placing new java jar files in the appropriate Pathways distribution directory Pluggable components include sampling clustering core algorithms and visualiza tion normalization data analysis image formats microarray descriptions and web interconnec tivity New plug ins can be created by ResGen third party software vendors or end users 1 6 Compatibility with Previous Versions of Pathways Pathways 4 represents a significant advance in technology over Pathways 2 0 The algo rithms for autocentering imported images sampling and normalization have been revised and improved As an improvement on Pathways 2 Pathways 4 includes the following analysis capabilities Statistical analysis Clustering Multiple modes of visualization Furthermore Pathways 4 stores microarray data in sharable files whereas Pathways 2 0 stored data in Microsoft Access databases These improvements mean that microarray images tha
51. Network updates are the initial default To change this setting simply select the CD option and Pathways will attempt to update from a CD when the update tool is invoked CD updates are recommended only for those with internet access problems ESTE Applicaton parramewcek General Re searrrer ohn Smh Look and fogl f System C Java Load last onyect Hide clone cursar Auring snapshots F Internat Lindate Sources Network CO Browser tani ce ha Lise proe After setting the update source to CD CD updates may be accessed from the Tools Update Pathways menu item When the user selects Tools Update Pathways a file dialog will be displayed Select the CD drive that contains the Pathways Update CD by single clicking on the appropriate drive letter as shown below Click Ok to start the update process 127 RGMA10011 rev B After the CD update has started the dialog for selecting and updating data files and programs files is identical to that of the Network updater Pn Look in File name Files of type Ali Files d Cancel When either a network update bad connection update server is down et cetera or a CD update unreadable disk incorrect cd location fails Pathways will display an Update Interruption Failure dialog as shown below This dialog contains four choices The user may retry the update change the update source to CD change the up
52. Path Hormalizascn YO Harmnalizeabon This nonmalization algontien doesnt mqure any add anal inputs If items require modification click Back and make necessary modifications Clicking Finish creates a single microarray project and opens a comparison analysis window 7 6 Two Microarray Comparison Projects A two microarray comparison project offers a mechanism for generating a project that com pares two microarrays The Project Wizard steps for the two microarray comparison project are identical to those for the single microarray except for the microarray selection page 81 RGMA10011 rev B fe alain Tiers ramh raters Librai Barana LIII uririgbis J Cana de J Sora amp Timeii_i s Time E mon T E onmi I amp Boh r Mom J impari duo B bon Tha Ja jE 25 4 BCT XC E Bod J EUIS Legien image mmp gprz225 1 COH Fieri m T uh gau Acamph pl ld a ali TE mnm Eumipiiag ipi Far hr reu First sid Le Pr Be HP ea DET 119 pes Lidl beconj amr x Prog iph e Fra beer q5 pa Sha Each microarray for the comparison is selected by clicking on the desired microarray and then clicking the Add button for the first array A second array can be selected similarly If two arrays are added and the Clear button is clicked the second array will automatically move to the first slot Once the wizard setup is complete a two microarray project is created and a comparison analy sis window is displayed 7 7 Empty P
53. PeoDa Brand OT Gp Cid WeumutpAmes o sz wes 2j area Rete att her ann mm Aievitelions ean be entekegd eit The first time a spreadsheet layout 1s being used a profile of the layout must be created The program remembers this profile so that future microarrays using the same spreadsheet layout may be imported without resetting the parameters The first screen of the Import Spreadsheet wizard contains fields where the microarray Brand File Layout and Type can be set The File Layout 1s the most general descriptor it contains information about the layout of the spread sheet The same File Layout may be used for many different Brands Similarly the same Brand may contain many different Types In addition Experiment and Researcher names and Annotations may also be entered on this screen Once the layout has been created click the Next button to continue 32 RGMA10011 rev B If the file layout has been created the next two steps may be skipped by clicking the Finish but ton The microarray is imported without changing the layout Clicking the Next button will generate a window asking if the user wants to Edit or Copy the layout Rs Question x 3 A layout Affymetrix already exists Do you wantto edit ar make a copy af the layout Cancel Set the layout of the spreadsheet on the second screen of the Import Spreadsheet wizard A jmpa Sponedabeet Step 2 al T Ths nage
54. Stage After the layout information is specified the Array Designer window appears This window creates a view of the microarray from the layout information in the file The fields along the top of the window allows the user to adjust certain attributes of the microarray and of the importing process Trim size allows the user to set the amount of space to trim from the edge of the image If the user specifies a Trim size trimming occurs after centering during the import process The des ignated Trim size is cropped from the edge of the imported image The units used to calculate this space are the same as the units in the input file e g pixels Spot size allows the user to set the size of the microarray spots using the same units as the input file Spot shape allows the user to set whether the microarray spots should be circular or rectangular Both Spot size and Spot shape are considered during the import process after centering has been completed 51 RGMA10011 rev B The Autocenter mode allows the user to set the mode to use to find the centers of spots When a microarray is imported the program calculates a seed position for the center of each spot However many factors can introduce noise into the image causing the positioning of centers to be slightly off When the microarray image is clean and uniform the Autocenter mode can be set to None to keep the positions of the centers where they are in a rigid grid pattern There are two
55. TWARE PROVIDED CLICK CANCEL BELOW AND RETURN THE MEDIA CON TAINING THE SOFTWARE AND THE ACCOMPANYING ITEMS INCLUDING WRITTEN MATERIALS AND PACKAGING AND ALL COPIES THEREOF TO THE LOCATION WHERE YOU OBTAINED THEM FOR A REFUND PLEASE NOTE THAT YOU WILL BE REQUIRED TO REGISTER THE SOFTWARE PROVIDED WITH THIS AGREEMENT PRIOR TO USE 1 LICENSE GRANT Unless otherwise authorized by ResGen under a separate agreement ResGen grants you a limited non exclusive non transferable license to use the SOFTWARE on only one 1 computer Further you agree to not load the SOFTWARE on a file serve with out first obtaining permission to do so from ResGen You also agree that you will only copy the SOFTWARE into any machine readable or printed form as necessary to use it 1n accordance with this license or for backup purposes in support of your use of the SOFTWARE This license 1s effective until terminated You may terminate it at any point by destroying the SOFT WARE together with all copies of the SOFTWARE modifications of the SOFTWARE and all supporting written materials and packaging and certifying such termination in writing to ResGen Also ResGen has the option to terminate this license if you fail to comply with any term or condition of this Agreement You agree upon such termination by ResGen to promptly destroy the SOFTWARE together with all copies of the SOFTWARE modifications of the SOFTWARE and all supporting written materials a
56. alifier f Start with row 5 r Header row 4 Delimiter Delimiter Comma Ignore cons Data preview LPositive LHegative F airs OFP airs Used Pairs InAvg CF ds AFFX MurlL2 atC5LT 2002001 900 250 0 8900 7054 AFFX MurlL10 atLBr5rearguarmasrp 45rm ssrmn arms i AFF MurlL4_at o OS e 0 A S 00 20 00 17 ON 302 blio AFFs MurFAS ath OOS DAO e A S 00 50 02 24 0 3 096 0 EP ET 0 35 00 1561 In the Delimiter field select the character used to separate columns in the spreadsheet The Affymetrix spreadsheet in this example is separated by the Tab character If the Ignore consec utive delimiters box is checked Pathways treats two consecutive delimiter characters as one Delimiter Delimiter Tabl Ignore consecutive delimiters Data preview irs Pairs Used Pairs In amp vg Pos Fraction APP sur at 20 19 0 25 19 0 45 2 AFFX MurlL10 at Ping 20 18 0 20 3 AFFA MurlL4 at 20 34 RGMA10011 rev B Once these fields have been selected click the Next button to continue The third screen of the Import Spreadsheet wizard appears ML 5 pucadishew Step 3 ul 1 This nage wal leben sign 3 sp ps B 9 interes ror mete cate fo each coum rag must designe ene cand onte one inten eg column 3nd a piirit key Berk re fi eius Cmumn karen aimi Faim Li ge zi Fairs inisg z Dati Sree zs Pare Pars Used Pareinteg Pos Fraction FFL m 2D H tg D 25 4 AFF N MuriLI0_at 3 3 mG 1g D 45 d AFFH MonLA wi 3 2 tg D 20 A
57. ample when two GF200 GeneFilters are present in a condition and the microarray address method is selected the first clone in the first GF200 microarray is averaged with the first clone of the second GF200 the second clone in the first microarray with the sec ond clone in the second microarray et cetera standard deviation values for the repeated clones are calculated for use in the analysis process This type of averaging does not average multi ply spotted clones in a microarray if a clone appears in multiple locations in a microarray the clone key is the same but the microarray address is different TT RGMA10011 rev B A clone Key is a unique identification that is present for each clone in a microarray The clone key is usually the accession number of the clone although the key could follow other naming conventions for biological materials that are not in the public databases for example ResGen uses the string tgDNA to identify total genomic spots The option to identify repeats by clone key searches for repeats of a clone key and groups these together Averaging by clone key enables statistical analysis for repeated clones in a single microarray and also enables Statistical analysis of the same clone across different microarray types When it is undesirable to average repeated experimental results place the microarrays in different conditions 7 3 Grouping of Data There are four ways of grouping data in a new analysis w
58. around the microarray The algo rithm finds the clone locations as before but it does not look outside the cropping window The cropping rectangle is available only during interactive importing In batch mode it is impossible to specify the cropping rectangle refer to Chapter 6 for more information about these importing modes With template centering drag a template a sketch of the microarray layout on top of the exper imental image and adjust alignment points until they match the experimental image exactly The centering algorithm uses the template to identify the clone locations rather than searching the entire experimental image The most common use of template centering is when the autocentering algorithm fails during importing In addition an alternate microarray product might not lend itself to autocentering for example when there is no periodic identifiable pattern to the clone layout In this case the alternate microarray product would rely on the template mode for image importing 5 5 Sampling Microarray Data When the clone locations on the image are known Pathways samples each clone s intensity and background In general the sampled data for each clone includes an intensity and either an overall background intensity or a background intensity per clone The values of the clone and background intensity are determined with the sampling plug in to allow sampling algorithms to be added 59 RGMA10011 rev B 5 6 Pat
59. arriprzizas rcx Ea amirarad hara cinder i Shree error Shore peat add ie emere The output format printer PDF file HTML file CSV file can be changed through the Output to selection For the printer output format a Preview option is available The Researcher 99 RGMA10011 rev B Project and Description fields are editable The Series section contains options for printing the Selected experiment or All experiments Selecting All shows data for all experiments in a single table and should only be done when outputting to a CSV file The check boxes on the right of the dialog box allow customization of the report to include the data source intensity ratios et cetera and meta data accession title et cetera The Show error option on the Report Wizard adds a report column for the standard deviation of a condition s intensity The Show point validity option adds a report column for whether or not points are flagged as invalid If the user wishes to exclude invalid points from the report apply the invalid clone filter before generating the report When the report is configured click the Print button When the output is being sent to the printer a Print Preview window appears from this window the document can be sent to the printer When the output is being sent to a file a file chooser dialog box appears The report formatting that generates the printer PDF and HTML output is time consuming with large data
60. ation 0 0 0 0 eee ene 85 8 2 Pathways Normalization Algorithms 0 0 0 ccc ee eee nes 85 8 3 Normalization Groups 3 2 dox hsdcuaghianbpeds V 39 93 39 13 ORC RC 309 RR eee 87 Chapter 9 Data Paths and Filters 89 9 1 Analysis D ta C 89 O 2 Data Filenin AM V 90 DO OUICLOCUING uoo vals ado doh ah R UAE ennt bo ES RASSE e ERE XAR ET ues 4 T5 9 9 4 Simple Data Filters CTTr 92 9 5 Statistical Data Filters piu thangs nah headend dub ron SF VOU P Ear E dr RO CPC RR did ent 94 Mc D 94 9 7 Creating a New Path vawetichernmheuete oe Gh REN P Era E Sob oh et eee e 95 DIS Editing PAVAS 2668424 65465540 604 54h ae 922222 292255525445 9224253 49 2 9 96 9 9 Path Filf rning oo sua ne ows hh ede pha ehadeed thaw ee eee bee eedewh ae bah ome oF 9 10 Invalid Clone Filtering uus aos we dec bandas donde eben bebe e een tines nee ee 98 5 RGMA10011 rev B Chapter 10 Reports and Exporting Data 10 1 10 2 Pathways Reporting 2 0 0 eee eee ees Repost Wid nm Book V Pathways Analysis Chapter 11 Comparison 11 1 11 2 11 3 11 4 11 5 Introduction to Comparison Analysis 0 0 0 cece eee eee ene Comparison Toolbar ua du uS bees e ESSERE ER Ps eur dst dca d NUS V vadI I ire Microarray rrr eeunmddu PETC
61. ation see http www corning com CMT Products CM TGeneArray asp Compare two microarrays This button on the Quick Start Palette initiates a Two Microarray Comparison Project Conditions Conditions represent states of previous imported microarray data in an experiment Each condition can contain one or more sampled microarrays that do not have to be of the same type e g GeneFilters GF200 could be grouped with GF201 Control points Control points are the landing lights or positive controls that are used for ori entation and in the Pathways alignment process for ResGen GeneFilters Cropping rectangle A cropping rectangle is a rectangular box that is used in the importing process to better identify the location of a microarray in an image Difference In a Microarray Pair or Condition Pair analysis window difference is the numerical value resulting from the subtraction of the Normalized Intensity of one clone on the first data set from the Normalized Intensity of the same clone on the second data set Filter Pathways data filters reduce the amount of microarray data that is displayed in analy sis windows or shown in reports Data filters can restrict the displayed clones to a range of intensities or ratios and or to a minimum level of statistical significance and or to be mem bers non members of selected paths Framework A Framework is a collection of modules that work together to provide a method of importin
62. ation and cluster ID it would be entered as http someplace com mylink UG build_version amp CLUSTER cluster_id Determine the appropriate pattern for a web site by visiting the link directly in the Pathways browser or any other web browser and observing how the URL changes when for example different clones are examined online To edit an existing link highlight the link and click Edit The Link Name and Link URL fields are activated for editing until Abort or Apply is clicked the Abort and Apply buttons are present during the editing process To add a new link click New and enter a link name and URL When finished click Add or Abort to add the new link or abort the edit the Add and Abort buttons are present during the editing process Certain web links require a specialized plug in For example a Unigene cluster search requires the organism and cluster ID to be separated the link looks like http amp ORGzorganism amp CIDzclusterid The Unigene cluster plug in splits up the cluster ID e g Hs 2 to yield the desired web link e g http ORG Hs amp CID 2 124 RGMA10011 rev B 14 3 Introduction to Pathways Updates Pathways data and plug ins can be updated on a regular basis from the ResGen data server These regular updates offer the following enhancements Data updates description files for each GeneFilters microarray updated to reflect for example a new
63. available update service to the ResGen Data Server Pathways 4 has the flexibility to accommodate other commercial or custom microarray formats rather than ResGen GeneFilters microarrays alone This software supports other image formats Pathways Tiff and Fuji image formats may be imported and there the software can be extended to include other image formats From image format and sampling to normalization and data analysis Pathways 4 can be customized Along with many others these changes allow Pathways 4 to perform as a complete package for the analysis of differential gene expression using microarray data II 2 Image Import and Alignment Image format and microarray format capability Pathways 4 supports multiple image formats including Pathways Tiff and Fuji formats Because different researchers have access to different scanning equipment other image formats can also be accommodated Unlike Pathways 2 0 Pathways 4 can be used for microarray formats other than ResGen GeneFilters microarrays The software can be customized to analyze microarray products marketed by other vendors or even on custom arrays through the use of the array designer The analysis tools featured in Pathways 4 Universal are made avail able to researchers no matter what type of microarray they decide to use for their experiments These features are unavailable in Pathways 4 GeneFilters9 Batch import Data from Pathways
64. cally created around the edges of the image when the layout is designed The bounding box serves as a global template for an array layout It tells the pro gram where to look for other templates When it is not properly aligned the user may create a new one manually 52 RGMA10011 rev B To create a new bounding box click on two places on opposite corners of the area to be bound ed The bounding box appears The bounding box provides Pathways with a rough sizing esti mate for any templates that may be present 53 RGMA10011 rev B The second button in the upper left section of the Array Designer window allows the user to create a template 7 Array designer m Trim size 4 Select four corner points of template or press ESC to restart Sometimes microarrays are laid out in sections Templates allow the user to mark the bound aries of these sections so that the program recognizes that the microarray is not uniformly arranged for more information on templates refer to Chapter 6 and to Sections 4 3 and 4 4 To create a template around a section of a microarray click the four corners of the section The template appears around the section The third button from the left in the Array Designer window allows the user to delete a tem plate P Array designer CEB Trim size 4 0 E Select a template to delete 54 RGMA10011 rev B To delete a template first move the cursor over one of the e
65. cated and comprehensive set of tools for the analysis of differential gene expression using microarray data The following highlights are included Batch importing for rapid automated importing of multiple microarray images Support for multiple image formats including Tiff Fuji and Pathways image formats Multiple views of each data set and analysis results including scatter plots tables and synthetic images of the microarray Statistical analysis of data sets including unrelated t tests and ANOVA Isolation of genes in large data sets by filtering based on user specified criteria Multiple clustering algorithms including KMeans Hierarchical clustering and SOM An embedded browser enabling hyperlinks between a clone and public sites like the National Center for Biotechnology Information s NCBI s GenBank and Unigene databases Updating of default data files and plug ins from the ResGen Data Server to ensure that data for each clone is always current subscription service Java architecture for ease of cross platform use Multiple pluggable components allowing Pathways functionality to be extended by ResGen third party vendors or end users RGMA10011 rev B 1 3 Hardware and Software Requirements Processor Pentium II 400 MHz or better Memory 256 MB RAM Hard drive Core program 45 MB including Java runtime environment and two sample images Operating system Windows 95 98 2000 or NT Linux Solaris
