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ChipInspector 2 User Manual

1. 1 16223896 4 78 ONM 001042704 5 88 1 16255514 ah 5 33 inm 000085 6 13 1 47422073 25 487 2 NM_005764 6 28 1 47422134 25 5 36 3 NM_005764 6 64 1 47423263 25l 5 24 4 NM_005764 6 4 1 47423314 25 5 81 S NM_o005764 6 16 1 47425604 25 4 64 6NM_005764 6 72 1 58814109 25 5 17 7 ENSTOO000371225 5 95 1 58814291 25l 5 27 8 ENST00000371225 5 93 1 71250641 25 5 01 alnm 198719 4 571 1 71285428 25 4 96 10INM_198719 5 07 1 77655941 25 5 78 11 ENSTOOOO0354567 5 49 1 77756895 25 6 13 12 ENSTOO000354567 5 68 1 77797280 25 6 05 13 NM_174858 5 36 1 110947700 25 5 37 14 AK290061 4 96 1 1110947738 25 5 32 15 ak290061 4 94 1 149306331 25 5 43 16 NM_006818 5 01 1 153427110 25 4 82 17 ENSTOO000338684 4 67 1 161310779 25 5 62 18 xM_001 174405 5 25 9 3 The Transcripts Window The result list lists the significantly regulated transcripts and their probe coverage i e the number of significant probes that map to the transcript exons from left to right e the NCBI GenelD the official gene symbol the RefSeq Accession Number e the coverage of this transcript by significant probes e the average fold change of all significant probes for this transcript Clicking the accession number will update the Alternative Transcripts window and display all alternative transcripts at this locus If there is sign
2. Steps 1 Choose Action 2 Choose Files to Import 3 Microarray Data Import Genomatix I Steps 1 Choose Action 2i Genomatix ChipInspector Choose Files to Import Microarray Data lLexample GSM51679_13_0 CEL a 2 ALLexample G5M51680_17_24 CEL i 2 4LLexample G5M51682_17_0 CEL Y b m HG U133_Plus_2 Human v Protocol Files Browse Created Files roarray DatalGSM51679_13 O cif roarray Data GSM51680_17_24 cif roarray Data G5M51682_17_O cif b Chip Type Microarray Data Import Please wait while the data is imported to your project data folder This may take a few minutes Importing GSM51676_24_0 CEL to G5M51676_24 O cif eT 17 Importing microarray data files Import in progress Stop lt Back Finish Cancel Help x Choose Action gt S Project MyTestproject v Categories Actions Treatment Contral Pairing Time Course Titration Experi Presence Absence Calling 32 Description Performs a one class statistical analysis 4 single sided permutational T test is calculated and each probe is assigned a significance score This analysis is suited For the x Finish Cancel Help understanding gene regulation Provide a name for the analysis Click Next The data files for the project are presented in the central file list Select the control files and as
3. Full Screen Alt Umschalt Eingabe v Help Memory Projects Small Toolbar Icons Reset Toolbars Customize Click the memory monitor to free unused memory t ChipInspector 2 0 File Edit wiew Navigate Tools Window Help EETL lA alle Projects 4 x Welcome x 2011 Genomatix Software GmbH 46 GenomatiX Chipinspector understanding gene regulation 10 Projects and Analyses 10 1 Creating a New Project To create a new project select File New Project from the menu or click on the New Project toolbar button A form is displayed which asks for a project type Currently only one type of project is supported the New Project Click Next An input form is displayed asking for a project name Enter a name for the new project and click Finish x Steps Choose Project 1 Choose Project Categories Projects Qe an New Project Genomatix Me Description 4 new project is created Data can be imported into this project for subsequent mutiple analyses eis Finish Cancel Help Steps Name and Location 1 Name and Location Project Name MyTestProject Project Location fand SettingsihahniGenomatixProjects Browse Project Folder ahn GenomatixProjects MyTestProject Genomatix Cancel Help 2011 Genomatix Software GmbH 47 Genomatix ChipInspector understanding gene regulation 10 2 Opening an
4. EFI understanding gene regulation wien Factor Binding Sites FOr comments questions or bug reports p W006 All sights reserved For more information please contact Genomatix Software GmbH Bayerstr 85a 80335 Munich Germany Phone 49 89 599766 0 Fax 49 89 599766 55 Email info genomatix de WWW http www genomatix de 2011 Genomatix Software GmbH GenomatiXx ChipInspector understanding gene regulation Table of Contents Ts troduction t CHIDINSDSCIOF sscscncsmnssnycsencsnncennniepenensveheyopadcbaveraverenesitsuinveansalwstaisnusimnelnincessunssians 4 1 1 What is Genomatix Chiplnspector 00 0 ceccccsscecceecesssseeeeeesessseeeeeeesessseeeeeesesseaeeeeeees 4 1 2 Which Pages in the Manual Should Read First ceeeescecssseeceeseneeeeesseeeeeseeeeees 4 2 Theoretical Background eieiaeo teseadatnd amaueinalspencainsaainaueis ia svoanctideetasanidebosesasebuscsecens 5 2 1 Rationale of the Single Probe Approach ccceessscccccsssssseeececsssseeeeeeesessseeeeeeseeesseeeeeees 5 2 2 Normalization and Fold Change Calculation cccescccceesssecessneeceessneeeeseneeeesssneeeees 6 2 3 Statistical Evaluation seoan aS EE dem vert EAEE E 7 2A Fie CombNaUONS seirian a E ENN O AO OR 9 2 5 One Class Analysis Experiment versus Control ccccccccsscccessteceeessteeeesssseeeeessneeeees 10 20 Mu ulicl ss AMAL SIS sorcisti A seiceuanduenncndsnnedeutieetiaantuctsacataatlaccseemsaust 10
5. Back ii 4h Home x Bookmarks L mozilla org L mozillaZine L mozdey org Genomatix GenomatixPortal Switch to encrypted login page Please log in Username bmustermann Password f ra free of charge evaluation account if you haven t done so yet Lost Click here to retrieve your account info if you lost your username your password and or password For comments questions or bug reports please contact support genomatix de Genomatix Software GmbH 1998 2006 AH rights reserved License Agreement OY Ge CCS oar 2011 Genomatix Software GmbH 22 Genomatix ChipInspector understanding gene regulation After login you will see the following page Click on Password B Genomatix Welcome to Genomatix Suite Bernd Mustermann Mozilla Bile Edit View Go Bookmarks Tools Window Help z Ft Eee P K Nf gt v a Hi AX http portalt 0 genomatix de cgi bin welcome welcome pl s Fa7eesbd4236dd7e87F 1 c2d5eadd cd3 v S4 X m 2 z Print Back al Reload ji Ah Home W Bookmarks mozilla org wozilazine mozdev org Genomatix Pecani S New ee ut GenomatixPortal ElDorado Gene2Promoter BiblioSphere Sequences Protocol GenomatixPortal GEMS Launc r Login information 2006 03 17 Welcome to Genomatix Suite Bernd Mustermann 6 new messages Our database contains no records about y