66. cess 1s beyond the scope of this guide The following sections are an introduction and overview of clustering analysis Clustering data space is based on the analysis grouping and a variable For example a cluster analysis of normalized intensity of three microarrays might group clones that are close together in a three dimensional space defined for each clone as intensity in first microarray intensity in second microarray intensity in third microarray An effective clustering algorithm groups clones that are bright bright bright across the three microarrays Likewise clones appearing as dark bright dark dark dark dark bright bright dark et cetera over the three microarrays are grouped together Therefore this analysis seeks clones that have similar expression profiles across the three microarrays As another example a time study could be clustered based on ratios between each time point and the control which is at time zero Assuming four time points plus the control the cluster space would be four dimensional time 1 control time 2 control time 3 control time 4 controlj Therefore the clustering algorithms would group together clones that have a similar upregulation and or downregulation pattern over the course of the study 13 2 Clustering Algorithms Clustering algorithms differ in how they group points together in data space For example the KMeans algorithm groups data into a specified number of clust
67. complete when the Pathways sample and image files are written With a description file for a microarray these Pathways files contain information neces sary to analyze microarrays in Pathways 4 In interactive mode it is necessary to interact with the import process in Steps 2 and 4 Other steps are automated by Pathways In batch mode Pathways performs steps automatically The Trim option automatically trims the boundaries before storing the image in Pathways image format Trim reduces the file size of the stored image 5 2 Supported Image Formats Phosphor imaging systems store data in image file formats the data represent intensities for each pixel in the image The intensity values may be encoded scaled to make the image files smaller Tiff Fuji and Pathways image formats are supported in Pathways 4 mage readers are pluggable in Pathways so the program can be extended to include any other image format 57 RGMA10011 rev B The Tiff option reads grayscale Tiff images tif or gel with or without square root encod ing The Fuji option reads the Fuji Bas scanners img inf file combination see Appendix II for details The Pathways image format is a proprietary image format that stores raw image data image encoding parameters and computed clone center locations after the import process The format is described in more detail below Users of Molecular Dynamics and Packard phosphor imaging syst
68. conditions may be selected to overlay the data comparison analysis or analyze the data over a range of experiments pro filing and clustering analysis 78 RGMA10011 rev B Condition pair grouping is similar to microarray pair grouping except that the ratios and dif ferences represent the differences or ratios between averaged normalized intensities across the microarrays Multiple condition pairs may be selected to overlay the data comparison analysis or to analyze the data over a range of experiments profiling and clustering analysis 7 4 Creating Projects in Pathways Create a new project in Pathways 4 by selecting New Project from the File menu This selec tion launches the Project wizard which presents a guide for the creation of new projects In the first panel of the wizard there is a prompt to select a single microarray project a two microarray comparison project or a new blank project EU ri Project This wizard will walk you through the steps af creating a new Pathways project To get started selectthe kind of project you d like to create C Analyze intensities for a single array Next Cancel Each of these options is discussed in this chapter 7 5 Single Microarray Projects A single microarray project is a quick route to analyze normalized intensities for a single microarray After selecting this option click the Next button The wizard prompts input for the project and t
69. d answering a few questions about how to map the data in that file to a microarray data model 3 4 The Spreadsheet Framework One way of adding a new microarray to a project without using an image import tool 1s through using the Spreadsheet Framework The spreadsheet framework derives intensities and meta data and may optionally derive geometry from a delimited text file In this example an Affymetrix GeneChip file is imported though the process is the same for any spreadsheet To add a microarray from a spreadsheet file select the project condition to add the GeneChip experiment to and select Add microarray s from the Edit menu P Pathways 5ample pwp File i8 Comparison Profile Cluster Project Ctrl F Normalization Ctr z Add condition Ctr c Rename condition Remove condition Add microarrays Cirl A 31 RGMA10011 rev B The Add Remove Arrays window appears Select Spreadsheet from the Framework field at the top of the window Add Remove Arrays Available j Framework ETTEN Library Biri GEML K Lookin Pathways Switch to the Browse tab to locate the GeneChip file Click on the file and then click on the Add button The Import Spreadsheet wizard appears A jmp Spemmisheet Sia l EK This pape val elyi sel the raa Nope e Grey and apermigmalsp tar he hiesa dihe fi mpat Heh are using a a Fle layout ply tes p Hees 2 ani nd chek Tinah harp Prop eri bs Ecuiren
70. d of microarray from that product line e g GF200 A specific brand may contain many different types Once the Array Designer creates a layout for a particular type of microarray Pathways stores information about that microarray in description files These description files are located in the installation directory in the folder Descriptions Universal lt Brand gt where Brand is the user inputted name of the product line The user needs to design a layout for a particular microarray type only once After that the user can import images of this microarray type multiple times without having to design a new layout 4 2 Concepts Importing Microarray intensity levels must be measured before a microarray experiment can be analyzed The importing process begins with a raw image Tiff Gel Fuji etc of a microarray and ends with sampled intensity levels for each spot present on the microarray 39 RGMA10011 rev B The Array Designer allows the user to provide Pathways with the information necessary to import and sample microarray images This information includes the physical location of the spots on a reference ideal layout and descriptive information meta data for each spot Once the array design has been provided to Pathways a description of the design will be stored on the computer s hard drive and this description will be used in importing and analysis of the given brand and type Before the spot intensity can be samp
71. date source to network or the update can be cancelled If the user elects to retry the update Pathways either tries to re establish the network connection or presents the user with a file dialog box depending on the update source The second and third choices temporarily change the update source and try to update from that new source This change is only temporary and does not affect the update source as defined in the settings dialog box Finally if cancel is selected the update process 1s aborted and the user is returned to the Pathways main window Update Interruption F ailure Update from CD C Update over the Network C Cancel the Update Ok 128 RGMA10011 rev B Chapter 15 Examples 15 1 Introduction to Example 1 This section is a quick reference for a basic application using the Pathways framework com parison of two microarrays to analyze the intensity ratios and differences Parts of the process such as importing have been explained in detail in previous chapters The aim of this example is to provide a guide for a simple project from start to finish GeneFilters microarray images are used in this example In this example there are three steps in the Pathways Quick Start palette 1 Import new microarray images 2 Use the project wizard to create a new project 3 Compare two microarray images and print a report Interactive importing of two GeneFilters microarray images in the template mod
72. dd the experiment to and select Add Microarray s from the Edit menu The Add Remove Arrays window appears Select GEML from the Framework field at the top of the window 7 Add Remove Arrays Available Framework esi Library gi 2 Preadsheet BEML Arrays Pathways 37 RGMA10011 rev B Switch to the Browse tab to locate the file in the system The GEML framework can automat ically decompress ZIP and GZIP files in addition to opening GEML files so make sure the Files of type field is set appropriately Hinana Aapa Awailatda Conkol Framewnrni CEM lima O14 Ti E i Library Browse ime D Lex iP 4 fj ce xm ES Descriptions _ Fe ib 1 Paths Plugin Took _ Uunimstallertiaa Updates File mame Files of We JI Filgz amp GEHL Ales gem DF rings Map IGZIP fias MID Select the GEML file click the Add button and then click the Ok button to continue A win dow will appear asking for confirmation DU 7m 32 Add 1 new array s To finish importing the microarray from the GEML file click the Yes button 38 RGMA10011 rev B Chapter 4 The Array Designer 4 1 Introduction Previous versions of Pathways imported a single microarray type ResGen GeneFilters Pathways Universal introduces the Array Designer feature which facilitates the import of any microarray type as long as certain
73. dges of the template The selected template is highlighted Click on the template to delete it Once the bounding box is set and necessary templates have been created click the Ok button to finish designing the microarray layout The microarray is ready to be imported into Pathways 55 RGMA10011 rev B Book III Core Concepts Data Flow and Importing Images RGMA10011 rev B Chapter 5 Data Flow From Experiment to Analysis This chapter describes how the Pathways Framework imports and stores data Other Frameworks may use alternate methods to import and store data 5 1 From Experiment to Analysis The Importing Process Microarray intensity levels must be measured before analysis of the microarray experiment can begin The Pathways 4 image importing process begins with a microarray image and ends with sampled intensity levels for the clones There are five steps 1n the process of importing an image file 1 Loading the image Pathways reads the image file and displays it on the screen 2 Aligning cropping template Adjust the image orientation and optionally apply a cropping window or manually specify the grid of clones on the microarray 3 Computing centers Pathways automatically computes the centers of the clones on the microarray 4 Verifying centers After computing the centers Pathways displays the computed points for review and any necessary adjustment 5 Writing output The import process is
74. e are outlined in this example Finally printing a report of the project 1s demonstrated The Pathways Quick Start palette appears at start up unless it is disabled by checking the Do not show at start up box at the bottom of the palette Alternatively the Quick Start palette can be selected from the Tools main menu and clicking on Quick Start or pressing Ctrl K 15 2 Example 1 Step One Import Microarray Images From the Quick Start palette select the Import microarray icon Pathways Quick Start 129 RGMA10011 rev B When the Import dialog box appears perform the following steps 1 Select the images to import from the appropriate directory folder file from the Look in field 2 Complete or select fields in the import dialog box to specify microarray name and brand image format sampling type file type output directory et cetera 3 Click the Add button and Ok to continue importing Uncheck the Batch process box for interactive importing In this example Tiff images named Sample 91225 l tif and Sample gf225 2 tif are selected from the Pathways 4 folder The output file is selected by clicking the Browse button lipe liii Dclke Leekim 1 Patres mi er za amp j E Image fonat ITif image jel E E 3 UninstaligrO ala Micro Ba GF 225 Lea Untates ee ionnan C Batth processes jF F ename Esmpie g 225 1 Amd il Remp r Files offype Tiit nage af
75. e data set based on a specified set of criteria to generate a more man ageable set of data Data filtering can take various forms Simple data filters establish a threshold for the data An example of a simple data filter is a requirement that the ratio of normalized intensity be greater than 2 0 or less than 0 5 thresholding a level of up and down regulation Statistical data filters reduce the data set by eliminating clones that lie outside a specified significance level Path data filters reduce the data set by requiring that clones be either members or non members of a specified list of clones a path Each filter type is described in detail in this chapter Invalid clone filters reduce the data set by allowing the researcher to show all clones only valid clones or only invalid clones The data displayed in the analysis window are those that have not been filtered out they are data that meet the criteria The default behavior for all filters is to not filter any data until the user inter acts with the filter by for example adjusting a histogram A check is next to each active filter in the current analysis window When the cursor is inside the filter selection window the current status of the filtered data is displayed as a status message Invalid Clones Fath name v Intensity 7 9 3 Strict Setting Two data filtering options are available for the case when multiple data sets multiple microar rays microa
76. e meta data table below the thumbnails Condition s A thumbnail for each microarray that contributes to the current point is stacked on the thumbnail view A spinner control flips through each thumbnail if this data point does not have any repeats then the spinner is disabled The average normalized intensity for the currently selected clone is displayed in the table below the thumbnail Condition Pairs s Condition pairs are displayed as two stacked condition thumb nails The average normalized intensity for each condition is listed in the table below as are the ratio and difference between the pairs Because each condition can contain clones from different microarrays a check box next to each condition label indicates the condition from which to derive the meta data in the table The ratio is displayed in a ratio format meaning that upregulation B gt A is displayed as positive B divided by A and downregulation B lt A 1s displayed as negative A divided by B 17 RGMA10011 rev B Examples of each detail view are shown below Microarray Microarray Pair Condition Condition Pair aeb Links bat 3255 E354 E 12 Ds The intensity background and normalized intensity values are displayed to the right of each thumbnail image The signal intensity and background intensity represent the sampled intensity values that were measured during the importing process The normalized intensity 1s
77. e od Be BE ee ee ee a Be PTE E II rm Book II Universal Concepts Chapter 3 Frameworks 3 1 22 3 3 3 4 Do Introduction to Frameworks eeee RR RI Res Influence ob Frameworks cc adeuctageuaneeeee ceeee SIRE X ER A EC SE NE The Pathways Eram WOrk 2 200 ur 82 3303 97 3 9 1A ES HRCR RUBER SERO PR AUR 2 9 Ra The Spreadsheet Framework sou a ders uu ER RS PRICE ERRORES EE RA EAS EE dS The GEML Framework 2ezee iesessacswus s mk 60908468 6 ed oe EON UE SL woes Chapter 4 The Array Designer 4 4 2 4 3 4 4 4 6 4 7 4 8 4 9 IMCOOUCHOM c Concepts nuUi AM tn 0 24a4e oo he bs Gee maei Oke Aaaeeeaa DSE Concepts Auto Crop Mode ios inae ace ice spe 4h Sn beech ems ee ewe BU as Concepts Template Mode 2444 40 45 29 9 23 ROS dhe gees he HOSE EY wee Oo Reading from a Spreadsheet File 0 0 cece teenies Reading from a GEML File iusserunt bake HARD s Kaw b HES RSE Owe x ed Reading from a Clontech Atlas Array Gene List File Reading from a Corning CMT Map File l l 4 10 The Array Design Stage lt 2 4 lt 30 acude omm AG abe wee eee Mans dhe we bee RGMA10011 rev B Book III Core Concepts Data Flow and Importing Images Chapter 5 Data Flow From Experiment to Analysis 57 5 1 From Experiment to Analysis The Importing Process 0005 57 5 2 Supported Image Formats 10g 440 c804 004 2s doe eead soba bG eee S ge E RR d
78. each data point 11 5 Chart Properties Right clicking on the plot area generates a menu that allows the user to zoom in out on the chart view to Reset the axis limits and to display the Chart Properties dialog box The Chart Properties dialog box allows customization of the current graph 106 RGMA10011 rev B T L kal Peet pri Ghob Seren Axa Char Tie Porta Stork Contal Coma am iz Tima Panig CHEIR Barapa The Global tab has options for the chart title and background and foreground colors 3 keg eel et Giaa Parian agma TIRE werd E Cariri ex Horii 7 D Wen ueet iie bounds Tiik Go AzkgnU E M y ual mere bounds CO koe user definesd bods Hi O Am m The Axes tab allows the limits for each axis to be specified explicitly the default option auto matically scales the axes To apply the modifications click Apply To exit the dialog box without applying changes click Cancel To exit the dialog box and apply changes click Ok Control drag pans the chart on the screen Shift drag creates a region for zooming 107 RGMA10011 rev B 11 6 Table The table view for comparison analysis presents the microarray data in spreadsheet like format 7 Compaen Condition par Chry aeilress g te fft m Len index comme Kay C7 Address Metadata i Al Control vs Manth 1 Control v Month 2 R43334 R36571 R51835 R38B 2 R607313 R45183 R51346 Clone number Y E 2 50
79. ed in the Data pulldown menus in the profiling toolbar Data Ratio Pog Analysis Data Clone number Intensity If the current axis variable contains no negative data then the axis can be changed to a log scale using the log button The error bar button displays the error bars representing the standard 111 RGMA10011 rev B deviation of the data points These deviations represent each state s average value This result is not the same as error bars for repeated clones that are displayed in other analysis windows Right clicking on the bar chart displays the standard chart controls including the Chart Properties dialog box This version of the Chart Properties dialog box is slightly different from the version for condition comparison The bar color has been added to the Global tab in the Chart Properties dialog box and the Series tab is not present because no additional specifica tions are necessary for each series 12 5 Table The table view presents the profiled data in a spreadsheet format F Peitr Condition paw jy actors imm 2 eM Inde column prm C Aaddesk 7 Weeesis Cine nussser IMANEN i Intensiv Rato Difeeenca Cutiens Conruive Wosih 1 Comm ve Mosh 1 Cental ye Nosh Coniai vs Morin 24 Control evi Imma nee m IEEE SOR OR me Gb ZG MN N 20 OD b E Um meor rm cm n m ee c mmn b mnn mcr I Om mmc m m m m NECEM DOM oe A o0 185 amada Marat AB Fo BAD 13
80. ed on the remaining data after the active fil ters are applied The report icon is the sixth icon on the toolbar 102 RGMA10011 rev B The find icon binoculars locates clones in the current data view The find dialog box appears and allows searching for clones by entering a clone key microarray address or path The find icon is the last icon on the toolbar Fired cines by clone larg Paih Hama Era act Career Fipa tinpe Dy recr ara meta Find clones brgh Search Aa mdis DE225 419 GE325 BD4 DF325 B74 DEFI25 BHIA DE325 Bg DrE25 bph GF125 1311 Detail views are available in data views by clicking on a data point In addition the clone selection can be moved around in the synthetic microarray view using the arrow keys 11 3 Synthetic Microarray The first data view for comparison analysis is the synthetic microarray view 7 Comper is Contin par iiy address i tz mim cx J Paintar Fata Brightness eymipelic view 1 C arigis Menk 2 A synthetic microarray shows analysis data as colored spots that are arranged in the same pat tern as that of the microarray When multiple microarrays or multiple conditions are presented the different microarrays appear in tabbed panels as shown in the Comparison Condition pair window 103 RGMA10011 rev B pom The synthetic microarray view is not available if the framework being used does not supply geometry X and Y coordinates The coloring on synthetic views de
81. ely In Pathways 4 users can select how ratios are repre sented Pathways 4 plots the true ratio not the gt convention but it allows the ratios to be displayed when filtering ratios As in Pathways 2 0 Pathways 4 offers the ability to filter large sets of data to certain areas through the following techniques Manipulating histograms Designating paths or lists of clones Keyword or string searches II 5 Viewing Data The filtered data can be viewed in various different graphical representations such as scatter plots histograms and clustergrams Of the different graphical representation offered each is interactive A clone can be selected from the graph and a detailed view of that clone from the original image appears The clone title accession number cluster ID and more are also includ ed The tables graphs and reports generated in Pathways 4 can be saved and printed for use in papers posters and presentations refer to Core Concepts in Data Analysis Reports II 6 Data Management and Updates Pathways 4 does not employ the Microsoft Access Database therefore it allows users to share data files After microarray images are imported into the software the images and data are cata loged in a library They are organized and saved by microarray type along with the information pertinent to each microarray For example the library displays the original image file image type import date and any ex
82. ems Images originating from Molecular Dynamics and Packard brand phosphor imaging systems use a special encoding of the TIFF standard characterized by a GEL or TIF extension While Pathways software is capable of reading these specially encoded files do not open them in another application first Opening the images in a graphics application such as Adobe Photoshop strips out critical information used to decode the image leading to incorrect pixel intensities in the image 5 3 Microarray Description Plug Ins The variety of available microarray products differ in geometrical layout biological contents and manufacturing materials e g glass versus nylon membranes The differences in each product potentially require different algorithms to describe the geometry determine the location of clone centers and sample the data Pathways 4 has a generic Microarray Description plug in option the GF Description plug in that allows any microarray product to interface with the Pathways suite of tools The GF Description plug in has the following responsibilities Providing Pathways with a description of the product geometry for example describing the arrangement of the oligos or clones on the supporting material Providing Pathways with meta data accession description et cetera for each clone on the microarray Providing Pathways with the location of clone centers on an experimental image Miscellaneous customizable
83. ems can be analyzed by Pathways software Unigene This is a system for partitioning GenBank sequences into a non redundant set of gene oriented clusters containing sequences representing a unique gene and its associated meta data It is located at http www ncbi nlm nih gov Unigene 166 RGMA10011 rev B Analysis Data 89 Annotation 71 74 133 ANOVA 90 94 AntiPath 86 Architecture 10 Array Designer 15 27 39 Auto Crop 67 Autocentering 10 59 73 151 160 Automated search paths 95 Background 18 160 Bar Chart 111 Batch Importing 57 61 73 160 Chart Properties 106 111 119 Chen test 90 Clone key 78 160 Clone key paths 94 Clone number 89 160 Cluster filter 115 Clustergram 121 Clustering 113 142 Comparison 102 135 138 Condition grouping 78 89 Condition pair grouping 79 89 138 Conditions 76 77 82 137 161 Contrast Controller 21 64 Control Point Normalization 85 Cropping rectangle 59 161 CSV File 99 Data Point Normalization 85 Detail View 17 Difference 17 161 Empty Project 82 137 Error bars 106 111 Examples 129 Filters 90 92 94 97 153 161 Frameworks 22 27 80 104 129 Fuji 155 Gel 58 161 GEML 27 37 49 GenBank 123 153 GeneFilters 162 Global 69 131 Glossary 160 Graphical User Interface GUI 12 Grouping of data 78 Help 25 Hierarchical clustering 113 116 Histogram 92 162 HTML File 99 Hyperbolic tree 121 Image Formats 57 61 Image Import dialog
84. er from the Tools menu or click the bottom button in the Quick Start menu Help Window show Quick Start Path editar Browser Ctrl B Edit web Links Ctrl ev a O lhe pmpa wend pede you Sg Se Bengh the craton of amp zer prope Update Pathways Aayar relaie pieni values ofa Array Designer HE ite ii iritas Tana and A Cento demr kib ef genes beck on pur cuim ram Dengn eu nkroacmr m s useg fhe a d foray Designer tool The Array Designer Data Source window appears e UTI HNSICPEETO IS The Bite designer ol proide g an neracie arsironmeant sor designing naw microarray layouls foe use weth Pallas To begr chog one of ihe folieedng sources far Ihe inilial mitroaray iiiu inlnrmalinm Road bayou information fram x Spreadsheet ie Read ane informaiton from s GEML fie Read iaou information fram a Clontech Gere Les file Read layout information from Comeng Map file 44 RGMA10011 rev B WARNING Pathways must be restarted if a microarray description which has been loaded into the current Pathways session e g the description has been used within a project or within the importer is modified Changes made in the Array Designer will not take effect until Pathways is restarted If a layout has already been defined for the type of microarray layout being designed select Read layout information from a Previously created design and click the Next button A window appears and it asks
85. ers by finding the center of the cluster and assigning data to the nearest cluster center in an iterative fashion KMeans is a par titional clustering algorithm because the clustering process consists of finding the best cluster partition for each clone Hierarchical clustering algorithms work by finding the two closest clones and calling this a cluster The cluster is assigned a position in the data space that 1s based on the linkage method Next the closest two entities clone or cluster are found and grouped into the second cluster This process repeats until all clones are grouped into clusters The self organizing map SOM clustering algorithm finds clusters in an input data set by map ping the data onto a two dimensional array of nodes Each node contains a reference vector that records the value associated with that node The data points represent successive expression levels of a clone The map is constructed by comparing each point in succession with the refer 113 RGMA10011 rev B ence vector of each node The node with the reference vector that is nearest an input vector is updated with a weighted combination of the reference and input vectors This process is repeat ed over many iterations The number of iterations in each stage and the initial values are user defined There are many ways to describe the Distance between points in data space The traditional distance calculation is a Euclidian calculation The squared Euclidia
86. ert 57 5 3 Microarray Description Plug Ins 0 eens 58 5 4 Finding the Location of Clone Centers 20 0 0 0 cee ees 59 5 5 Sampling Microarray Data 42s coset 9 9 55 9 9 3 09 935 93 9 319 oad donee eee eee Oey 59 5 6 Pathways Sample and Image Files llle 60 Chapter 6 Importing 61 6 Imace Import Dialog 122a achdeace 9 9 39 cO TA RC Ee Sca ecd d eoe A d 9 d e Yea 6l 6 2 Introduction to Interactive Importing 0 0 0 cee ee eee 64 6 3 Interactive Importing Auto Crop Mode 0 0 nee 67 6 4 Interactive Importing Template Mode eese 69 6 5 Reviewing Alignments and Saving scelere 71 60 Invalddtmbo q Clone oerrgirirricr 8e n aba aes FU ie uri ea a dos eee es T2 O7 ceno D sospire ndean ER E EE R E E EEE 73 Book IV Core Concepts Pathways Data Organization and Management Chapter 7 Pathways Projects 76 nM 76 peni MMF T TI 7 3 Grouping of Data 223222 92933 308 975 2 2 202 2 372 H hs ek Sed deed 78 7 4 Creating Projects in Pathways 2 0 0 cee eee 79 7 5 Single Microarray Projects oiseau ares koh apr aw RR ER eens dawn eae ed wars 79 7 6 Two Microarray Comparison Projects 0 0 cece eee eens 81 JF Empi Project 2252093 3723 930 75 2 2 23 3 32 be he rene 13 3E ee den d 82 Chapter 8 Normalization 85 8 1 Basic Concepts in Intensity Normaliz
87. es in mean intensity values obtained under different experimental conditions are statistically significant ANOVA compares the variance of the data calculated within conditions to that across conditions If the variances are not the same then it is an indication that the means are different The t Test is a special case of ANOVA for two conditions The algorithm here is formulated in way that allows the experi menter to create and install a Java plug in to exploit experimental design ANOVA in Pathways has been designed to accommodate different experimental designs through the use of plug ins A plug in is provided for One Way Analysis If the data will sup port an analysis three or more conditions and two or more samples for at least one clone in each condition then the ANOVA entry will appear in the data filter window The filter control sets confidence levels of 99 9 99 95 90 75 and 50 and any which shows all points At a given confidence level the filter will remove from the display those spots whose value of the test statistic is less than the critical value for the selected confidence 9 2 Data Filtering Microarray experiments generate massive data sets A single microarray can hold 5 000 or more clones and even the simplest experiments uses two or more microarrays In general these large data sets must be reduced before the underlying significant data sets become apparent 90 RGMA10011 rev B Data filtering reduces th
88. est values into equally sized bins The number of items in the data set that fall into each bin allows a graphical representation of the distribution of the data set Housekeeping genes Housekeeping genes are genes whose expression is required for normal function of the cell In general their expression is consistent regardless of stimulus Import microarray Selecting Import image from the File menu or clicking this button on the Quick Start Palette begins the process of importing a new microarray image into Pathways software 162 RGMA10011 rev B Importing Importing is the process of loading an image from a phosphor imaging system file into Pathways software In this process the image must be loaded into Pathways the loca tion of each clone must be determined and the clone intensities must be sampled The results of this process are stored in two files the Pathways sample file pws extension and the Pathways image format file pwf extension The results are then imported into Pathways database aligned and intensity data 1s recorded into the database Intensity Intensity is the numerical value assigned to the level of expression of a gene or ORF through the import and sampling process Pathways analysis uses normalized intensities and Pathways normalizes raw intensity values before proceeding with analysis Inverting an image Inverting an image reverses the background and foreground colors of an
89. etch the cropping rectangle drag any of the handle points on the side or corners of the rectangle To rotate the cropping rectangle press the Ctrl key and drag any of the corners the center of rotation is the opposing corner To remove cropping rectangle click anywhere outside the rectangle When satisfied with the image orientation and with the positioning of the cropping rectangle if used click the Next button to invoke the Pathways autocentering routine 68 RGMA10011 rev B 6 4 Interactive Importing Template Mode The template mode allows more precise specification of the alignment of the arrayed spots in the image The template mode overlays a template skeleton representation of the microarray layout on top of the experimental image When the template is lined up properly with the experimental image the clone centers can be determined by referring to the manufacturing lay out of the microarray The template mode involves the following two stages 1 Global alignment 2 Adjusting alignment points with or without magnifier The Global setting drags and rotates and stretches a global rectangle that has the microarray template type attached to it The function is similar to that of the cropping mode discussed above but the objective is to use the global rectangle to align the template on top of the microarray data rather than to place the rectangle outside the microarray data points After the templates are adjusted usin
90. ettings for Pathways 4 are found under the Edit gt Settings menu rbr AFArea 7 eral Pannen bapi or ta Lo el el prt Hide Hae curt dag bier ieee Lindi faite 7C Hikari Co CO S ES TRITT ET BI Liss proe auis ran General settings allow entry of a default Researcher name for projects and imports the pre ferred look and feel for Pathways and whether to load the last project when Pathways is started The Look and feel option can be set to System the program would for example look like a Windows application when running on a Windows operating system or Java a charac teristic look for Java applications that appear the same on any operating system The Load last project option when checked loads the most recent project The Hide clone cursor during snapshots option when checked hides the clone cursor while snapshots are being taken Internet settings allow specification of the built in web browser and for online update services The default options for Update Source may be set to either Network or CD The default Browser font size and Proxy settings can be entered For computers behind a firewall Pathways supports proxies and proxy authentication To enable proxy support check the Use Proxy checkbox and enter the host and port in the Proxy host and Proxy port fields respec tively Microsoft Proxy users must complete the IP address of the proxy and not the windows domain name for examp
91. ew cluster s components and the entity in question Unweighted and weighted averages calculate the new distance as the average of the previous distances weighted aver ages weights this average by the number of subentities in a cluster Unweighted and weight ed centroids use the average of the cluster location in data space to calculate the new distance weighted centroids weights the average by the number of subentities in a cluster When the properties are set click Ok to proceed with the cluster analysis 13 7 SOM Clustering SOM Clustering is a third option for clustering analysis a Clustering Clustering Algorithm Cluster Variable Cluster on Lag Properties Distance Output Layer Dimension Output Layer Y Dimension Ordering iterations Initial Rate Ordering Initial Radius Ordering Convergence Iteratians Initial Rate Convergence Initial Radius Convergence Kernel Selection Gaussian 117 RGMA10011 rev B After the cluster variable has been selected SOM clustering requires the following information Distance plug in calculates the cluster distance Output Layer X Dimension number of dimensions for nodes on the X axis Output Layer Y Dimension number of dimensions for nodes on the Y axis Ordering Iterations number of iterations during the ordering phase Initial Ra
92. fidence evel af Cisnbution Tepe amA Eudent t 9 6 Paths The three Path types offer the ability to include or exclude clones from an analysis window Microarray address paths identify a clone number on a microarray type For example a Microarray address path might specify clones 30 45 1000 and 2010 on GF200 This path type is specific no members of this path are on a GF211 filter for example Create microarray address paths manually through the path editor or automatically based on analysis window clones Microarray address paths routinely exclude certain clones from analysis For example if the thumbnail image of a clone shows an experimental or sampling error a microarray address path could be created with the address of this clone and then the clone could be filtered using a path filter Clone key paths are similar to microarray address paths except the clones in this path are list ed by the Clone Key the accession number when it is available or by a unique identifier such as tgDNA Clone key paths are not microarray specific and they are therefore a preferred means of identifying clones Create clone key paths manually through the path editor or auto matically based on the clones in an analysis window Clone key paths identify a set of clones for further research For example if an experiment shows that a set of clones is consistently upregulated then these clones could be isolated into a clone key pat
93. for the gene list file The Array Designer currently supports Clontech 1 2 Arrays Trial Arrays Small Arrays and Large Arrays see http www clon tech com atlas index shtml for more information about these array types The Brand is auto matically set as Clontech and the Type is derived from the filename Click the Browse button to locate the file Once the file is located click the Finish button to proceed to the array design Stage 4 9 Reading from a Corning CMT Map File To design a microarray layout for a Corning CMT Map file open the Array Designer and select Read layout information from a Corning Map file from the Array Designer Data Source window Then click the Next button A window appears and it asks for the file location and for the Array Type of the file Array Designer Corning Information Please enter the full path to the file containing a Corning Map text file ar click the Browse button to locate this file using a file browser dialog box Also specify array type corresponding ta that file Filename Browse Array type BHLRSEEESEET TITENE I Cancel 50 RGMA10011 rev B At the time of printing Corning supports only CMT Yeast Gene Arrays Future plug ins will provide support for other Corning formats as they become available Click the Browse but ton to locate the file Once the file is located click the Finish button to proceed to the array design stage 4 10 The Array Design
94. for the location of the description file F Biray Ve zin Description fa neither Piegse ender fie full path bo the file cantsrimg a Patras Liners decirighon fle or Click the Browse button to locate fnis fle using a file browser dialog bor Filenarna S a c Bark Cancel Click the Browse button to locate the description file in the system Pathways creates a description of the attributes of every microarray layout designed with the Array Designer The program can then access these files quickly for future use Description files must be stored in the Descriptions Universal lt Brand gt folder in the Pathways installation directory where lt Brand gt is the entered name of the product line 4 6 Reading from a Spreadsheet File The Array Designer provides customized microarray layouts for GEML files Clontech Atlas Array Gene List files and Corning CMT Yeast Array Map files When the microarray data 1s in none of these formats a layout for the microarray must be designed from a spreadsheet file 45 RGMA10011 rev B To design a layout for a spreadsheet select Read layout information from a Spreadsheet file from the Array Designer Data Source window and click the Next button A window appears and it asks for the file location PEUT Desig fin nee Fipase ontore full path p na Tia conan Ina ina cro ey recul iefpemabun b clic Iha Browza buis io orata this dig zszing al brewer dalcg bas _
95. g microarray data from various sources Frameworks are only available in Pathways Universal GEL file This type of image is a form of TIF file that is acceptable for Pathways analysis See the TIF File GEML This is an XML based tag set that provides a method for exchanging gene expres sion data and related annotations It is used for transmitting data independent of the methodolo gy used to collect that data GenBank This is an annotated collection of all publicly available DNA sequences located at http www ncbi nlm nih gov Genbank 161 RGMA10011 rev B GeneFilters microarrays This is a reusable microarray system that can be probed by stan dard auto radiographic methods offering low cost entry into the microarray arena Developed in conjunction with Pathways analysis software GeneFilters microarrays simplify gene expression analysis and take advantage of the combination of GeneFilters membranes and iso topic detection GeneFilters Human microarrays These microarrays consist of a single membrane contain ing up to 5 184 non control spots The membrane 5 cm x 7 cm also contains controls genomic DNA monitors the homogeneity of the hybridization and a series of housekeeping genes is included for orientation and alignment Each of the non control spots on the membrane contains at least 0 5 ng of insert DNA from an Integrated Molecular Analysis of Genomes and their Expression Lawrence Livermore Nationa
96. g the global rectangle the location of the alignment points can be further refined using fine adjustments to alignment points in the template These points can be adjusted either with or without a magnifier a feature that locally enhances the image for more accurate adjustment of the alignment points Template mode is selected using the template button at the top of the import window 32 V Adjust Global Use Magnifier Adjust the global setting Select Adjust Global initially selected by default Drag Resize Move Rotate the global rectangle as with a cropping rectangle instructions above As the global rectangle 1s dragged a template is attached to 1t Use the global rectangle as with a cropping rectangle to align the template on top of the experimental 1mage After the global alignment uncheck the Adjust Global box and refine the location of the tem plate alignment points 69 RGMA10011 rev B To adjust template points with a magnifier present perform the following steps 1 Deselect the Adjust Global option and select Use Magnifier 2 Move the pointer over each alignment point and a magnifier window appears an alignment hint picture also appears on the left panel 3 Click anywhere in the magnifier window and the alignment point snaps to this location 4 The magnification can be increased or decreased using the up or down arrow keys while the magnifier is present Perform the following steps to ad