6. at this point the analysis is stored for later inclusion into a batch job i e when more than one analysis is run in one continuous process The time required for an analysis depends on the CE x r St Run Analysi size of the microarray the mes ee 1 Choose Action Please wait while the analysis is performed This may take several size of the data set and ysis is p y 2 Name and Location minutes the hardware 3 Analysis Design 4 Combinations and Store values i Normalization Type 30 The program gives an 5 Run Analysis Performing analysis MyFirstAnalysis 1 1 pa 8 G e lg Oo m a t i xX Analysis in progress Stop overview of the progress When the analysis is finished Click Finish Finish Cancel Help Please refer to page 38 for a description of the various data views which are presented by the program 2011 Genomatix Software GmbH 56 Genomatix understanding gene regulation ChipInspector 10 8 Committing an Analysis for a Batch Job Right click the project or select New Action from G New Action l 3 Ea Steps Choose Action 1 Choose Action Project ExampleProject z the menu bar to display the gt possible actions and select Analysis Batch Job from the list Genomatix Click Next Actions EJ Cel File Import ff Tabular File Import Treatment Control Pairing ES Time Course Titration Experiment p Presence Ab
7. permutation T test analysis is performed Presence Absence Calling Choose this option if you want to measure expression values relative to the average expression on the chip e g for gene expression values in one specific tissue In this case a permutation T test analysis detecting probes which are significantly above the experiment average is performed Biological replicates with n gt 2 are still required Click Next 2011 Genomatix Software GmbH 53 Genomatix ChipInspector understanding gene regulation Provide a name for the analysis Click Next The data files for the project are presented in the central file list If Treatment Control is selected two windows are displayed to which the respective files are assigned Select the control files and associate them with the Control list 1 Choose acti 2 laa Lm MyFirstAnalysis 3 Analysis Design 1 G snomatix ix New Action Steps 1 Choose Action 2 Name and Location 3 Analysis Design 4 Combinations and Normalization Type 5 Run Analysis Genomatix Fimisti Help New Action Steps 1 Choose Action 2 Name and Location 3 Analysis Design 4 Combinations and Normalization Type Run Analysis 2011 Genomatix Software GmbH 54 Genomatix ChipInspector understanding gene regulation Select the files that xi represent the experimental Steps
8. 2 7 Absolute Expression Value cccssssscccccssssssssssnssssccccceecssssssssssseeeceeeseessesessssaaaasaeeeees 10 PO POE OaE A eee ee AA OA N O A E E O OEA 10 3 Supported Microarray Platforms ccccsscccsssssssececcesssssseeeeeesseesseeeeeseesseeeeeeseeesaeeeeeeesenaeees 11 3 1 Affymetrix Microarrays eeseessesesssssesssssesssseessssressssreesssrresssreesssessseresssseressetressereessereesse 11 3 2 Agilent Microarrays eeeeseeseeesssesseresrsesreesrssserresrsssstresrsesseresesessressessetrernsesseresrsessereeesee 11 AUIS MN A Sa eee oa sane ERNEA EAEE A A AE eased ANE AS 11 3 4 Platforms and Probe Numbers ccccssscccssssssssecccesssssseseeecsssssseeecesssssaaeeceessssseseeeeees 12 A File IREQUIRGCINGING wiceradincmaniveravivernniverasaaaiawansareareasetimaestetenieauivioncivemnenuenndes 14 5 Technical REQUIFEMENS ccccccccccccccsssssssssnssccccceecsesssssssseeessecececesesssssseaessseecceseseseeessesenes 14 5l Operating SS eene E EAEE ERA EEEE E a aa aaas 14 92 Java Runtime ENVIFOMIMEN Tj scccssrmnnnencnnsunere orasncsnownbnaanenvonssvoptsnsanenthiienssdnddienssiveweshondacssenusees 15 6 Installation and Configuration Of Chiplnspector 0 0 cccscsssteeeeeeesseeeeeeeessseeeeeesssseaeees 17 6 1 OWT E aE O EE ENEE REEE AE OERA 17 6 2 Get Login and Password sesesessssesssssessssresssseessssrresssrresssreesssteesseeessseersssereesssreessereesse 19 6 2 1 ESES 241 2 L EA EEA
9. A AA ee ee A A E A T 19 6 2 2 Change Password scree E TEENE EE E EEEE 22 6 2 3 PassWord Ta ete eee petieiay averse aineric EENE aai ieai 24 6 9 gt 2 1 9 6 ee eenner ene earn ree Creer cree eee ee eet ee ee eee eer 24 6 4 Configuration Of CHIPINSPECHOS ee ccccceessssseeeceeeseseseeeeeeeeseseeeeeeeseesseeeeeeseeseeeeeeeees 26 6 4 1 Proy COnN NOUTA UO sisia A medecetaccducer cannes 26 6 4 2 Configuring automatic updates ccc cecessecccsssneecesesseceeeseeeeeesneeeeessteeeesesneeeeeseas 27 6 4 4 Selecting a Data Server ec cccsssccsssssecccssssseccssssesecesssssecsssesecessseeeesesssnseceseanes 28 6 4 5 Configuring the Java memory settings ssseseessssesesssseerssertssseresssereessereesssrreessee 29 7 Step by step Performing a Chiplnspector ANnalySIS ccccccsssccessssseeeceesssssseeeeessssssaeees 30 8 Variant Chromatin IP workfloW cccccccssscccssssssececeesssssseeeeessessseeeeeseeesseeeeeessessaeeeeseesenaeees 35 9 The Chiplnspector Workspace Alrea ccccccccsssssccccssssssseceessessssseeccsssaseeceessessaseeeeesesssanees 36 9 1 The Projects Window sesesssesessssssesssssessssressssressssrrssssrresssseesssessseesssserssseeresssreesssreesse 37 g2 The Probes WnNdOWseeeieiiiesciessiiiiunan eiun NINE NENE OSESE 37 GS The Transcripts WINDOW siucsiinniacvsavriinriiarecunecssancsaraiancnenavancsacetecesniavdvenvenetiaetantianriasciaets 38 9 4 The Genome Brows