97. generate the condition analysis win dow The first view shown is the scatter plot Specify a 99 confidence interval for the t Test filter the points shown have a 99 likelihood of being differentially expressed to ensure a statistically significant difference in expression Cone mem m ii fie m iE heat Y it nienti ta F inicira iy i fh Significant Up Down Kegulatian gt Contin Tees xa 1 Mani Trias Como Triak s 1 on Trials Centri Times 3 Mon Tub Pos wl T E im imira Fg d Confidenra Lived Flor intem abo rar I HE a LD ke pan bee bean Hr becca dar maur alert oe nel eater delice ad a roxiklarza evel d OHtTitutan Tar iia i fidens A title has been added to the plot and error bars are enabled these options are established in the chart customization dialog box which is generated by right clicking on the chart 15 7 Example 2 Profiling Analysis After reviewing the differential expression for the experiment focus on how the genes that are associated with cancer behaved in the study To analyze expression levels rather than differen tial expression choose the Condition s grouping for the analysis from the Profile menu IMiUII M Cluster Tools Micraarravtrs Microarray pairis Condition si Condition pairts 139 RGMA10011 rev B F Profile Conditions Control Trials 1 Month Trials 2 Month Trials 3 Month Trials Average by Microarray Address
98. ges autocenter without prob lems assuming the orientation is as shown below In general first try the autocentering without a crop ping rectangle click Next in the alignment window If Pathways cannot find all the clones in this mode add a cropping rectangle If Pathways still cannot find all the centers use the template mode aT ZH EHIBHHIBHIDHIBHS psp Ho ate etalabtetartelar atabinbardeterietstartalaetelartelartelabie PIPERS HEIL Pera rver ATRAE I48 AE bo et Rae b Bini NETH TA od A m aa La ab ial Ba in miibinid iB bimbibd GHEE LIEISHIETEI EIE noe EIEE R amp LIEIZLILIZ dutiinilnnidi Toor TE PIP MPS EPE ee ee ers ee eee BPE PUPP MIDLET MI PIs IIA nlna nn rieirrintipretiristireratirisiteertetsi trie eidniipaiiihadrisH isa fittata ian AEEA MEIRE a ETE TTEIETL m EEE EH Hea SHE inner EIL TEDIETETILas isiat eisiiki Oo ef E FERNE arr x SHEEN prraEg3 EE E p kisLim 2 3 HHEBBEPIT L a 2 Li ZEI Hi H BEd bi miaii kha iad oo Ux rir I ia nin ia ri FIFINI ianialz Ere Miriam rine not Itti EES BIESFTERIHEJ Virieirte Li Ll ari EIER Li H H Li t ijs m ui As baie ofa te zin EI FIEIRFIFIS p Li TE H LES Li H L oo ni OF L aha um HE H HE mia After the initial alignment setup click the Next button Pathways calculates the clone centers When Pathways is unable to calculate the clone centers
99. h for investigation on further experiments Likewise if a researcher has a set of clones on which their efforts are focused then a clone key path would be created with keys for this set of clones 94 RGMA10011 rev B Automated search paths create a dynamic path based on keyword searches Specify the fol lowing information a rule a field to search the keyword for which to search location in the field start beginning in the text whether to match or mismatch the keyword Multiple rules can be added to a path and it is possible to select whether to match clones that satisfy the rules or to match clones that satisfy one or more rules Automated search paths isolate a research concept For example create an automated search path by requesting clones that have the keyword cancer in the clone s description field Search paths are dynamic Whenever a GeneFilters microarray description is updated through the Pathways update service automated search paths may change For example if Pathways data files are updated and new clones have the keyword cancer in their descrip tion they are added to the path automatically 9 7 Creating a New Path The Path editor can be accessed from the Quick Start palette from the Path creation button in an analysis window or from the Path filter view When creating a path in the Path editor or in the Path filter window the New Path dialog box appears a Crealn nem path Fa
100. he Add button or double click the file icon to the left of the file name Add multiple files by following the same procedure they must be of the same microar ray brand and type and image format Remove files from the list by highlighting the desired file s and clicking the Remove button The default output file name for the imported data is the same base name of the input file but with a different extension To change the name of the output files edit the output name col umn by double clicking in the selected file table and typing the new file name Output name W Sarnple_gf225 1 Sample gf225 2 To change the output directory for the imported files click the Browse button next to the output directory field and select a directory from the output directory dialog box A directory must be selected in the directory dialog box for the Ok button to register the new directory Output director C aAPragram FilesiResGeniPathiways 4 In attempting to import an image with the same output file name and directory as an existing Pathways file a dialog box appears and requests verification before overwriting the existing file Dverwrite Press OF to overwrite the following files CProgram FilesiResGeniPathways disample gr zz5 1 pmws Image format Microarray brand Microarray type and Sampling type are plug in choices that may have additional specified properties As an examp
101. he process of creating a project involves creating a condition and then adding microarrays to it To add a microarray to the currently selected condition choose the Add Microarray menu item from the Edit menu or right click on the condition name The Add Remove Arrays dia log box appears T Adif ormaren fingar Framas F han Library Bizwin gusrefbber g a fin E aw gll amp Pere gls 7 X Dnrizs Fare i Tara 1 Mir Mir Meri i Horii i F Mari d Min D o2 a a a di a E EN Lane ad Ch BEL EE M H NE C MAD BE MA 30 RGMA10011 rev B The Framework field allows the user to choose a framework in which to work Add Remove Arrays Available Framework Pathways ue Library Spreadsheet GEML Pathways Arrays The contents of the rest of this dialog box depend on the current framework The Pathways framework allows the user to add microarrays to a condition by selecting them from the library of previously imported microarrays or by browsing the file system for additional microarray files that are not in the library Chapter 15 contains a detailed example of adding a microarray using the Pathways framework For frameworks that do not provide an import tool or a library of previously imported files the only way to add new microarrays to the project is to browse the file system In this case the user s responsibilities are reduced to selecting the file an
102. he profile view shown above has isolated a cluster using a cluster number filter set to show the cluster of a clone associated with cancer The Cluster Information button generates a dialog box with the details of the clustering algo rithm that generated the current results The Cluster Information button is available for all cluster visualization techniques The left and right arrow keys moves a selected clone to the left or right data point for the cur rent clone For example if the currently selected clone is in the 1 Month condition above then the left arrow moves the selection to the same clone in the Control condition and the right arrow moves the selection to the same clone in the 3 Months condition Right clicking on the plot generates the chart menu Chart Properties Zoom In Zoom Out Reset see Comparison Analysis for a detailed explanation of the Chart Properties dialog box Control drag pans the chart Shift drag creates a region for zooming 119 RGMA10011 rev B 13 9 Tabular The tabular plug in displays cluster data in a spreadsheet like format Cisim Fass Cor ity Fic is Date A cuarton manon role inte conn Rey Addesk O Meteata o Chaat z 5 5 i P 4 4 k E 4 i un uni e Conia Woh Caonmiwz onim 2 Carnmaiws Monin3 METE IHI 21551433 XJ5 H8B 15324533 11 3B 4B 3I65578 PETTITT E 181 B 157 14014177 J 1384503 14223387 J 2 n J TBI A
103. he researcher s name 79 RGMA10011 rev B New Project EJ Please enter your name and provide a name far the new project then click Next to move to the next step Project Details ame Day 5 Array 1l Researcher Lohn amith zs Back Next Cancel In the next window a prompt appears for the selection of a microarray for the project Hes Fhungel E3 Ballabig Framework Fataar z Litriri Errera Anus infirmam qq GpneFiln gy Gini Tima 1 Tima G_2 Wenth 1 1 Month 1 2 Tim hd 1 L6 O4 DT 2001 C IP regm Pilas aar 9a eA Palmera temple eft mE Image type THT lig Samping type An z EE Selected Aer C Program FilpsiR prem ohen Comammple g 2z5 1 ps es Bark aia e The Framework field at the top of the screen allows Pathways Universal users to select the appropriate framework from which to obtain the microarray data refer to Chapter 3 for more detail on frameworks 80 RGMA10011 rev B Click on a microarray and then click Next A prompt appears for a normalization technique use the default value or refer to the Normalization Chapter for more details F Hire Prigeck Finally select a noemalzaton alec I use tor nomislizing tha sampled imensitias T u rg nat sure which one Ia rings just use Ihe gatul semna Note Hal Some n rrmiizalian algeriims require addib nal mipuls Pioni lesion Aigo ntti lontra Point Hommaan Data Pori Homala
104. hways Sample and Image Files Once the microarray data is sampled Pathways stores the imported data in two files a Pathways sample file and a Pathways image file The Pathways sample file pws extension contains the sampled clone intensities The Pathways image file pwf extension contains the calculated clone locations and a full resolution version of the original image The image may be rotated and cropped depending on the alignment of the clones on the original image Pathways 4 uses the Pathways sample file for data analysis The Pathways image file pro vides thumbnail pictures of the currently selected clone in an analysis window The Pathways image format allows rapid file based access to the raw image data for clones The advantage of file based access is that the entire image need not be loaded into memory to view clones thereby dramatically decreasing the memory requirement for thumbnail views in the analysis modules Pathways 4 does not require the Pathways image file to perform data analysis If a sample file pwf is loaded and there is not an accompanying image file Pathways issues a warning and then proceed with the analysis The only noticeable difference is that a question mark icon is displayed in place of the selected clone thumbnail Having two files means that the sample file is small and can be shared between researchers whereas the image file pwf being a full resolution image
105. hways program from file man agement to data analysis The workspace contains active analysis windows for the current proj ect The project tree shows the current project s microarrays The detail view shows relevant information about the currently selected clone s in the active analysis window including a thumbnail view of the clone in the original experimental 1mage The filter view displays data filters that are available for the currently active analysis window Each of these sections is dis cussed in more detail in the following text 12 RGMA10011 rev B Each section of the GUI is divided by borders that can be resized to allow more space for a sec tion of the GUI To change the size of a section Move the cursor over the border that needs to be moved A double headed arrow appears Click and drag the border 2 2 Menus The File menu commands create and save projects import images and exit the program A Pathways New project B es Comparison Profile Cluster Tools Help wind New project Ctrl M Open project Ctrl 0 Save project Ctr 8 gave project as A impart Image 1 ClRamalPathway Output FilesiSample pwp 2 CaARamalPathway Output FilesiTest Froject pwp New Project Opens a new project dialog Open Project Opens a dialog box from which a saved project can be loaded Save Project Saves the current project Save Project As Saves the Current Project as a new file name Import Image Opens a d
106. ialog box for importing a new microarray image Recent Projects A list of recently opened projects appears here Exit Exits the program 13 RGMA10011 rev B The Edit menu offers a variety of choices for editing the current project and allows general pro gram settings to be adjusted 7 Pathways Sample pwp File pmi Comparison Profile Clu Project Ctr P Normalization Ctrl z Add condition Ctrl C Rename condition Remove condition Add microarrayis Ctrl A Settings Project Edit project properties such as project and researcher name Normalization Edit the normalization groups and methods for the current project Add Condition Add a condition to the current project Rename Condition Rename the current condition active only when a condition is selected in the Project Tree Remove Condition Remove the current condition from the project tree active only when a condition is selected in the Project Tree Add Microarray Add a microarray to the current condition active only when a condition is selected in the Project Tree Remove Microarray Remove the current microarray active only when a microarray is selected in the Project Tree Settings General program settings see Settings section The Comparison Profile and Cluster menus represent the core analysis capabilities of Pathways M Comparison analysis is used to analyze data for an entire set of clones e g deter mination of
107. icroarrays as the project type 3 Enter Project name and Researcher s name 4 Choose the two microarrays 5 Select the normalization type in this example Data Point Normalization 6 Click Finish to exit the wizard 134 RGMA10011 rev B 15 4 Example 1 Step Three Comparison Analysis amp Report Generation After exiting the Project Wizard the workspace displays the comparison data for analysis Showing the intensity ratios in a red green overlay the synthetic view is the default setting Data can also be viewed as a scatter plot or table Plots and tables may be saved as images In this example the data appears as a scatter plot below 135 RGMA10011 rev B To generate a report perform the following steps 1 Click the Report button to generate the report wizard 2 Select how to output the report e g printer 3 To include information that appears in the report check the boxes on the right and complete the description fields 4 Click the Print button to generate a Report Preview window that allows the report to be previewed and printed RGMA10011 rev B 136 FOEI apliki feta 3a subaudi l be d peospriar 11 XI EDI SAG TTS Deal zm homo D eefrely E mariar 5 A AJ TE actam aelig prh mp 3l roaches end actin fissa ree mites poston E BAS ITO Harz sapere chet AG i ra H amp eee F ARTS SEDI espd cei protete T SHSOLBL AAMT aial peptidase copier LERDS A A11
108. ied of your claim of defect ResGen will refund to you the amount you paid for the SOFTWARE Any replacement SOFTWARE will be warranted for the remainder of the original Limited Warranty period 7 LIMITATION OF WARRANTIES AND LIABILITIES EXCEPT FOR THE EXPRESS LIMITED WARRANTY IN SECTION 5 LIMITED WARRANTY ABOVE THE SOFT WARE AND MEDIA ARE PROVIDED ON AN AS IS BASIS AND NEITHER RESGEN NOR ITS SUPPLIERS WARRANT THAT THE SOFTWARE OR MEDIA ARE ERROR FREE RESGEN AND ITS SUPPLIERS DISCLAIM ALL OTHER WARRANTIES WITH RESPECT TO THE SOFTWARE AND THE MEDIA EITHER EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION THE WARRANTIES OF MERCHANTABILITY FIT NESS FOR A PARTICULAR PURPOSE AND NON INFRINGEMENT OF THIRD PARTY RIGHTS THE ENTIRE RISK IS BORNE BY YOU AND IF THE SOFTWARE PROVES TO BE DEFECTIVE YOU AND NOT RESGEN ASSUMES THE ENTIRE COST OF ANY SERVICE OR REPAIR SOME JURISDICTIONS DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES OR LIMITATIONS ON HOW LONG AN IMPLIED WARRANTY MAY LAST OR THE EXCLUSION OR LIMITATION OF INCIDENTAL OR CONSEQUEN TIAL DAMAGES SO THE ABOVE LIMITATIONS OR EXCLUSIONS MAY NOT APPLY TO YOU THIS WARRANTY GIVES YOU SPECIFIC LEGAL RIGHTS AND YOU MAY ALSO HAVE OTHER RIGHTS WHICH VARY FROM JURISDICTION TO JURISDICTION 8 SEVERABILITY In the event of invalidity of any provision of this license agreement the parties agree that such invalidity shall not affect the validity and enforceability of the remaining
109. ighted in the upper left corner Contrast slider and color options are available throughout the import process to enhance the dis played image The contrast and color have no effect on the data sampling instead they enhance the displayed image Traditional gray scale images are displayed in 256 shades of gray False coloring makes 256 values available for each of the color channels red green and blue and it can display over 16 million colors for the same image See the graphical interface overview chapter for instructions on adjusting the image contrast 64 RGMA10011 rev B The first window that appears is the image alignment window fies fees mem F z Ly F g r mee keya a mo arr rase in sil ala a m mas wq The image alignment window allows rotating the image and optionally applying a cropping rec tangle auto crop mode or template overlay template mode The Auto Crop mode invokes the fully automated centering routine while the template mode uses a template overlay to locate the centers Use the four rotation buttons to rotate the 1mage by 0 90 180 and 270 degrees respectively fx vlc 88 h ate 90 degrees Rotation allows the image to be aligned Array images created using the Array Designer must be rotated to correspond with the same image orientation used in the Array Designer 65 RGMA10011 rev B ResGen GeneFilters Microarrays Users Most GeneFilters ima
110. image that is if looking at dark spots on a white background inverting the image allows the researcher to view light spots on a dark background Java Java is a programming language that creates programs that run on multiple operating sys tems Windows Linux et cetera Java is fully object oriented and it is ideal for writing appli cations with plug ins graphical user interfaces GUIs and internet connectivity KMeans clustering This is a clustering algorithm that groups data into a specified number of clusters by finding the center of the cluster and assigning data to the nearest cluster center in an iterative fashion Known genes This is a microarray that contains only genes with known functions as the data points Catalog GF211 Main menu At the top of the Pathways window this is the bar that reads Files Edit and Help The Quick Start Palette offers a short cut to items in the main menu Meta data Meta data is the auxiliary data such as accession cluster ID title et cetera that is associated with each clone Meta data is typically read from a description file for the appropri ate microarray Microarray address This is an identifier that associates a clone with a spot on a microarray e g GF200 Clone 500 The microarray address identifies the spot by location whereas the clone key identifies only a clone type on the microarray For example there are multiple tgDNA clone keys on ResGen microarrays
111. ind clones by path Search Results 143 RGMA10011 rev B Finally save the results from the study to a text file for later reference by opening the Report dialog box from the cluster window toolbar RGMA10011 rev B abu mr Dupri ia Ea Fiki Deacnpien Tms that cluster with AALKLAAS Aid butcher Lu an Bari Tra Crue Busy Eampaung 4 144 Cr o Adi Mote dala hb Du ve tEn iH rimm pusier id ratius rai gt j Diii mei sr Chose hiris CIgeierag dats Bhreare Bhorwsbprn sali Book VI Appendices 145 RGMA10011 rev B Appendix I ResGen GeneFilters Microarrays I 1 Introduction Being able to profile gene expression patterns of tens of thousands of genes in a single experi ment cDNA microarrays have generated great interest in the past few years When combined with the Pathways 4 software package ResGen GeneFilters microarrays offer the scientif ic community an opportunity for low cost entry into the microarray arena without compromising experimental quality By offering a reusable microarray system without the need for complex and expensive laboratory setup this powerful tool is affordable to every laboratory involved in genomics research This Appendix includes an overview of ResGen GeneFilters microarrays technology as well as brief descriptions of each microarray product GeneFilters Mammalian microarrays human rat and mouse GeneFilters Yeast microa
112. indow by Microarray s by Microarray pair s by Condition s and by Condition pair s Cluster Tools Microaraytsy Microarray pairts Conditions Condition pair s Microarray grouping examines a single microarray to analyze normalized intensities expres sion levels Depending on the analysis type synthetic arrays line plots tables and other tech niques view the data When multiple microarrays are selected in a comparison analysis then the data from the microarrays are overlaid synthetic microarrays and tables appear in tabbed windows while scatter plots show different colors or symbol types Profiling and clustering use multiple microarrays to analyze data over a range of experiments Microarray pair grouping examines the ratios and differences of normalized intensities between a pair of microarrays differential expression These data may be viewed in a fashion similar to that of the non paired option If multiple microarray pairs in a comparison analysis are selected then the paired data is overlaid in the analysis windows Profiling and clustering use multiple microarray pairs to analyze upregulation or downregulation over a range of experi ments Condition grouping is similar to single microarray grouping except that the analysis uses con ditions rather than microarrays Therefore this analysis looks at a condition s average normal ized intensity for repeated clones across the microarrays Multiple
113. ings allow determination of trends in the expression levels of the clones from one experiment to the next The microarray pair s or condition pair s groupings allow determination of trends in the upregulation or downregulation of the clones between pairs of experiments 12 2 Profiling Toolbar The profiling toolbar is present for profiling views The first three buttons represent the plot bar chart and table views for profiling analysis these buttons are discussed in more detail below The remaining four toolbar buttons are the path save image report and the find buttons Refer to Chapter 11 for details 109 RGMA10011 rev B 12 3 Plot The plot window displays a variable intensity ratio difference et cetera on the Y axis while the X axis displays labels for the microarray microarray pair condition or condition pair depending on the data grouping type Profile Coran org aibi ss i wlm dja A aean 0 s Brezst Path Time Study Canil Mii T The foregoing plot used a path filter to isolate only certain clones Path filtration is advisable when using the plot view because the plot becomes overcrowded with large data sets Clicking the left and right arrow keys moves a selected clone to the left or right data point for the current clone For example if the selected clone is in the 1 Month condition above then the left arrow moves the selection to the same clone in the Control conditio
114. is checked Pathways treats two consecutive delimiter characters as one RGMA10011 rev B 47 Once these fields have been selected click the Next button The next window of the Import Spreadsheet wizard appears Baray Designs pinaidzhect Dados E3 This page will lel you assi a dala type eg X roondimabe or Premarg Kei 30 earn calumn Tou musi designar one column as the primary b beton finishing Background awaragag Background sanded desia ss i j P MCoodinaies Y haal v F we Data preview A Fifertgpe Filter description Tae Background lav 1 GFz0U0 Resaarrh Genetiweta Dat kt k Human GE 200 2 3104207515 z E sF 2700 Research Genetics Genet ier Species Human GF 2005 2 3104 2707515 j OF 200 Wesgarrh Genetics Gener iter Species Human OF 200 2 3104 2075 15 4 laGFzU Resnarrh Genetics GpneF iter Species Human F200 7 3104 707515 5 GF z t Research Genetics Genef ter Species Human GF 2005 23104207515 5 OF zb Research Genelirs Genet iter Spates Human GF 200 23104207515 of x Back Finish Cancel In the Column Identification section a Data Type and a Label for each column may be entered When designing a layout for a microarray image with the Array Designer the user must indicate which columns are to be used for X and Y Coordinates When these columns are not specified the program lacks the geometry data that it needs to construct an image for importing One c
115. items The GF Description plug in accesses this file to provide Pathways with necessary information for each clone These description files can be augmented with additional data GeneFilters description files can be updated through the ResGen data server The user must select a microarray brand e g GeneFilters and a microarray name e g GF200 When the brand and type are specified Pathways automatically locates the appropri 58 RGMA10011 rev B ate microarray description plug in e g ResGen GeneFilters 5 4 Finding the Location of Clone Centers Once the appropriate image format and description for the microarray is established Pathways searches for clones on the microarray image Pathways has two methods for locating clones on an experimental image autocentering and template centering With autocentering Pathways searches for clones on the experimental image without user input The autocentering algorithm looks for patterns indicating rows and columns in the image and then focuses on these rows and columns to locate clones Experimental images are of vary ing quality and the autocentering algorithm may not locate all the clones or it may misidentify rows and columns Therefore visually inspect the results of the autocentering algorithm It is possible to give the autocentering algorithm a hint as to where the microarray is located on the experimental image by dragging a cropping rectangle
116. ities by dividing sampled intensities by the mean sampled intensity of all clones except the control points total genome spots identified by the key tgDNA Pathways 2 users Pathways 2 normalized spots such that the average normalized intensity for spots was 2 000 based on empirical observations of microarray intensities Pathways 4 normalization typically nor malizes the intensity to an average of 1 0 This normalization allows the Pathways 4 user to immediately recognize normalized values that are greater than the mean sample gt 1 0 or less than the mean sample 1 0 To compare Pathways 4 normalized intensity val ues directly with Pathways 2 normalized values divide Pathways 2 normalized intensity values by 2 000 Control point normalization normalizes sampled intensities by dividing each sampled intensi ty by the mean sampled intensity of the control points present on the current microarray Path normalization is a normalization technique that normalizes sampled intensities by divid ing each sampled intensity by the mean sampled intensity of a defined criterion path With GeneFilters Control Point Normalization is an example of Path normalization the path selects tgDNA spots When different types of microarrays are grouped together for normalization 85 RGMA10011 rev B only the elements of the path that are common to all microarrays in the group are used in the normalization process Gr
117. itions in the project Removing microarrays from the project Removing conditions or microarrays from the project may affect open analysis windows In this case Pathways requires closing the affected analysis windows before the Pathways session proceeds RGMA10011 rev B 84 Chapter 8 Normalization 8 1 Basic Concepts in Intensity Normalization Microarray experiments may yield sampled clone intensities that are brighter or darker than similar intensities for a reference image s due to experimental variations such as pipetting hybridization time et cetera To compare experiments adjust for global shifts in intensity lev els so that for example high intensity ratios truly represent upregulated genes Normalization algorithms correct for global intensity shifts across multiple experimental images The normalization process creates a set of normalized intensities which vary from the sampled intensities by a scaling factor These normalized intensities are the basis for Pathways 4 data analysis 8 2 Pathways Normalization Algorithms Four standard normalization plug ins are included with the basic Pathways 4 installation In addition normalization is a pluggable component and therefore new normalization algorithms can be added to Pathways at any time Data point normalization is the default normalization technique for Pathways 4 GeneFilters microarray analysis This technique generates normalized intens
118. ization 86 Zoom 106 111 119 167
119. just alignment points without magnification Deselect the Use Magnifier check box Click and drag the alignment point to move it With ResGen GeneFilters microarrays the template alignment points are located over 16 of the control spots for each microarray In Field 1 of the GeneFilters microarray the template alignment points are the upper right control spot in each grid In Field 2 of the GeneFilters microarray the template alignment points are the lower right control spot in each grid refer to Appendix I When the control spots are not visible e g in cross species hybridization then the alignment points must be in the approximate position where the control spots normally reside verify the accuracy of the alignment by viewing the overall template positioning 70 RGMA10011 rev B When dragging a template over a ResGen GeneFilters microarray image first click the upper right control point and then drag the tem plate to the lower left corner of the microarray Perform refinements resizing and rotation to the global template position with the lower left corner of the global template once the upper right control point is positioned 6 5 Reviewing Alignments and Saving The second screen in the import process is the verification window This window displays the imported image with an overlaid grid representing the detected clone center locations A detail viewer 1s shown in the left panel A tool palette at
120. l Laboratories I M A G E LLNL cDNA clone containing the 3 untranslated region end of a gene These clones were isolated sequenced and verified to be correct Insert DNA was denatured and UV cross linked to the positively charged membrane GeneFilters Yeast microarrays These consist of a total of 6 144 gene ORFs Open Reading Frames derived from the same SGD individually amplified and spotted onto two nylon mem branes The amplification reactions use specific primer pairs designed to amplify the entire open reading frame The primers were generated from unique sequences containing the start codon ATG and termination codon supplied by M Cherry at Stanford Genome Center Therefore the insert DNA consists of the complete open reading frame including the start and stop codons A robotic device spots approximately 1 10 of a microliter of the denatured insert DNA solution on a positively charged nylon membrane The DNA is then UV cross linked to the membrane A system of positive controls consisting of total yeast genomic DNA is printed on each membrane and it is used for orientation and alignment Hierarchical clustering This is a clustering algorithm that works by finding the two closest clones and then calling this a cluster It repeats this step until all clones have been assigned to a cluster Histogram A histogram is a graphical representation of the distribution of a data set Histograms divide a data set from the lowest to high
121. le WProxy is not a valid proxy name If the proxy requires authenti cation check the Authentication box and enter a username and password The proxy pass Word is stored in an encrypted format in the settings These proxy settings are used for browser windows and weblinks and the update service Contact the system administrator to determine whether proxy settings are required to access the internet 24 RGMA10011 rev B 2 11 Online Help The help menu allows access to two submenus Help Topics and About About offers general information on Pathways software including the license key Help Topics offers online access to the full Pathways manual a glossary of terms and a keyword search utility Falma LS A A 11 2 Clustering Algorithms Ojala Gp Chagkr T Guta T i a a T beirut en fo Cigre ape Caged pie P E Madras Ciia Fisrarh ralci Firg Cigtke Tabs Hea Peers Tek s Qj Chapter 1 lod Miei Lira arai Adda are r PE Bin TE F Linch re me 29 Cheaper i EzwTE Pein nem 1 kj AES ELRG AM mme ee ee oca ox aea RGMA10011 rev B iiri rton in i Eip 1 Ex Etara 1 EE Exnanpis D Crrrm m Tere CETE NEIT RI Elustaring algoritum r r tha wip thet fey greep prezir Wanha m dali part For dapi tac KAJAANI daii ipi dux porr nio p apai pumber of oea by fgg tle a of Tis Pheer mE angre cia parin do ikr nerez arar erim mTignu dast Dika Hidde d e a DE a pA D
122. le a sampling plug in might allow 62 RGMA10011 rev B changing the area around each sampled spot When additional options are present the button to the right of the plug in is enabled Clicking this button allows specification of additional options The default Sampling type for ResGen GeneFilters microarrays is called ResGen mean This algorithm visits each clone center and determines the mean image intensity inside a circu lar area surrounding the clone The background level is determined by sampling intensities in the gap between fields 1 and 2 of the GeneFilters microarray The ResGen mean sampling routine is set by default to sample 75 of a spot A pop up menu next to the sampling selection in the dialog box discussed below allows changing the default set ting ResGen recommends this default setting However if the default sampling area needs to be changed do not compare imported images with two different sampling areas If the sam pling area size is changed the new size is remembered in future Pathways sessions to help assure consistent sampling from ses sion to session The available sampling routines are dependent on the brand of Microarray e g GeneFilters are the only brand to use the ResGen mean The Basic sampling type is available for all microarray brands To change the sampling routine select the Basic sampling type Options Image format Tiff image w Microarray brand
123. lected clone This feature is powerful when it is combined with the Find Clones dialog box For example turn on the isolate cluster feature and use Find Clones to find each clone in a cancer path the result identifies the clones clustering with each cancer clone 13 5 KMeans Clustering Selecting an analysis grouping from the Cluster menu generates a dialog box The dialog box includes the current clustering algorithms in a pulldown menu A Clustering Clustering Algorithm Cluster Variable Cluster on Log Properties Distance Correlation Mo of Means 5 hax Iterations f Tolerance o o000000001 Cancel 115 RGMA10011 rev B The Clustering Algorithm is the first option in the Clustering dialog box Available selections are KMeans Hierarchical and SOM The next selection is the Cluster Variable Variables like intensity ratio and differences are available depending on the selected analysis grouping The Properties section of the dialog box allows entry of information that is specific for the selected clustering algorithm For KMeans specify the following options Distance plug in calculates the cluster distance Number of means the number of means clusters that the KMeans algorithm generates Maximum iterations the KMeans algorithm groups and regroups clones into clusters according to the distance from the clone to the cluster center The maximum
124. led it is necessary to determine the location of each spot on the experimental image There are two primary steps in the spot centering process 1 Determining seed locations for the spots 2 Refining the seed locations to more exactly match the experimental image Each of these steps is discussed in more detail below 4 3 Concepts Auto Crop Mode The Pathways importer has a built in capability for determining the skew angulation of microarray images and then searching for the rows and columns that are typically present in microarray layouts These rows and columns can be used to automatically generate seed loca tions for each spot based upon the individual spot s row and column in the reference geometry provided to the Array Designer This concept is illustrated below the crosses represent the seed locations which would be determined for these rows and columns 40 RGMA10011 rev B Seed locations will not necessarily find the exact location of the spot centers Therefore each seed location is independently adjusted using a user specified autocenter mode The three autocenter modes which are available in the current release of Pathways are Centroidal Profile and None These modes will be discussed in more detail later in this chap ter The Pathways importer allows a cropping window to be specified The purpose of this crop ping window is to reduce the region of the image which will be searched during the auto align
125. lone as invalid during the data analysis process the clone will be treated as invalid only within the cur rent project The user may also mark a clone as invalid during the image import process using the Pathways Framework importer If the user marks a clone as invalid during the image import process any project using the microarray will display this clone as invalid refer to Chapter 6 for more information about marking invalid clones during the image import process 98 RGMA10011 rev B Chapter 10 Reports and Exporting Data 10 1 Pathways Reporting Pathways supports reporting exporting data from all analysis windows Reports include clones that are not filtered in the current analysis window The report can contain any combina tion of data sources such as intensity ratios et cetera and meta data accession title ef cetera There are four options for output Printer send report to printer PDF file save report in Adobe Portable Document Format PDF HTML file save report in html format for web viewing CSV file save the report as a comma separated value CSV for export to spread sheets like Microsoft Excel 10 2 Report Wizard Pathways analysis windows support reporting in the toolbar by clicking the Report button El which generates the Report Wizard dialog box For comparison analysis the Report Wizard dialog box appears Oumpuro eer Fen rarar ono Erri Pree Ta ri Fred Liaurrizi1 Dam
126. malization technique to a group or groups of microarrays These groups enable the following options Assigning different normalization techniques to subgroups of microarrays in a project Applying the same normalization technique to different groupings of microarrays ina project used to selectively apply dependent normalization techniques By default Pathways 4 groups microarrays by their type e g GF200 GF211 and it applies the data point normalization technique mean of all points except controls To change normalization techniques and or normalization groupings select normalization from the project menu to generate the following dialog box P Mumalhr ali lanus Hprmalzaine prow pes Graup peop eris Dafault Gesup OF 10080 IZ Mormaii Fame T Ore 9 Dalah Group OF 225 atas Poin eerie amp Worth 7 Piarmasiskor Y 5 Normal gard on s Worth 11 eure Fa a Tie 7 Wohin eripere amp Woni d_a iiis iaaiiai e 1 amp Trad i Worth 2 Tarmunadon Crean h wi hd N rmalima using ak sputs amp Worth Fc altra uaina ari Ha p TI Mord 2 raid using anf the paih apale The window shown above was created from a project that had only one microarray type GF200 As discussed above initially all of the GF200s were grouped into a default group with data point normalization To change the normalization technique select this group in the Normalization Groups window and edit the group pr
127. meing Paes updates am available Tor dowriaad Please zalpct tha updos you wizh to downiaad at ires prie and elck LUpdale amp proceed Feel ike LUndales EET dii Airian Aaaa i ar Human Gane itera Micr amsys Reales vll OF JOT Human GanaFiters Memarnraye Ralesse tarpon Humen Bors Soacitic Geref here Microarrays CGFZ7 Paffwesys main program jal Hain Jar OngwWis Plugin KWnans Clusierirs Algorithm LL k Rhimgim3g mg Version Agr 24 2001 Hehe veh uf Seer Fal 3erzFibez Descartes Fikia 1 GFR Select items to update and click Update or click Update All When a large number of updates are available download times are reduced by selecting a single update rather than all of the updates When the updates are complete a dialog box appears and it indicates the status of the updated files Save the project and restart Pathways to use the updated data and plug ins 126 RGMA10011 rev B 14 5 Updating from CD For those customers whose Pathways installation computer does not have access to the inter net a CD based updating facility is provided The CD Updater will update both GeneFilters and program files giving the user access to the latest GeneFilters data as well as new program features and bug fixes Enabling CD updates is accomplished by selecting the Edit gt Settings menu item As shown below there are two updating protocols that are available to the user Network and CD
128. n ni NI a Add clone to key path I Add clone to address path P Add clone to new path If an opened analysis window is affected by such a change a dialog box appears and it lists rel evant analysis windows and prompts for an action _ Close analysis windows The following analysis windows willbe affected as a result ofthis operation Comparison Microarray by address Would you like ta clase the windows now You will need to do it by hand later ityou dont do it now Cancel Select Yes to automatically close relevant windows To mark several other spots as invalid select No Those windows must be closed and recreated later to take effect Spots marked as invalid from in a project are seen as such in the current project only Invalid clones are marked as such with crosses if the Mark invalid clones button in the upper right corner of the view is selected RGMA10011 rev B PS fel E3 Brightness quA x 105 11 4 Scatter Plot The plot view enables viewing the data set as a scatter plot with defined axes Cainer Bie Cia poor y ULTIIM ze m x wciM epenn enfin Ba Gorka vs Manth 1 Gora vs Mant 2 1 Idi s Analysis Data Clone number Intensity If the current axis variable contains no negative data then click the log button to change the axis to a log scale When a condition is viewed and the data points have repeats click the error bar button to superimpose error bars on