10. Analysis Design condition and associate Choose Action 1 2 Name and Location Treatment Microarray Data Control i 3 Analysis Design a EA G5M51674_24_24 cil e N 8 GSM51676_24_0 1 th em with the Treatme nt 4 Combinations and EJ csms1677_13_24 cil GSM51679_13_0 1 Normalization Type FE Gsm51680_17_24 cil s GSM51682_17_0 list 5 Run Analysis Genomatix Click Next If time Course Titration is New Action x S e e cte d J yo u a re Steps Analysis Design a d d iti on al ly as ke d to TAA Number of Experimental Classes p z 2 3 Analysis Design 4 Combinations and choose the number of Combinations a lormalization Type Treatment 1 Microarray Data Control 1 m y 5 Run Analysis KJ isMap Brain y 7 experimental conditions P F Eo which are relevant for this gt Dji mernans e s Treatment 2 TisMap_Kidney_02_v1_W Control 2 analysis R EJ TisMap Kidney _03_v1 o ai lt l Assign the files accordingly Treatment 3 control 3 control files may be A F le le B e l gt associated with more than Genomatix raran E one experimental class a is w a gt s el 3 while each treatment file Ra ita may be associated with only group Q Please add at least 2 treatments lt Back Next gt Finish Cancel Help Click Next New Action x Accordingly wh e
11. Existing Project To open a previously saved project select File Open Project from the menu or click on the Open Project toolbar button and select a project tree file from the dialog 10 3 Saving the Project Tree To save the project tree including all projects analyses and results select Save from the File menu or click on the Save All toolbar button 10 4 Deleting a Project To delete a project including all analyses and results right click the project in the projects window and select Delete from the context menu 10 5 Importing Data Files into a Project Projects 41 x Welcome x My To import data files into o T New Action button in kj HUEX x Set as Main Project HH Time Course Titration Experiment rs the task bar or right A MyFirst Close 15 Analysis Batch Job click the appropriate J MF Rename 2 aiaa t 5 bi dis Move project and select New amp Microart Other Action Opy from the context menu 3 newproject Delete 4 ChipIns Chip Properties Chip li Am Anahree Select Cel File Import from the context menu 2011 Genomatix Software GmbH 48 Genomatix understanding gene regulation In the next step the raw data files are imported The wizard opens this dialog automatically Depending on the type of data choose CEL File Import for Affymetrix or Tabular File Im
12. Window Help version of the Gea program is EUL available you will be notified by a symbol in the bottom right corner of the screen Click this icon to update Chiplnspector automatically or go to the Help menu and select Check c fi a x Welcome x ChipInspector Genomatix understanding gene regulation AVAILABLE MODULES ChipInspector Analyse Microarray Data GENOMATIX NEWS ul 8 2008 6 00 00 PM Deep Sequencing Study Reveals New Insights into Human Transcriptome oint project of the Max Planck Institute for Molecular Genetics and Genomatix takes the first step ul 2 2008 6 00 00 PM Next Generation Data Analysis for Next Generation Sequencing Data Genomatix Genome Analyzer released The Genomatix Genome Analyzer GGA is the first integrated har un 4 2008 10 15 00 AM Genomatix releases RegionMiner fast extensive analysis of genomic regions RegionMiner is Genomatix new tool for analyzing genomic regions coming from high throughput experim Mar 3 2008 12 00 00 AM NIA uses Genomatix software in Stem Cell Research Suggests Novel Transcription Factors for Stemness Linkage of Pluripotent Stem Cell Associated Transcripts to Regulatory Gene Networks Feb 26 2008 12 00 00 AM nen mena O ON E S EE mon mene Mian fen men Sn mannii Semone Binet mon meni ALL NEWS 1 update found Click here to make your application up to date For Updates 6 4 4 Selec
13. application you will need Java 1 6 or higher To test if you have an appropriate Java version already installed on your system type java version on command line Here is an example for windows users how to check the installed java version Click on Start All Programs Accessories Command Prompt see screenshot below Ca E mail wa Accessories a Accessibility gt i Jutlook Express Im Games gt i m Communications b r re i j b m i b e an a Microsoft Office Tools D Entertainment m Startup gt i T System Tools gt Internet Explorer UJ Address Book Microsoft Access amp Calculator wy Adrese Microsoft Excel cx Command Prompt KS Microsoft FrontPage Notepad rr Notepad Microsoft Outlook Y Paint f Microsoft PowerPoint Program Compatibiity Wizard Nero Burning Rom ee _ Microsoft Word Synchronize amp seinen WI MSN Explorer Tour Windows xP A Outlook Express LI Windows Explorer Server Manager pa Remote Assistance DN Windows Movie Maker windows Media Player A WordPad a 2 a QP Morpheus 2 0 F Event viewer G Windows Explorer 2 A Download Accelerator gt All Programs FA HP RecordNow 2011 Genomatix Software GmbH 15 Genomatix ChipInspector understanding gene regulation A command window will pop up cx C WINDOWS System32 cmd exe WINDOWS gt Type in java version and press Enter If Java is installed you will get an output like
14. apply For online demonstrations and unlimited access please contact sales genomatix de First name FO Last name Company Organization ee Department TF Affiliation academic commercial Address FO P o Box ZIP Postal code City FC State Province ee Country Please selectyour county x Phone number FO Email address FO How did you hear from us Please select gt The fields marked with are required fields Please provide your full First name and Last name yll and correct details above We offer free of charge special howrong or incomplete registration details will be cancelled without we allow for one free account per use arrangements for academic teaching co pre notice Your IP address is being mor Please fill in yo us After the registration your evaluation account information and password Reset Form For comments questions or bug reports please contact support qgenomatix de Genomatix Software GmbH 1998 2008 All rights reserved License Agreement 2011 Genomatix Software GmbH 20 Genomatix ChipInspector understanding gene regulation Check your e mail A mail with your login data should be sent to you right away Dear Bernd Mustermann your registration for a Genomatix evaluation account was successful Here are your account details login name bmustermann password nUDb Pn Please note that userna
15. center higher than the expected line Most of the data points will deviate little from the straight line only in the extremes of the set is a marked change in observed versus expected signal visible When the cutoff values Apos and Aneg are changed interactively by the user the program recalculates the set of deviating probes i e those regulated by the experimental condition Decreasing the A value yields more significant features at the cost of increasing likeliness to find false positives Increasing A diminishes the group of detected probes the resulting set has a higher stringency i e less danger of including falsely detected probes A slightly different picture is obtained when the analysis is run as multi class analysis Here the features cannot be separated into down and up regulated groups since the fold changes are calculated individually for each class Each feature may thus be up regulated in one and down regulated in another class e g at a different time point in the experiment The statistical score is therefore calculated as a deviation from the expected absolute behavior of the average feature there are no negative ratios in this case The resulting curve is shown in Figure 3 There is no magic number or recommendation for A or the resulting FDR This is an experiment specific value a trade off where the user has to decide on the stringency level 2011 Genomatix Software GmbH 7 Genomatix ChipInspector un
16. ewu This is especially helpful when the analysis is based on an older version of ElDorado therefore disabling the graphic view in Chiplnspector The ElDorado view can then still be used to see the genomic environment of the selected transcript 2011 Genomatix Software GmbH 39 Genomatix ChipInspector understanding gene regulation 9 4 The Genome Browser Window The Genome Browser shows the genome annotation at the specified locus It automatically repositions the view if a probe or a transcript is selected in another view The following elements are extracted from Genomatix ElIDorado database and displayed in the graph Element Color Element Description Primary transcript Exon Promoter 3 UTR Transcription start region based on CAGE tag evidence Up regulated probe significant non significant Down regulated probe significant non significant a i Oo a The elements on the forward strand are displayed above the black line the elements on the reverse strand are located beneath the black line The vertical size of the probe reflects the fold change in the experiment Tool tip texts give additional information about the elements The following graph explains the various functions e Da x alp 7 Uy E E E E J hromosome 3 From 52529676 To 52533585 2011 Genomatix Software GmbH 40 Genomatix ChipInspector understanding gene regulation A region
17. is feasible when not comparing individuals e g patient samples 2011 Genomatix Software GmbH 9 GenomatiXx ChipInspector understanding gene regulation 2 5 One Class Analysis Experiment versus Control This analysis is suited for the comparison of two experimental groups such as knockout versus wild type treated versus untreated or diseased versus healthy 2 6 Multiclass Analysis This analysis is suited for kinetic or titration experiments with more than two data points 2 7 Absolute Expression Value This analysis is suited for comparing individual probe signals against the average chip expression value 2 8 Projection The approach to microarray analysis in Chiplnspector is annotation driven In contrast to most other software packages Chiplnspector does not rely on the predefined probe to transcript assignment done by the chip manufacturer at the time of the chip design ChipInspector includes a new probe to transcript association performed by the Genomatix proprietary mapping pipeline based on an up to date genome ElDorado Significant probes are projected to transcripts using pre calculated mapping files As default value three significant probes are needed to detect a transcript as significant This figure of three probes was determined empirically via spike in experiments and proved to produce a low false positive rate while maintaining high sensitivity However the number of probes to define a transcr
18. m ate E TisMap_Brain_03_v1_WTGene1 cif ffTisMap_Kidney D1 _v1 wW 1102500 1102500 173 99 26 29 483 E TisMap_Kidney_O1_v1_WTGenel cif TisMap_Kidney_02_v1_W 1102500 1102500 215 85 24 18 117 aA TisMap_Kidney_02_v1_WTGene1 cif TisMap_Kidney_03_v1_W 1102500 1102500 174 76 25 17 686 W h eth e r th e raw d ata a TisMap_Kidney_03_v1_WTGene1 cif Expression of Unique Probes files in the analysis are on comparable expression levels 240 000 230 000 220 000 210 000 200 000 190 000 180 000 170 000 160 000 150 000 140 000 130 000 120 000 110 000 100 000 90 000 80 000 70 000 60 000 50 000 40 000 30 000 20 000 10 000 Number of Probes it 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1 000 Expression E TisMap_Brain_O1_v1_WTGene1 CEL W TisMap_Brain_02_v1_WTGene1 CEL E TisMap_Brain_O03_v1_WTGene1 CEL TisMap_Kidney_O1_v1_WTGene1 CEL I TisMap_Kidney_02_v1_WTGene1 CEL I TisMap_Kidney_03_v1_WTGene1 CEL I By right clicking the graph you have access to zooming functions 2011 Genomatix Software GmbH 45 CT Ty z tt A A y a 4 Forearm understanding gene regulation ChipInspector 9 12 The Memory Monitor Click View Toolbars ChipInspector 2 0 Memory to display a File Edit view Navigate Tools Window Help memory monitor in the F Fa Toolbars A v ChipInspector Analysis toolbar field Se eee