129. n and the right arrow moves the selection to the same clone in the 3 Months condition The y axis variable is selected in the Data menu in the profiling toolbar Data Ratio Analysis Data Clone number Intensity Intensity Path data is unavailable for plotting in profile analysis because there is little meaning in plot ting two values on the y axes Member Non Member versus a handful of profile state points 110 RGMA10011 rev B If the current axis variable does not contain negative data then the axis can be changed to a log scale using the log button When a condition is being viewed and the data points have repeats then error bars can be superimposed on each data point using the error bar button Clicking the connect lines button toggles between symbols with lines connecting the data points and symbols alone Right click on the plot to display the chart menu Chart Properties Zoom In Zoom Out Reset see Chapter 11 for a detailed explanation of the Chart Properties dialog Control drag pans the chart on the screen Shift drag creates a region for zooming 12 4 Bar Charts Bar charts provide the summary of a variable at each state in the profile analysis al HP P TD suraps Fabi m Conto va Mani Conia va Month 2 Cond va Month 3 The height of each bar represents the average value of a variable for clones that have not been removed by data filters The y axis variable is select
130. n distance places a heavier weighting on distances than the standard Euclidian calculation The correlation metric is Pearson s correlation coefficient proposed by Eisen et al 1998 and it is analogous to the vec tor inner product 13 3 Cluster Visualization Visualization of clustering is a challenging task Clusters can be viewed in one of two spaces data space or cluster space Data space as described above is defined by the variable and the grouping e g five conditions five dimensional space Cluster space is the distance from each clone to a cluster This representation of the data helps to display how close each clone is to an arbitrary cluster The number of dimensions is equal to the number of clusters Both data and cluster space tend to be greater than three dimensional space and they are impos sible to view in a traditional sense Therefore visualization techniques focus on displaying as much information as possible within the constraints of the two dimensional computer screen 13 4 Clustering in Pathways In recognition of the complexity of the entire clustering process Pathways clustering analysis has been designed to be highly modular through the use of plug ins Plug ins are available for the following major components Cluster plug in core clustering algorithms such as KMeans and Hierarchical Cluster Distance plug in calculates the distance between two clones or between a cluster and a clone based
131. nating from an image a spot can be marked as invalid during the image import process after spot centers have been identified by checking the Invalid clone box below the detailed view of a selected spot Contrast W Auto Invert Color Invalid spots are marked on the main microarray view with crosses 72 RGMA10011 rev B Such markings can be shown or hidden by selecting and deselecting the Toggle Invalid Clones button on the toolbar in the upper left corner of the main view Toggle Invalid Clones Spots marked as invalid during the image import process are seen as such initially during future uses of the resulting microarray file in projects Refer to Chapter 9 for more information about Invalid Clones If analyzed microarray data comes from a spreadsheet file clones with missing inten sity or background data are marked as invalid automatically 6 7 Batch Importing Batch processing allows for the rapid automatic import of multiple images From an algorith mic standpoint batch processing mode is identical to interactive mode without applying the cropping rectangle The autocentering algorithms in Pathways 4 are robust However the quality of experimental images varies and the autocentering algorithm can fail to locate all the clones or incorrectly identify rows and columns especially for images with relatively low signal levels even if they appear to be normal by visual inspection Theref
132. nd packaging Any failure on the part of ResGen to exercise its option to terminate this license shall not be found to be a waiver or estoppel of such right 2 UPGRADES This license is limited to the version of the SOFTWARE enclosed and does not include the right to upgrades except as provided in this Section 2 If you purchased this SOFTWARE directly from ResGen you are entitled a as to products other than Pathways software to download from our web site http www resgen com and use all upgrades of the SOFTWARE including filter libraries released during the one year period following purchase and b as to Pathways software to download from our web site http www resgen com and use all upgrades of the SOFTWARE released during the one year period following purchase You must in any event register with ResGen to receive all upgrades hereunder 3 COPYRIGHT The SOFTWARE including the source code file definitions and object code is protected by United States copyright law and international treaty provisions You acknowledge that no title to the intellectual property in the SOFTWARE is transferred to you You further acknowledge that title and full ownership rights to the SOFTWARE will remain the exclusive property of ResGen or its suppliers and you will not acquire any rights to the SOFTWARE except as expressly set forth in this license You agree that this license does not 156 RGMA10011 rev B allow you to modify
133. ng but it still does not appear to be correct select the check box to interactively import the image After previewing imports If one or more images are checked for interactive import click the nteractive button at the bottom of the dialog box to proceed with interactive import for the selected image s or Click the Done button If imports were successful it ends the importing process When any images are marked for interactive importing a warning is issued before closing the window 74 RGMA10011 rev B Book IV Core Concepts Pathways Data Organization and Management RGMA10011 rev B Chapter 7 Pathways Projects 7 1 Projects A Pathways analysis session begins with the creation of a project Pathways projects organize previously imported microarray data into conditions that represent states in an experi ment Each condition contains one or more sampled microarray data sets A simple project can consist of one or two microarrays Simple Project Mi hes Project Experiment iioii Experiment Condition Control gt Condition C omo gt Diseased gt Microarray GF200 Microarray GF200 A more complex project can consist of multiple conditions each containing multiple microarray types and or repeat data for the same microarray type Drug Time Course Study Condition E E ai Mont os bee me e OF ni GF21
134. of a spot on a microarray in a microarray pair or condition pair divided by the Normalized Intensity of the same spot on a second microarray Release plate row column These describe the location of the verified clone in the ResGen cDNA libraries Reimport Pathways image files can be read back into Pathways reimported without the need for finding the location of the clones Reimport allows changing sampling technique for a file without dealing with finding clone centers or to adjust clone centers if the center is found to be in error without having to deal with global alignments Reports Reports are printable documents compiled by Pathways software that allows the data specified in an analysis window to be viewed on screen printed out or saved to a text file Rosetta GEML Conductor This is a program that converts gene expression data files into GEML For more information see http www geml org conductor conductor home htm Sample image These are images supplied with Pathways software that allow the researcher to practice using the applications Sampling Sampling is the process by which raw image data generate an intensity value for each clone on a microarray A typical sampling algorithm averages the image data in a circular area of a clone center yielding a clone intensity value Settings From the Edit menu this selection allows the researcher to customize some of the applications in the software SOM
135. olumn must be set as the Primary Key The Primary Key is a unique identifier for genet ic material that Pathways uses to distinguish between microarrays The clone key is usually the accession number or image ID of the clone although the key could follow any other naming convention As discussed in Chapter 9 the key field is one way of grouping similar clones together for statistical analysis A Secondary Key mst be specified if the spreadsheet has any missing Primary Key entries Refer to Chapter 7 for more information on keys The user may edit the Label field for Primary and Secondary Keys Any column containing descriptive information can be set as Meta Data When a column s Data Type is set as Meta Data the column information for each spot is available during the analysis process The user may edit the Label field for Meta Data 48 RGMA10011 rev B The Data Preview section of the window contains the finished layout of the spreadsheet If the spreadsheet is improperly laid out click the Back button to change its parameters To proceed to the Array Design stage click the Finish button 4 7 Reading from a GEML File To design a microarray layout for a GEML file open the Array Designer and select Read lay out information from a GEML file from the Array Designer Data Source window Then click the Next button A window appears asking for the file location Click the Browse button to locate the file in the system Once the file has
136. on the locations in data space used in tandem with Cluster plug in Cluster Visualization plug in takes a cluster calculated by any method and displays the cluster The plug in establishes the format of the display graph or table Therefore the process of clustering consists of the following components 1 Selecting the desired clustering plug in and specifying any auxiliary information needed 2 Selecting the desired visualization technique Implemented plug ins are described in more detail below 114 RGMA10011 rev B A cluster filter is added to the filter view after performing a clustering operation imabd Cionas 7 Sew AB Clusters Numer af visible clusters f1 unti C Sw Chenier in Seectied Range Gwas s magg Beeeefmnupn clusters p Viewing chestar 1 Iscintm Cluster for Eelerinz tiora Paths The cluster filter has the following four settings Show all clusters no filtering Show clusters in specified range isolates one or more clusters with a series of specified cluster numbers e g 1 to 5 10 25 Sweep through clusters a slider bar reviews multiple clusters As the slider is moved from the left to the right the first through the last cluster is shown in the cluster visualization window If the number of visible clusters is set to greater than one the slider shows multiple clusters around the currently visible cluster Isolate cluster for selected clone shows only that cluster associated with the se
137. operties In addition to the group proper ties each normalization plug in may require additional data items e g maximum iterations for Y C Normalization which appear in the plug in properties window To add a new normalization group click New Group and edit the properties Individual microarrays can be moved between normalization groups by dragging them from their current folder and dropping them into a new folder Normalization groups can be removed by high lighting the group and clicking the Remove group button 88 RGMA10011 rev B Chapter 9 Data Paths and Filters 9 1 Analysis Data Pathways supports a set of analysis data that depend on the grouping method Microarray s Clone Number Intensity Paths Microarray pair s Clone Number Intensity I Intensity Il Ratios Differences Paths Condition s Clone Number Intensity Paths Condition pair s Clone Number Intensity I Intensity Il Ratios Differences Paths Microarray or Condition Microarray Pairs or Condition Pairs A Clone number Analysis Data Clone number Intensity Paths ta DNA The clone number is the index of the clone in the microarray the microarray address 1s the fil ter type plus the clone number e g GF200 100 For GeneFilters microarrays the clone number is ordered by field grid row and column Intensity is the normalized intensity for a clone This value represents an average for condi tions with repeats
138. or prepare derivative works of the SOFTWARE or written materials You agree that any copies of the SOFTWARE modifications and supporting written materials per mitted hereunder will contain the same proprietary notices that appear on and in the SOFT WARE 4 REVERSE ENGINEERING You agree that you will not attempt to reverse compile modi fy translate or disassemble or otherwise attempt to reverse engineer the SOFTWARE in whole Or in part 5 LIMITED WARRANTY For thirty 30 days from the date purchase ResGen warrants to the original purchaser as evidenced by a copy of the invoice that the media i e diskettes on which the SOFTWARE is contained will be free from defects in materials and workmanship which substantially affect performance Any failure that results from misuse abuse or a failure to follow the operating instructions 1n the accompanying written materials shall render this Limited Warranty inapplicable 6 CUSTOMER REMEDIES If the media does not conform to the limited warranty in Section 5 above Limited Warranty your sole remedy shall be to return the media and notify ResGen in writing within thirty 30 days of your claim of any defect including a description thereof The defective media in which the SOFTWARE is contained will be replaced by ResGen at no additional charge to you If you do not receive media which is free from defects and materials and workmanship within thirty 30 days of ResGen being notif
139. ore visually inspect the results of the autocentering algorithm To determine how well the autocentering algorithms work on experimental images before proceeding to a full batch mode import multiple experi mental images in the iterative auto crop mode To start Batch importing select Batch Process in the import dialog box and click the OK but ton The batch import dialog box appears and shows a table of files being imported and the status of each file in the import process This dialog box begins the import process for images that were selected in the import dialog box When the import process is completed the status for images is listed in the second column If the autocentering algorithm does not locate all the clones in an image then the Interactive box is checked next to the image indicating that Pathways proceeds with an interactive importing session for this image When the files have been imported the images and alignments can be previewed either in a slide show fashion by successively clicking the Next or Previous buttons or by selecting files from the table 73 RGMA10011 rev B Annotation information Researcher Comments can be added by using the Annotate button The image contrast and color scheme can be adjusted by a menu that pops up after right clicking on the image see the section on image contrasting in Chapter 2 The grid can also be turned on and off through this menu If an alignment passes autocenteri
140. oup properties Mormalization Fath Mormalization Plug in properties Fath name taDNA Use anti path E Subtract background p Minimum intensity 7 f Path normalization has a checkbox selection labeled Use anti path An anti path represents all spots except those contained in the path Anti path normalization therefore normalizes the data using points that are not contained in the path With GeneFilters Data Point Normalization is an example of a Path normalization technique in which the clones not in path option was selected normalize by points not in the tgDNA path Path normalization also has a checkbox selection labeled Subtract background When this box is checked the input to the normalization algorithm will be the difference between the intensity and the background intensity intensity background intensity for each spot rather than intensity alone A Minimum intensity level for the normalization algorithm may also be specified This value will be applied either to the intensity alone or to the difference between the intensity and the background intensity if background subtraction is enabled The minimum intensity value is typ ically used to assure that all inputs to the normalization algorithm are greater than zero If zero or negative intensity values are present in the normalized data sets then ratios will be disabled for microarray pair and condition pair analysis Pathways disables these ratios
141. pends on painters selected from the toolbar Painter color k Brightness 1 1 Color White on black Hlack on white For microarray s or condition s the synthetic array shows expression levels Three painters are available 1 Color highest normalized intensity is bright red lowest is dim blue 2 White on black highest normalized intensity is white lowest is black 3 Black on white highest normalized intensity is black lowest is white For microarray pair s or condition pair s the synthetic array shows upregulation or downregulation Two painters are available 1 Ratio upregulation high ratio is green downregulation low ratio is red brightness is relative to the normalized intensity of contributing spots 2 Difference upregulation high difference is red downregulation low difference is blue brightness is relative to the normalized intensity of contributing spots A Brightness slider increases spot brightness Moving the slider to the right allows viewing low intensity spots The synthetic microarray may also display invalid clones Once a microarray is added to a proj ect individual spots can be marked as invalid by right clicking on the detailed view of a select ed clone and selecting the Invalid clone option 104 RGMA10011 rev B web Links Time D 1 1 Intensity act i Auto Invert v Color build vai View original image Save thumbnail image chrom Update image locatio
142. perimental annotation associated with the microarray that 1s high lighted Select microarrays from this library for analysis Analysis performed on these microar rays is saved as a project which can be reopened later Project organization lets a researcher save work Once analysis is complete users can generate printable reports Other key features of Pathways 4 are hyperlinks and data file updates Clones can be searched for on public databases such as GenBank and Unigene through hyperlinks Users can also add hyperlinks to other web sites To ensure that the data for each clone are current ResGen offers a subscription service to help keep information up to date 153 RGMA10011 rev B II 7 Making the Change The easiest way to convert to Pathways 4 from Pathways 2 0 1s to use this manual The Pathways 4 manual contains detailed explanations and descriptions of the features contained in the Pathways 4 software Book V of this manual contains a chapter of examples to guide researchers through the basic steps of image importing and analysis This chapter illustrates how to simulate the functions of Pathways 2 0 and it shows detailed examples of how to use the new analysis features of Pathways 4 Questions about the Pathways 4 software can be answered by emailing the GeneFilters Pathways technical support group at ccu QG resgen com II 8 Migrating from Pathways 3 Pathways 4 is fully compatible with Pathways
143. pi c H 121 RGMA10011 rev B This tree 1s mapped onto a hyperbolic surface that displays the tree spiral starting from the root of the cluster tree out to clone nodes The tightness of the spiral 1s controlled by a slider on the top bar Clones can be selected by clicking on the clone or through the find clones dialog box Clicking the center clone button centers the current clone in the viewing window Clones are labeled using the Clone Key Microarray Address or available Meta data The tree can be magnified by creating a zoom rectangle shift left drag or using the up or down arrow keys zoom in or out The image can be panned using control left drag Right clicking generates an options menu for the cluster tree ee UuImr ELI D wA Wiarton Wetting Trees i iie A yA Lateie ii key 7 iea JC bite pa Ei 0 N l This menu allows for High quality drawing slows the drawing process down but produces higher quality images Label size selection Zoom In Out and Reset resets the cluster tree to the original zoom and spiral 122 RGMA10011 rev B Chapter 14 Pathways Data Updates The information on clones in a microarray may change daily as discovery in genetics progress es Likewise analysis techniques improve as the field matures Pathways ensures that clone data and analysis techniques are current with the following features An integrated web browser with links from each clone to critical sites such as