19. microarray experiment the raw data is normalized on the single probe level i e the values are adjusted to improve consistency and reduce bias Each value of an array is multiplied by a constant to make the mean intensity the same for each individual array Fold changes are subsequently calculated by probe wise division of the expression values from the experimental condition by the expression values from the control condition and finally a logarithmic transformation log2 is performed for each data point Because values are calculated for the single probes Chiplnspector calculates differing fold changes when compared to a probe set based analysis strategy The transcripts are deemed significant if they are detected by a user selected number of significant probes and the fold change value for a transcript is the mean value of the detected fold changes in the significant single probes not in the complete probe set This should be kept in mind when the resulting list of significant transcripts is further condensed by introducing a minimum fold change filter 2011 Genomatix Software GmbH 6 GenomatiXx ChipInspector understanding gene regulation 2 3 Statistical Evaluation The statistic algorithm in ChipInspector is a T test with a permuted artificial background It is based on and enhances the original SAM algorithm by Tusher et al 2001 Significance analysis of microarrays applied to the ionizing radiation response PNAS 98 9 pp 51
20. transcripts and promoters to achieve superior signal to noise ratios in microarray analysis It is unique in removing statistical and gene calling errors at the single probe level This annotation driven microarray analysis technology provides the basis for unmatched accuracy in significance analysis of microarray data It increases the number of significant features while simultaneously reducing false positive rates by an order of magnitude The resulting lists of significantly regulated genes from the experiment are directly usable as input for pathway mining tools such as Genomatix BiblioSphere PathwayEdition ChipInspector includes only a rudimentary tool set for quality assessment of microarrays Low level quality assessment of the hybridization and thus the success of the experiment can be performed with a number of good and cost free tools such as the Affymetrix TAS software Agilent s Feature Extraction or Illumina s BeadStudio The Genomatix approach puts its focus on the biological knowledge that is accumulated and stored in the annotation The annotation is made independently of the information and the intention at the time of design of the array Thus the data taken from the experiment is reduced to information about hybridization signals the biological implications are calculated on the basis of the most current state of the genome 1 2 Which Pages in the Manual Should I Read First Chapter 7 takes you through a step by step exam
21. your Genomatix account l credentials If you have no Server https www genomatix de account with Genomatix User Name yet please refer to page z a C 20 of this manual for chit instructions on how to Cancel Please provide your Genomatix login details activate an account Enter username and password and click Ok On the welcome page a EEEE EEN wizard is provided to guide Se 1 179 you through the creation of Peas 40 x Welcome x gt izin a project the data import Genomatix understanding gene regulation and the analysis All steps are also accessible via the program interface Chipinspector AVAILABLE MODULES GENOMATIX NEWS Mi 04 Jun 2008 i i Genomatix releases L Analyse Microarray Data RegionMiner fast extensive analysis of genomic regions If you previously deactivated the Welcome page you can re enable it in the Help menu Click the Chiplnspector logo in the Available modules column Regionhliner is Genomatix new tool for analyzing genomic regions coming from high throughput experim Mo 03 Mrz 2008 NIA uses Genomiatix software in Stem Cell Research Suggests Novel Transcription Factors for Stemness Linkage of Pluripotent Stem x In Step 1 an input form is displayed which asks for 1 Name and Location a project name Project Name MyTestProject Project Location fand Settings hahnGenomatixP
22. 16 5121 This algorithm creates artificial background data by randomly permuting the array results Each probe has a score on the basis of its fold change relative to the standard deviation of repeated measurements for this probe Probes with scores higher than a certain threshold are deemed significant This threshold is the Delta value The permutations of the data set are then used to estimate the percentage of probes identified by chance at the identical Delta Thus a relation of significant probes to falsely discovered probes can be given for each Delta threshold This relation is the False Discovery Rate FDR a stringency indicator The FDR is a confidence value giving the user an idea of how many probes in the result group are possibly falsely selected The ChipInspector algorithm calculates these numbers independently for the sets of positive and negative data points where applicable i e in a one class assay The resulting curve is displayed in Chiplnspector It represents an indication of the experiment s success in showing the amount of probes which deviate from the signal background Figure 2 shows the observed real values in the Y axis over the expected background values in the X axis The line pictures the expected values The single data points are ordered by their individual significance score values deviating downward are displayed left of center lower than the expected values values deviating up are displayed right of
23. 264 40 1050_4 4 G_Signal 264 A 650626 58155 6 720 562 61846 3 113 9 5720398 55914 9 1472 726 61667 059 2 4 540110 55190 7 144 468 56394 7 1301 9 5 1940161 55110 1 1181 451 62731 8 892 47 6 5810685 53729 1594 197 60625 1 1255 9 5960026 53060 7 067 279 48034 7 434 51 8 1170372 52262 1 221 438 56735 3 243 0 9 2940767 52214 7 053 392 53312 2 034 51 Genoma ti X 10 6280576 51904 8 937 309 35328 6 289 51 T gt 11 7200600 51821 1 908 314 58754 8 729 591 12 1980470 51535 6 1150 564 60739 939 54 13 280682 51004 3 443 951 62050 4 139 14 2900730 50975 5 430 277 56321 5 014 7 15 4050739 50513 3 78 115 57475 2 184 0 gt Please make sure that the file Format was recognized correctly before you continue Please make sure that the File format was rec lt Back ne gt Finish Cancel Help Choose the separator which marks the columns identify the first row where data is located and finally select those columns which hold the relevant data Only one column may be designated as holding the feature ID Chiplnspector expects the identifier for the unique and singular tag sequence 2011 Genomatix Software GmbH 50 Genomatix ChipInspector understanding gene regulation The unique identifier for Agilent probe sequences is the featureNum entity which is displayed in column 2 of the Agilent scanner output If the
24. 3_31_24 CcEL fj csms1694_37_0 ceL fj csms1704_20_ gt FO Open Files of type Microarray Data File cel X Cancel 49 Genomatix ChipInspector understanding gene regulation The program will new Action E x attempt to recognize the Stes Choose Files to Import j 1 Choose Action chip type 2 Choose Files to Import Microarray Data 2 al Lexample G5M51679_13_0 CEL a 3 MictoafP Detaimport 2 ALLexample G5M51680_17_24 CEL 2 4LLexample G5M51682_17_0 CEL Y gt G enoma t j xX Chip Type 0 HG U133_Plus_2 Human vV Protocol Files Browse You may have to select the chip type manually if it is not recognizable from the data file In this n Created Files roarray Data GSM51679_13_O cif amp case please select the roarray Data G5M51680_17_24 ciF X roarray Data GSM51682_17_O cif Y chip type from the list of E gt gt supported chips Click Next lt Back Next gt Finish Cancel Help If non CEL files are imported a preview step is interposed allowing you to select the data rows and columns which are relevant for the subsequent analysis New Action x Steps Import Preview 1 Choose Action 2 Choose Files to Import Separator 3 Import Preview Tab C Space Comma Semicolon Other 4 Microarray Data Import First Data Row 14 Text Delimiter I v Preview Expression Value 264 20 1050_F 4 G_Signal
25. 941 0 77 Probe Fiters 25 1 65 0 65 2 NM_052941 1 24 Transcript Filters 25 1 64 0 66 3 NM_052941 1 3 25 1 13 0 38 4l 0 73 Close Window 25 1 23 0 44 5 AKO27499 1 02 Maximize Window 25 1 08 0 31 6 NM_001357 0 97 ji h t P b 5 t bl Undock Window Alt 5hift D 5 Ta pene n p e robes table Gane Dan 25 11 0 32 9 NM_016343 0 85 igi 25 1 72 0 58 10 NM_016343 1 17 shows the signifi cant Close AllDocuments 25 1 15 0 39 11 AKO5S915 1 38 Close Other 25 1 48 0 58 12 AKOSS915 1 55 single pro bes Bocinas 25 1 22 0 43 13 AKO55915 1 41 25 1 11 0 32 14 AK222860 0 68 detected on the chi Reset Windows 25 1 22 0 38 15 AK093946 1 01 p Treo 25 1 17 0 39 16 NM_014911 0 76 2 69723860 25 1 17 0 4 17 NM_014911 0 86 2 73022767 25 1 4 0 53 18 AK094205 0 85 O rd e red by 2 105752745 25 1 32 0 49 19 ako21497_ 1 3 2 105752935 25 1 11 0 37 20 AK021497 1 28 ch romosomal 2 105752955 251 1 23 0 43 21 AKO21497 1 3 2 127522387 25 1 13 0 38 22 AK127325 1 09 iti 2 127522606 25 1 13 0 38 23 AK127325 1 05 position 2 182060253 25 1 23 0 38 24 i 0 98 2 215917827 1 15 0 33 25 AK076091 The program allows various views on the data In section 9 each view is explained in detail Click the Window menu item to see a list of display options 8 Variant Chromatin IP workflow ChipInspector can be used for the eva
26. Co gt java version java version 1 6 6_04 JavaCIM gt SE Runtime Environment Cbhuild 1 6 6_64 b12 gt Java HotSpot IM gt 64 Bit Server UM Chuild 16 6 b19 mixed mode If Java is not yet installed on your computer or if you have a Java version older than 1 6 0 please follow the link http www java com to download and install the newest version of Java at least version 1 6 0 2011 Genomatix Software GmbH 16 Genomatix ChipInspector understanding gene regulation 6 Installation and Configuration of ChipInspector ChipInspector is a JAVA program which must be installed locally on your computer Please proceed for download and installation as follows 6 1 Download To download ChipInspector please follow the following steps Create a folder on you hard disk where you want to store the installer Switch to http Awww genomatix de products ChipInspector ChipInspector6 html Choose your operating system from the download Click on the download button next to your operating system download File size re Windows XP Vista etownono 55 MB ae YS MacOS x 10 5 25 MB J MB Clicking on the download icon will result in the following screen Opening InstallGenomatixA pplications exe The file InstallGenomatixApplications exe is of type aa application x msdos program Anwendung and Mozilla does not know how to handle this file type This file is located at http www ge
27. View shows the various alternative transcripts at the specified locus It automatically repositions the view if a transcript is selected in another view The following elements are extracted from Genomatix ElIDorado database and displayed in the graph Element Color Element Description Primary transcript Exon yy Promoter 3 UTR Spe Transcription start region based on CAGE tag evidence Up regulated probe significant non significant Down regulated probe significant non significant The elements on the forward strand are displayed above the black line the elements on the reverse strand are located beneath the black line The vertical size of the probe reflects the fold change in the experiment Tool tip texts give additional information about the elements The NCBI GenelD and the RefSeq accession number are shown for each alternative transcript the Genomatix LocusID and the gene symbol are shown at the bottom of the window The following graph explains the various functions x Locus ID 152635 Symbol COSL y EiDorado 2011 Genomatix Software GmbH 42 Genomatix ChipInspector understanding gene regulation 9 7 The Significance Curve Window For an explanation of the statistical algorithm which is Significance Curve x applied in ChipInspector please refer to page 9 in this manual The resulting significance curve is displayed for each analysis The observed ratio va