144. rFX MurFAs mb 3 2 Z 1a Dao MFFM HiGE S ai 8 1 I 1g Dao AFFM BIGE M mi 1T q 1 iJ nas s il aj ZECE Finsh Cancel In the Column Identification section a Data Type and a Label for each column may be entered One column must be set as the Primary Key The Primary Key is a unique identifier for genetic material that Pathways uses to distinguish between microarrays The clone key is usually the accession number or image ID of the clone although the key could follow any other naming convention As discussed in Chapter 9 the key field 1s one way of grouping similar clones together for statistical analysis A Secondary Key mst be specified if the spreadsheet has any missing Primary Key entries Refer to Chapter 7 for more information on keys In this example the first column is used as the Primary Key for Affymetrix spreadsheets Column Identification Data Type Label Primary Key Probe Positive Negative Fairs Pairs Used Pairs InAvg 35 RGMA10011 rev B One column must be set as the Intensity The average difference Avg Diff column is used as the Intensity setting for this example Column Identification Data Type Pos Fraction Ignore Log Avg Ignore FosiMey ignore Avg Diff Intensity Abs Call Inc Data preview irs Pairs Used Pairs In amp vg Pos Fraction 1 pFFeMurlL2 at x Coordinates 2l 18 0 25 2 AFFX MurlLid_at fr Coordinates 14 045 Weta Data
145. rk The detail viewer may not display thumbnails with certain frameworks Refer to Chapter 2 for more information about thumb nails Additionally the synthetic microarray view is unavailable if the framework does not pro vide geometry Refer to Chapter 11 for more information about the synthetic view 29 RGMA10011 rev B 3 3 The Pathways Framework The Pathways framework is the standard framework used to import microarray data for analy sis Before data reaches the Pathways framework it must be processed by a Description plug in Description plug ins provide a format for geometry meta data and other information that Pathways can use for analysis ResGen GeneFilters microarrays pass through the GF Description plug in before the Pathways framework utilizes them Other data types pass through the Universal Description plug in after the Array Designer processes them refer to Chapter 4 for more information on the Array Designer Pathways stores a list of previously imported microarrays in a library file organized by microarray brands and types This library is similar to a collection of recently accessed files The user needs only to import the data for a microarray once After that the data 1s available in the library or by browsing directly for the imported file Use this library to set up new projects in Pathways A project consists of one or more conditions which in turn consist of one or more microarrays T
146. roject The empty project option in the new project wizard creates a project without any microarrays or conditions This option is used when setting up more complex projects that have multiple conditions and or multiple microarrays in each condition A wizard prompts the user to enter the researcher and project name and creates an empty project To add conditions to the project select Add Condition from the Edit menu or right click in the project window outside any conditions or microarrays that have been added 82 RGMA10011 rev B Pathways New project File Edit Comparison Profile Project Add condition To add a microarray to a condition either select the condition and select Add Microarray from the Edit menu or right click on a condition in the project tree Pathways Mew project File Edit Cnomnarison Profile Condition Add microarrays Rename condition Remove condition Selecting the Add microarray option causes the Add Remove Arrays window to appear For the Pathways Framework this window has two tabs for a full description of Frameworks refer to Chapter 3 The first tabs allows adding at least one microarray to the current condition based on records in the library file This file contains information on imported images and or information on files that have been used in a Pathways project The second panel allows browsing the file system to locate additional Pathways files
147. rray pairs conditions or condition pairs are selected for an analysis window Strict data filtering eliminates a clone from the analysis if the corresponding clone in any of the mul tiple data sets falls outside of the range of the filter Non strict data filtering eliminates a clone from the analysis only if the corresponding clone in all of the multiple data sets falls out side the range of the filter As an example consider an analysis window where two microarrays are displayed If a clone has a normalized intensity of 1 in the first microarray and 2 in the sec ond microarray a data filter specifying a minimum intensity of 1 5 with the strict setting on fil ters the clone while a non strict setting does not filter the clone 91 RGMA10011 rev B Strict data filtering is the default To change the data filtering type right click on the filter list and select deselect the strict option Invalid Clones Difference Path 9 4 Simple Data Filters Simple data filters use histograms and min max value key ins to limit a variable to a specified range of values With variables like normalized intensity and intensity ratios simple data filters reduce the data sets by eliminating data that the user regards as uninteresting Invalid Clones Kewin Intensity Wn izz Intensity Il 1044 733 NUNG N Difference NH First bin im x Hi c
148. rrays and MyArray DNA The many dif ferent applications of microarray technology such as differential gene expression gene discov ery genotyping pharmacogenetics et cetera can greatly accelerate genomic research I 2 The GeneFilters Microarray System Templates for genes are obtained from ResGen cDNA libraries arrayed in multi well culture plates The insert DNAs are prepared and quality control checked before being printed onto positively charged nylon membranes using an automated robotic system The DNA is then UV cross linked to the membranes These printed membranes are the ResGen GeneFilters microarrays In a typical experiment Fig 1 total RNA from both control and experimental samples are reverse transcribed and simultaneously radioactively labeled by incorporation of o 33P dCTP in the reverse transcription reactions These labeled probes are then purified and used in parallel hybridization experiments Expression levels of the genes on the arrays are observed as hybridization spots A phosphor imaging system detects the intensity and the image data set is then imported into Pathways 4 for analysis Pseudo colored images are created in Pathways 4 for each data set When comparing expression patterns between control and experimental samples the two pseudo colored images are merged to show intensity differences and ratios Information related to each clone including gene name clone ID accession number et cetera
149. s a few large bins and multiple small or empty bins due to one or two data points at the extreme end of the data scale Another fix for a skewed histogram is to lump a certain amount of the data into the first or last bin and use a lin ear scale for the rest of the histogram O72 282 405 FOF Oe 10 30 1942 0553 1765 19 77 21 88 24 00 26 12 28 23 30 35 ad LESEN z BH Mir i bee h Sk gt f DOE RS ER E EXM Fisihin bs r Dm 3 Bm a 1 57 255 4 19 5 85 11 20 16 15 VON MU i 0 24 135 PT ais BT 10 of Data in Last Bin 93 RGMA10011 rev B 9 5 Statistical Data Filters Statistical data filters reduce the data set by eliminating data falling outside a specified statisti cal confidence level The unrelated Student s t test analyzes each clone in a condition pair to determine whether the difference in intensities is statistically significant this test requires at least two measurements per clone Likewise the Chen test determines whether the difference in intensity of each clone in a pair is statistically significant but it does not require repeated measurements Finally the analysis of variance ANOVA test determines whether the means of three samples differ significantly from one another Each statistical test may have more than one distribution type as shown below Confaance Lowel Filler infomation URN LO of DOE OS beckons parm have Geen filtered becomes Chem mean intense ee nol eistutialhr chit si a con
150. sets ResGen recommends including no more than 250 clones in the formatted reports The CSV file does not require any special formatting and it can be used with an arbitrary number of clones The profiling and clustering versions of the Report Wizard dialog box are slightly different from the Comparison Wizard dialog box Oumurtio Press Fesranur ono Err Pree Teri Freis Liaurriz Daarriprizags rci be amirarad hara Dana LAS Fe ER FH rere era Jins potency For profiling and clustering a single index column is selected followed by a data source selec tion The index column can be the clone key microarray address or any meta data field A sin gle data column value can be selected for the report This data column is displayed for each item microarray microarray pair condition or condition pair that 1s included in the analysis Other functionality is as described for the comparison report dialog box 100 RGMA10011 rev B Book V Pathways Analysis 101 RGMA10011 rev B Chapter 11 Comparison 11 1 Introduction to Comparison Analysis Comparison analysis reviews the data in a single condition or microarray or compares data between two conditions or microarrays A simple comparison analysis would be to display a synthetic image of a single microarray such an analysis would allow a determination of the peak expression levels in the microarray A more complex comparison analysis might involve a
151. sley PA4 9RF UK Tel 44 0 141 814 6100 Tel Toll Free 0800 5345 5345 Fax 44 0 141 814 6117 E mail eurotech invitrogen com 159 RGMA10011 rev B Glossary ANOVA This is an acronym for Analysis of Variance ANOVA is a statistical analysis plug in that compares the variance of the data calculated within conditions to that across conditions If the variances are not the same then it is an indication that the means are different Array Designer The Array Designer is a tool available to Pathways Universal users It allows any type of microarray to be imported by creating a layout for associating data with an image of the microarray Atlas Array Gene List This is a microarray type developed by Clontech Laboratories Inc For more information see http www clontech com atlas index shtml Autocentering Autocentering algorithms analyze microarray experimental images and deter mine the location of each clone in an experimental image The process for locating the clone centers does not require user input and is therefore automatic Background The background display shows the computed background intensity for the image This display is useful when evaluating low intensity points Spots with intensities at or near the background average could be noise Batch The batch importing mode uses the autocentering algorithms to automatically import multiple microarray images Brightness Brightness adjusts the viewed intensity of
152. st be supplied to the Fuji plug in manually in the properties view see Locating the inf information below Obtaining images in Windows ImageGauge 3 12 The Windows standard format is already in the two file 1mg and inf form and so is ready to be read by Fuji to Tiff Converter9 The two files must be in the same directory when opened in the converter Obtaining images in other ImageGauge versions In File Export search for an option RAW or Fuji Exchange Format If RAW is selected the inf information must be supplied see Locating the inf information below When Fuji Exchange Format is selected the two files are output the img and inf files and both files must be in the same directory to be read by the converter Locating the inf information This information can be directly viewed from the file with the inf extension just open the file in a text editor If this view 1s 1mpossible the information can be obtained from Image GaugeQO under File File Info The appropriate encoding parameters can be entered in the properties view for the Fuji plug in in the Pathways image import dialog box 155 RGMA10011 rev B Appendix IV License Agreement STOP READ THIS INFORMATION CAREFULLY USE OF ANY OF THE SOFTWARE PROVIDED WITH THIS AGREEMENT THE SOFT WARE CONSTITUTES YOUR ACCEPTANCE OF THESE TERMS IF YOU DO NOT AGREE TO THE TERMS OF THIS AGREEMENT WITH RESPECT TO ANY OF THE SOF
153. subject to restriction as set forth in subparagraph c 1 11 of the Rights in Technical Data and Computer Software clause at DFAR 252 227 7013 or as set forth in the particular department or agency regulations or rules which provide ResGen protection equivalent to or greater than the above cited clause Contractor Manufacturer is ResGen 2130 Memorial Parkway SW Huntsville Alabama 35801 Should you have any questions concerning this license agreement or if you desire to contact ResGen for any reason please call 800 533 4363 fax 256 536 9016 or write ResGen 2130 Memorial Parkway SW Huntsville Alabama 35801 ResGen is considered a Supplier for purposes of this License ResGen is a branded product line of Invitrogen Corporation 158 RGMA10011 rev B Appendix V Technical Support For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 USA Tel 1 760 603 7200 Tel Toll Free 1 800 955 6288 Fax 1 760 602 6500 E mail tech service invitrogen com Japanese Headquarters Invitrogen Japan K K Nihonbashi Hama Cho Park Bldg AF 2 35 4 Hama Cho Nihonbashi Tel 81 3 3663 7972 Fax 81 3 3663 8242 E mail jpinfo invitrogen com European Headquarters Invitrogen Ltd Inchinnan Business Park3 Fountain Drive Pai
154. t From this window spots can be manually centered 132 RGMA10011 rev B ELS 878 EM FUEGO TYT Pees ed To add the research name and notes on the project click the Annotation button on the toolbar Yield a better picture of the alignment by zooming in and out on the detail viewer with the up down arrow keys 133 RGMA10011 rev B To manually invalidate a clone check the Invalid Clone box in the Detail View area Click Done if the alignment 1s satisfactory or click Back to realign the image The importing process for the first image is complete When the next image is loaded automati cally repeat the preceding steps The importing process is completed and the Quick Start palette reappears on the main screen when no more images are selected for importing 15 3 Example 1 Step Two Create a Project Using the Project Wizard The Project Wizard icon on the Quick Start opens a wizard that guides project creation Creating a project allows a researcher to organize the data for analysis save the data and reopen the data later Setting up the project includes specifying the kind of project and choos ing the microarray images from a list of imported images In this example a project is created to compare the intensity ratios and differences of the two imported images To start a new project perform the following steps 1 Click the Project Wizard icon in the Quick Start palette 2 Select Compare intensities of two m
155. t popiti Hane Fore Patr Fin Copy impor Tms Ad a be Fer M Aiden Cante This dialog box allows creation of an empty path copy an existing path or import a path from a text file key and address paths only Start the new path creation by typing the new path name in the Name text field To create an empty editable path select the appropriate type in the New 95 RGMA10011 rev B tab of the import dialog box and click Ok To copy an existing path and then edit that path select a path from the Copy tab and click Ok To import a path key or address select the Import tab select a file and path type and click Ok To import a key path create a text file with a single column of data representing a list of clone keys To import an address path create a three column text file representing a list of microarray brand type and clone numbers e g GF Description GF200 2 for the second clone in a GF200 microarray Separate the columns in the file by commas To create a new path from any analysis window click the New Path button on the analysis tool bar The new path contains the clones that are not filtered in the current analysis window A window appears and prompts the user to enter the name for the new path and whether the path is stored by clone key or by microarray address 9 8 Editing Paths Once a new path has been added or an existing path has been selected for edit the Path Editor dialog box
156. t were previously imported into Pathways 2 0 must be reimported into Pathways 4 The improved import process allows rapid reimport of previously imported data 10 RGMA10011 rev B Pathways 4 is fully compatible with all Pathways 3 projects and image files 1 7 Comparison of Pathways 4 Universal to Pathways 4 GeneFilters Pathways 4 Universal supports multiple microarray products while Pathways 4 GeneFilters software is designed specifically for ResGen GeneFilters microarrays Features exclusive to Pathways 4 Universal include the Array Designer and Frameworks which allow for analysis of other microarray products or previously analyzed array intensities Refer to Book II for more information about the Array Designer and Frameworks Whenever this symbol appears the text following it refers to features exclusive to Pathways 4 Universal 11 RGMA10011 rev B Chapter 2 Overview of the Graphical User Interface This chapter offers an overview of the Pathways Graphical User Interface GUI Detailed descriptions of importing project creation and data analysis are presented in later chapters 2 1 Layout of the Graphical User Interface The Pathways GUI has five primary sections Main Menu Workspace Project Tree Detail View Filter View Main Menu NN Project Tree Workspace Q Detail View Jm Filter View The main menu contains menu items for all aspects of the Pat
157. te Ordering initial rate of calculation during the ordering phase The initial rate decreases monotonically over the duration of the phase to small values near zero Initial Radius Ordering initial radius of calculation during the ordering phase The initial radius decreases monotonically over the duration of the phase to small values near zero Convergence Iterations number of iterations during the convergence phase Initial Rate Convergence initial rate of calculation during the convergence phase The initial rate decreases monotonically over the duration of the phase to small values near zero Initial Radius Convergence initial radius of calculation during the convergence phase The initial radius decreases monotonically over the duration of the phase to small values near zero Kernel Selection type of kernel used for calculation The Cylindrical option weights all nodes equally while the Gaussian option decreases the weight with increased distance from the updated node When the properties are set click Ok to proceed with the cluster analysis 118 RGMA10011 rev B 13 8 Profile The profile plug in displays the cluster data as a connected series of lines similar to the profile analysis technique E Cluster K mans Clustering Results Visualization Method Profile cluster visualization 9 im im a a dq a in Oo Control vs Month 1 Control vs Month 2 Control vs Month 3 T
158. tgDNA is spotted in multiple locations but a tgDNA spot has only a single microarray address Molecular Dynamics Storm This is a phosphor imaging system available from Molecular Dynamics 163 RGMA10011 rev B Normalization Normalization is compensation for global intensity shifts across multiple microarray experiments It is possible to make two images similar enough to reasonably com pare them by using either the control points data points paths or other methods currently installed as normalization plug ins Normalization groups Normalization groups assign an explicit normalization technique to one or more groups of microarrays Packard Cyclone This is a phosphor imaging system available from Packard Instruments Paths Paths are bookmarks or shortcuts to certain genes Paths are specified in one of three ways 1 by microarray address location on a microarray type 2 by clone key unique name of a clone usually accession number 3 as a search string s with each clone s auxiliary data e g contains cancer in the clone description Phosphor imaging system This is a high resolution scanner used with radioactive probes hybridized to ResGen GeneFilters or other microarrays Plug ins A plug in is a modular programming unit that adds or modifies key algorithms in the Pathways analysis process Pathways supports plug ins for image formats microarray type data sampling normalization statistical anal
159. the alignment window is redisplayed and there 1s a prompt to adjust the alignment 66 RGMA10011 rev B On successful alignment the alignment verification window appears Mpm eee eae TI eS Lares gree Teoria I r Paap ro n la Im LS on P When the centering process is not satisfactory click the Back button to revert to the previous window and adjust the centering parameters see discussion below on cropping rectangles and templates Click Done to write the Pathways sample data and image files and to complete the import process The following sections describe the interactive importing windows in more detail 6 3 Interactive Importing Auto Crop Mode The auto crop alignment mode invokes an automated algorithm that finds clones in the image A cropping rectangle can be dragged over the image to better define where the arrayed spots are located The crop auto and template buttons are located to the right of the rotation buttons Crop Automatic Use a cropping rectangle when multiple microarrays are within the image or when Pathways cannot find all the centers in the fully automatic mode 67 RGMA10011 rev B The cropping rectangle lies outside of the arrayed spots without overlapping the spots Perform the following operations to use a cropping rectangle Click anywhere on the image and drag for the cropping rectangle to appear Click anywhere inside the cropping rectangle and drag to move it To str