28. a w I 03 o4 05 O68 OF O8 0p Expected Ratio 2 4 File Combinations Due to the fact that the statistical evaluation of the experimental data is carried out with the data from single probes replicates are needed to create enough data points ChipInspector cannot be used with only one replicate We strongly recommend a minimum of three replicates per experimental point although it is possible to work with two replicates If only 2 samples and 2 controls are available the array parameter for file combination can be set to exhaustive pairing to create 4 combinations S1 C1 S1 C2 S2 C1 S2 C2 This is statistically tolerable and will be sufficient to run the analysis The results will still have less confidence higher False Discovery Rates than when 3 samples are used Exhaustive matching of the experiments introduces a second level of data volume enhancing This means it is using the complete set of available data points by matching the values from every condition experiment to the values from every control experiment With pair wise matching only a subset of this data is used Since the background data volume is thus increased the individual outliers from each experiment are leveled out and therefore signal is separated more sharply from background noise This gives the results greater confidence and therefore increases the number of values that are recognized as significant higher number of significant genes with lower FDR This
29. am Zuletzt Netzwerkumgebung analysis in verwendete Dokumente Genomatix RegionMiner or other annotation programs It will show the File name Janalysis1 Save oo Eigene Dateien significant Files of type BED bed v regions e Chromosome e Start position e End position 2011 Genomatix Software GmbH 58 Genomatix ChipInspector understanding gene regulation 10 9 3 Transcript List Export Export Options x If the table of transcripts Export whole table is exported and the fold Include single fold changes for every combination done in the analysis changes for every data combination are required Export data required for a pathway analysisin BiblioSphere additional columns for these data will be included in the output file Export Cancel Alternatively the transcript list can be exported in a format which is compliant with Genomatix BiblioSphere and PathwaySystem 2011 Genomatix Software GmbH 59
30. ap_Spleen_O3_v1_WTGenel cif Only the menu items with a meaningful function for the current state of the object will be activated 9 2 The Probes Window ChipInspector displays a table with the analysis results The Probes table shows the significant single probes detected on the chip ordered by chromosomal position For each single probe the following information is displayed from left to right e chromosome number e start position of the probe on the chromosome e length of the probe e strand where the probe is mapped to e statistical significance score of the probe in the experiment e significant region into which this probe is grouped e the transcripts to which this probe is assigned e the fold change log2 that this probe shows in the experiment When a table cell is selected the corresponding information is updated correspondingly in the other windows The list is filtered according to the filter settings in the Probe Filters window Clicking the transcripts tag in the table header toggles the view to display the transcripts that are associated with the probes in the list 2011 Genomatix Software GmbH 37 ChipInspector Genomatix understanding gene regulation TreatmentControlAnalysis res Editor 0 x TreatmentControlAnalysis res x gt ol Probes Transcripts Number of Probes 506 Crancsana Poston at Fold Change 092
31. array is Coverage detected by one or more single probes This filter is used to select the number of significant single probes that detect a transcript before this transcript is deemed significant The default of 3 should be set to 1 for non Affymetrix arrays Minimum 3 Fold Change Upper Cutoff 06 Lower Cutoff lo This filter is also used to restrict the number of transcripts in the result list by a minimum fold change The parameter refers to the average fold Defaut change of all significant probes which detect this transcript 9 11 The Unique Probe Statistics Window By selecting one or more imported data files in the Projects Window and right clicking you can select the action Show Unique Probe Statistics and view a low level assessment of the raw data iix Fie Edit View Navigate Tools Window Help Ld Ga or amp L FH Projects a x f Unique Probes Statistics x lt i gt zo ExampleProject Number of Rows 6 s P k TrestmentControlAnalysis File Name Total Unique Probes Legible Unique Probes Expression Average Expression Minimum Expression Maximum T h e d S p aye d Va u e S S Microarray Data TisMap_Brain_O1_v1_WTG 1102500 1102500 366 15 27 26 797 EA TisMap_Brain_01_v1_WTGenet cif fffismap_Brain_02 v1 WTG 1102500 1102500 317 85 28 17 958 i E TisMap_Brain_02_v1_WTGenet cif Tismap_Brain_03_v1_WTG 1102500 1102500 293 02 28 19 204 g ive a n esti