160. the calcu lated normalized intensity for this clone in the current project The background value is the raw intensity useful when evaluating low intensity points Genes with intensities at or near the background average could be noise Meta data such as cluster ID accession number and cDNA ID 1s also listed in the detail view This data can be updated as new information becomes available through the ResGen data server for details refer to Chapter 14 RGMA10011 rev B 18 Right clicking on the thumbnail image generates a menu with options for contrasting the image see Contrast Controller below viewing the original image and saving the thumbnail images Image and data files are stored separately and if a data file is moved the location of the image file must be updated The menu includes an option updating the location of the image Additionally options for adding the current clone to an existing or new path or invalidating a particular clone appear in the menu m d t i EU ETE Comit oa bof tee Rr T Ba Em F Gub dmend gt Co Subd verge image Sea Mimina irme Lied aha image D r aiia emit ciens dd dioe Vo ke paih id cone p eis m NE E riliji DE ings Md cune ko rere pah 2 6 Filter View The filter view displays available data filters for the current analysis window Esai Sane I 143 ao 0 etek bites nn 5 is 8 d AIRE S WN in LE Ab ee x Taek php bg Ln Lei a i fy Mails s LR
161. the contrast of thumb nails for other microarrays that are being displayed each image must be contrasted separately 21 RGMA10011 rev B Thumbnail images are framework dependent Some frameworks may not supply the geometry necessary to construct an image of the microarray In such cases the thumbnail image will contain the text N A as shown below Web Links PivotData 1 Intensity 554 1 Background Morn 0 436 Probe AFFA MurlL2 at Sort Score 10 Additionally right clicking on the detail view window area generates a different menu Web Links PivotData 1 Invalid clone Add clane ta key path Add clone to address path P i Add clone to new path Probe AFFR MurlL2 at Sart Score QIRI x The options for invalidating a clone and adding a clone to a new or existing path are present but the thumbnail options are not 22 RGMA10011 rev B 2 9 Progress Bars Progress bars are shown in Pathways when lengthy operations are performed to indicate the progress of the operation The progress bar on the lower right of the GUI indicates the progress of a basic operation like loading a file or calculating clone centers in the importing process A drawing progress bar appears in the upper left corner of a panel when a time consuming drawing operation is underway such as drawing a synthetic array or an experimental image 23 RGMA10011 rev B 2 10 General Settings General s
162. the spots in the SynFilter window If the spots cannot be seen easily the brightness is low it can be increased by using the Brightness Slider If the spots appear to be the same the brightness is high it can be decreased by using the Brightness Slider Brightness slider This tool adjusts the brightness or intensities of the spots in the Synthetic Microarray Windows Sliding the brightness slider to the right increase the brightness sliding it to the left decreases the brightness level Chen test This test is a statistical analysis plug in that determines whether two sampled inten sities are different based on a desired confidence level Clone key A clone key is a unique identification string that is present for each clone in a microarray The clone key is the accession number when available but it could be any identifi er such as tgDNA for total genomic DNA Repeat spottings in a microarray must have the same clone key to be treated as repeats for statistical analysis The clone keys are contained in the microarray s description file Clone number The clone number is the index of the clone in the microarray the microarray address is the filter type plus the clone number e g GF200 100 For GeneFilters the clone number is ordered by field grid row and column 160 RGMA10011 rev B CMT Map File This is a Corning Microarray Technology Map File A microarray type designed by Corning Incorporated For more inform
163. to Pathways Updates lllllle ene Launching the Updater ua u nuedechebadset ab OUR SOFAS E Esa RO doro eho doe did eleg Updating from CD au os eee bee taeda hehehe ae bebe een tae ee ae es oe Chapter 15 Examples 15 1 15 2 15 3 15 4 15 5 15 6 15 7 15 8 Introduction to Example 1 245446054540 9 2 RUE bee aoe aed pa eee ER Example 1 Step One Import Microarray Images llle Example 1 Step Two Create a Project Using the Project Wizard Example 1 Step Three Comparison Analysis amp Report Generation Example 2 Complex Time Study 3 5x aa PROPRE TUI eee Det 1 8 2 eg Cad ee Example 2 Comparison Analysis 222 ise ek dex RR RRLRE GR 3 m9 EE vs Example 2 Profiling Analysis auos acp ice ROTE 9 9 dox qr xh Ede CR CPU PR eee Example 2 Clustering Analysis 24 2 acte x cho Rede e Coe dose oe ods RGMA10011 rev B 99 99 99 Book VI Appendices Appendix I ResGen GeneFilters Microarrays LT TntrOdUCHOH 243 dudo Rer cee ne RUE aoe eee eaciu es dean eas L2 The GeneFilters Microarray System hoszaca s ku d eg do d d I 3 Layout of GeneFilters Microarrays 4s ciek ea RE SPERA RUP ME Appendix II Migrating to Pathways 4 Universal UN iS 2254560 lt ou tune ene ane ee Aad ooo S eee ech e nee es IL2 Image Import and Alignment 0 0 000 IL3 Grouping of Data and Complex Analysis IL4 Normalization and Data Filters 0 2 0 0 000006
164. to prevent divi sion by zero errors and ambiguous ratio values e g a ratio of 1 could result from an intensity of in the first spot and 1 in the second spot Y C normalization is a normalization technique adapted from a manuscript by Yidong Chen et al Chen Y et al J Biomedical Optics 2 4 364 374 October 1997 ISBN 1083 3668 This manuscript covers several important topics in microarray analysis including an iterative nor malization technique that normalizes a pair of microarrays such that the mean ratio between the microarrays is 1 0 A number of assumptions derive the normalization technique review the manuscript before using this technique 86 RGMA10011 rev B As the technique is iterative it does require as input the maximum number of iterations Max Iterations and a Termination Criterion Five iterations are sufficient to ensure that the mean ratio between the arrays is 1 0 although it 1s possible to select a larger number of iterations The algorithm stops the iteration process 1f the difference between the calculated mean ratio and 1 0 1s less than the termination criteria Group properties Normalization Bue o aidaa Fes Ta Plug in properties ax Iterations 5 Termination Criterion 0 001 f Normalize using all spots t Normalize using only the path spots The normalization technique has been extended to normalize more than two microarrays by set ting the first microarray in a multiple microarray set
165. to the description of the workspace 2 3 Workspace During a Pathways analysis session the Workspace may contain multiple analysis windows The current analysis window completes the appropriate information in the detail and filter views the browser for example does not use the detail and filter views 15 RGMA10011 rev B When an analysis window is open the Window menu options on the main screen are active The four options Cascade Maximize Tile Horizontally and Tile Vertically allow manage ment of multiple windows simultaneously mama Cascade Horizontal Vertical Maximize In addition each current analysis window is displayed in this menu An analysis window displays a title bar at the top of the window along with minimize maxi mize and close buttons The title bar displays the type of analysis It also gives details of the averaging method microarray address or clone address refer to Chapter 7 for more informa tion used for data analysis 2 4 Project Tree The Project Tree area displays the current Project as a tree of conditions and their microarrays Right clicking in the Tree window generates menus that serve as a shortcut to much of the func tionality in the Edit menu EE Control ode Time 1 04 Time 2 7 3 Month 1 0 04 Month 1 1 Condition Bea Mor r1 Right clicking outside any conditions generates a menu that allows adding a condition Right clicking on a condition
166. ts allow statistical data analysis 137 RGMA10011 rev B 15 6 Example 2 Comparison Analysis Comparison analysis obtain a broad overview of the data by displaying data from entire microarrays or conditions in a single plotting window with different series overlaid or with mul tiple table separated synthetic microarray or table windows For the current study clones that are up or down regulated are determined across the time study in comparison with the Control condition A comparison view is first created by selecting Compare gt Condition Pairs from the main menu Profile Cluste Microarravis Microarray paire conditionis Conditions are used for this analysis rather than microarrays because both microarray types including repeats will be reviewed across all conditions A condition pair is chosen to observe the upregulation and downregulation of the clones relative to the control condition Therefore pair each condition in the time study with the control condition to establish differential expres sion 7 Compate Conebloon Pairs Pairs Canty Triak vs 1 Month Triat Contro Thaks ve 7 Monilh Triss Canto rtas vn 3 Month Trma Becia First Corto Thais Second f3 Month Trias Conini Trials Average by Micriamran Address uoces Trialg C Awerage by Clone Key 2 mori Trials 3 Mor T ace ok 138 RGMA10011 rev B After selecting the condition pairs click the Ok button to
167. u wall be able to see a primii of the imm oried file im the window below Lavo Larout nare oF 200 ano ul Test Qualfigr i Stewahrgw R 3 Headerrae f1 E Dielrmiler Delim er Tat hgnanecnnescumes disti mers niew FM ANCEENT eA 1 oFz00 Research Genetics Gener ater Species Human OF 200 2 31 i 4 GF JU Hesmarch Genetics Gane ffgr Species e Human GF 200 2 3104 707515 1 3 GF z Research Genetics Genet fier Species Human GF 2005 2 3104 207515 4 OF 200 Wesaarrh CGenelicz Genef iter Speries Human OP 200 2 9104 207505 GFFO0 Research Genetica Hangt ier Species Human GF 200 Z 3104 ror 404 amp GF z t Resarch Genetics Genet iler Species Human GF 2005 2 31042074315 z Ci The Layout name field contains the name that was entered in the File Layout field on the pre vious screen In the Text Qualifier field select the symbol used to delineate text from the rest of the spreadsheet Typically this is either an apostrophe or quotation marks Select None if the spreadsheet does not delineate text In the Start with row field enter the row on which the data begins In the Header row field enter the row on which column headers for the spreadsheet are located or enter None when there is no header Typically the header row is one row above the first row of data In the Delimiter field select the character used to separate columns in the spreadsheet If the Ignore consecutive delimiters box
168. uisuig apre beeen He ches pe h of irig e beri cherie Cpurbiicn fer euch clone Hiarerchital cizzxrg porte work try rekzg Dur bam clan Ek and Dien aiig the d cher The Ce ad ee a paliken gi dais spare which in based upon ihe fime metic Hos fir ciun tere miter cler ar chiriei acr frend and ground is She pxondc srr Thi pra res jiegecabs ubl dd cheres ae grougsl diis rkurimr There ure mor wer do describe the digiti Perens pointe gr dais mmcc Tir iri era Abart calzzhebon iur a Fuckin caben Tor square Exc dai Giaa e rca hretrer esee apon dhe er ihe darrii Ezriimr cxxubiim 11 3 Cluster Visualization 25 Book II Universal Concepts RGMA10011 rev B Chapter 3 Frameworks 3 1 Introduction to Frameworks Pathways Universal introduces the concept of frameworks Frameworks are collections of modules that work together to provide a method of importing microarray data from various sources Previous versions of Pathways came with one framework that limited the importing of microarray data to ResGen GeneFilters image files ResGen GeneFilters microarrays were imported into the Pathways program using the standard Pathways framework Sophisticated image processing techniques then transformed the raw image file into a computa tional description of the microarray Pathways used this description in its analysis of the microarray Create Project Normalize Data GeneFilter Raw l Image Analyze
169. vailable in Pathways 2 0 refer to Importing Images II 3 Grouping of Data and Complex Analysis There is a dramatic improvement once images pass the importation steps and move into the data analysis process Using the terminology microarray s microarray pair s condition s and condition pair s is one of the new concepts of Pathways 4 Once researchers become familiar with these new concepts of data grouping they will value the global and complex analysis options Pathways 4 has to offer refer to the Core Concepts in Pathways Data Analysis grouping of data section In Pathways 2 0 single GeneFilters microarrays are analyzed for intensities by selecting Analyze GeneFilter from the tool palette To look at the ratios of normalized data from two GeneFilters select Compare GeneFilters These same functions can be performed in Pathways 4 but the terms that describe them are different In Pathways 4 these analysis tools are found under the Comparison menu Analyze a single GeneFilters microarray by selecting Microarray s Analyze two GeneFilters microarrays by selecting Microarray pair s from the Comparison pull down menu In addition these options are on the Quick Start menu which is similar to the Pathways 2 0 tool palette Multiple microarrays can be examined simultaneously For example by adding more than one GeneFilters microarray when selecting microarray s from the Comparison
170. wb ely sgum the deletes starting er and den quabfier tar your fie Yo vad be abis In See 3 piara of me imported Sis m me wind i be rw Lapai Lyriniai Ted Susie T Start with rw i Header row Mon Dednder Demie omma ignore cons ecutive dekmkers Chap Mee BOUT DE p NEAT MG AACE MiTVMES MOLITIAT El az MPa Ss si d CP otte ON egotet OP sire OP aire ed OP ars nag or FrictiencLag amp egLrFastre AFFE MorlLz iD OP Oe BED 25CHD BBETI 77 554 1PEACETELD eo 1 DEEC AFP Mol sBLBLDOCDUTTI S00 45L EGCTI BCP 5B aOR CeCe T ULT werg f The Layout name field contains the name that was entered in the File Layout field on the pre vious screen In the Text Qualifier field select the symbol used to delineate text from the rest of the spreadsheet Typically this symbol is either an apostrophe 7 or quotation marks Select None if the spreadsheet does not delineate text For Affymetrix spreadsheets quotation marks are used as the text qualifier Text Qualifier Header row Mone LIFILIE E LIF SE CLE TE Te 33 RGMA10011 rev B In the Start with row field select the row on which the data begins In the Header row field select the row on which column headers for the spreadsheet are located or select None when there is no header Typically the header row will be one row above the first row of data For this example the data start on row 5 and the header row is row 4 Layout Layout name Affymetrix Text Qu
171. would yield clones that are in both the Breast and Cancer paths 9 10 Invalid Clone Filtering During microarray analysis some clones do not provide valid sampled data Multiple factors including less than optimal experimental parameters a damaged microarray or the subjective opinion of the researcher may render some clones unusable for the analysis process Pathways provides several tools to flag and keep track of such invalid clones An individual clone marked as invalid will not be automatically excluded from the analysis process but can be excluded using the invalid clone filter If an invalid clone is used for the calculation of an aver age intensity during condition analysis the resulting combined spot will also be marked as invalid Invalid Clone filtering allows including or excluding specific clones from the data set The Invalid Clone filter window offers three options for showing all clones showing invalid clones only or showing valid clones only Invalid Clones Filtering Options Intensity show All Clanes Intensity Il v Ratio Show Invalid Clones Only Difference t Show Valid Clanes Only w pathc Clones marked as invalid from in a project are seen as such in the current project only Invalid clones are marked as such with crosses if the Mark invalid clones button in the upper right corner of the workspace is selected There are two places where the user can mark clones as invalid If the user marks a c
172. y Designer bh ah d Once this template is overlaid on the experimental image Pathways will generate the seed positions for each spot based upon a comparison of the template in the ideal coordinates versus the position of the template in the overlay in the experimental image Often the manufacturing process for microarrays introduces known offsets in spot positions based upon the mechanical devices that create the microarray For example each subgrid in a microarray layout with multiple subgrids might be offset from the other subgrids due to the manufacturing process 42 RGMA10011 rev B If this is the case then it 1s advisable that each subgrid be assigned its own template in the Array Designer in the figure below templates are shown in red global bounding box in black In general the smallest number of templates needed to adequately describe the geometry should be used because each template may require further individual adjustment by the user during the import process Regardless of the number of templates the final portion of the template importing process is identical to the auto crop mode The seed positions which are generated from the template overlay are fine tuned according to the autocenter mode choice For more information on the autocentering and the importing process refer to Chapters 5 and 6 43 RGMA10011 rev B 4 5 Opening the Array Designer To open the Array Designer select Array Design
173. ylon membrane Each membrane is cut in the upper right corner for orientation and the DNA is on the labeled side of the membrane Figure 2 illustrates the general format of a GeneFilters Mammalian microarray membrane Each membrane is divided into two fields Field 1 is at the top and Field 2 is at the bottom Each field is then further divided into eight grids Grids are laid out right to left A through H in each field and then organized into 12 Columns and 30 Rows Columns are numbered 1 to 12 right to left in each grid Rows are numbered 1 to 30 from top to bottom in all grids in both fields Control positives are in Column 1 Rows 1 3 5 7 9 11 25 27 and 29 and in Column 2 Rows 1 3 and 5 in each erid Also in each grid the housekeeping genes are in Column 1 Rows 13 through 24 Fig 2 and Fig 3 The spacing between each spot is 750 microns from center to center Fig 3 148 RGMA10011 rev B Grids bk t Oke Oe 0 9g a ale ae ae le Ae 0 a ode ale ale aks Oe Oe E 90 oD ode ale ale ats Ee oem Mle Be leg oT Dy le ae ae ae RD ae le le a ae al le ls Ee A a ee E E ee a a a a a i Ti n PER nuns paggggms p gugsg Deed ee De De Dee E E Jane eeegaee E p a H mom ae RRATE Aa ye ae ee GF200 980930 48 IXJ2RRERESARSZZRRERSXZSRERDI32 Ho oe ee pl hoa ee doh a EIE E Wel alot td tt add tt dt i dt edt tt ed F SR i e aa aia eS i oe a a Ae Er Ceai wd aa le ee eo a a ac T id un n m
174. ysis and clustering Pluggable A component of Pathways is called Pluggable when a plug in is associated with the component e g clustering Ratio This is a convention for displaying the ratio of the normalized intensities of two clones that facilitates the recognition of upregulation and downregulation The ratio of clone A versus clone B is equal to the normalized intensity of B divided by A if the normal ized intensity of B is greater than A and otherwise the ratio is negative A divided by B Project Pathways projects organize previously imported microarray data into conditions that represent experimental states PWF file This is an extension for a Pathways image file that contains calculated clone loca tions on the experimental 1mage as well as a full resolution copy of the original experimental image this copy may be cropped and or rotated depending on the alignment of the original image PWS file This is an extension for a Pathways sample file that contains sampled data from a Pathways import session This file contains the data that is used throughout the Pathways analysis process Quick start palette This is the picture menu that reappears when a task such as importing analysis comparison ef cetera has been completed or exited This palette can be enabled or disabled in the Options 164 RGMA10011 rev B Ratios Ratios are the numerical value of the Normalized Intensity

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