32. culate the portion of RAM for the Java Machine If you have a x64 operating system and a suitable x64 Java installed then the maximum memory manageable by the JVM lies between 3000 and 3400 Megabyte If this is not the case the limit is between 1500 and 1700 Megabyte Leave at least 200 Megabyte for internal computer processes Step 4 Edit the file which starts ChipInspector accordingly Under Windows right click the ChipInspector Ux icon and choose Properties In the General Shortcut Compatibility Security Shortcut tab edit the last number in the G Chipinspector 2 0 Target box accordingly For example if you 7 decide that 1300 Megabyte of RAM can be Targettype Application allocated to the JVM the end of the line Taga ben tet should read Target spector 2_O bin chipinspector exe J mx1 300m chipinspector exe J Xmx1300m Start in a Shortcut key None Run Minimized isd Comment Genomatis Microarray Analysis Find Target Change Icon Advanced Cancel Apply Step 5 When the RAM portion size is not compatible with your computer setup the JVM and thus ChipInspector will not start Redo step 3 until ChiplInspector is functional again 2011 Genomatix Software GmbH 29 GenomatiX ChipInspector understanding gene regulation 7 Step by step Performing a ChipInspector Analysis Genomatix Authentication Request x When the program starts a login screen asks for
33. derstanding gene regulation that she requires for her results The user chooses the statistical significance of the calculated ratios from the statistics curve All the selected probes and thus the resulting projected transcripts are then significant with this FDR With increasing A the curve diverges away from the linear gradient Experience shows that there is often a point where a sudden distinct decrease in the number of significant features happens and this is usually a good point to set the cutoff It cannot be stressed too often that microarray analysis needs to be complemented by a biological evaluation of the results icance Curve X Jbserved Ratio 0 50 0 25 0 00 0 25 0 50 0 75 Expected Ratio Figure 2 Graphic output of the Chiplnspector statistical calculation for a Treatment Control analysis The plot shows the observed actual relative fold change ratios in the Y axis over the expected background ratios in the X axis The features with deviating values i e gt A are regulated by the experimental condition 2011 Genomatix Software GmbH 8 Genomatix ChipInspector understanding gene regulation Figure 3 Significance Curve x Graphic output of the 275 1591 Chiplnspector 2 50 f statistical calculation P T p P for a multi class 2 25 ir A he analysis 200 Pa I o 175 y z 5 or 1 50 Fhe lt i 735 9S o 1 25 o p aii T 2 ay I gt 1 00 i i
34. e Password to change your password Datei Bearbeiten Ansicht Chronik Lesezeichen Extras Hilfe lt z E th http www genomatix de eg binjpasswordipassword pi7smd2ezBGede MG Gh i A HE Getting Started Latest Headlines Genomati neater nse suena Da GenomatixPortal Change password for your Genomatix online account Your username is bmustermann Please enter your old password Pee Please enter your new password AAAAAAAAOK Please re type your new password Change Password L BesetForm Please note e password length must be at least 6 characters password must contain at least one non alpahabetic or capital character e password must not contain blanks or tabs For comments questions or bug reports please contact support qenomatix de Genomatix Software GmbH 1998 2007 AH rights reserved License Agreement Fertig 2011 Genomatix Software GmbH 23 Genomatix ChipInspector understanding gene regulation 6 2 3 Password Policy Genomatix password policy requires all passwords to be at least 6 characters long and must contain at least one non alphabetic or capital character No blanks or tabs are allowed 6 3 Installation Switch to the folder on your hard disk where the installer was saved Execute the installer see below and follow the instructions install Windows XP Vista doubleclick on the Genomatix install icon in the folder x MacOS x 10 5 co
35. e single probe signals is forfeited therefore decreasing the chance of combining signals which are independent of each other Consider Figure 1 to view the difference between the single probe and the probe set approach The above comments hold especially true for the new generation of tiling arrays and the subsets of exon tiling and promoter tiling arrays Here the single probe approach disregards the previously known annotation altogether thus allowing transcript specificity and the discovery of new splice variants when exons are assembled in a previously unknown combination A caveat at this point The tiling array probes are mapped strand specifically A tiling array approach using single strand RNA would lead to a loss of detection of transcripts The basis for Chiplnspector is the Genomatix proprietary probe to transcript assignment based on the mapping of all probes of a microarray against the most current version of the genome of interest The genomic data is available in the Genomatix gene annotation browser ElDorado Only probes which map perfectly and uniquely in transcripts of the genome of interest are used for the analysis This information is saved in mapping files which are an integral part of the program Mapping files are updated and provided for automatic download by ChipInspector as soon as an updated version of a genome becomes available 2 2 Normalization and Fold Change Calculation Initial to the significance calculation for a
36. elect Microarray Data Files Look in D ALLexample F A E My Recent Documents Desktop io Eigene Dateien SUESeetAeeeseeeeeeneseseeeesereesesenneseresenesaeeeesesereeeenel E G5m51676_24_0 cEL fj csm51686_32_24 cEL fg csm51697_33_ E GSMS1677_13_24 CEL fag G5M51688_32_0 CEL E Gs5m51698_40_ E GSMS1679_13_0 cEL fj csm51689_33_24 cEL fj Gsm51700_40_ EJ GSMS1680_17_24 CEL fag G5m51691_33_0 cEL E Gsms51701_43_ E GSMS1682_17_0 cEL E G5m51692_37_24 CEL fg G5m51703_43_ EJ GSM51683_31_24 CEL fg csm51694_37_0 CcEL fj Gsms1704_20_ gt Open Files of type Microarray Data File cel X Cancel File name 2011 Genomatix Software GmbH 31 Genomatix understanding gene regulation The program will attempt to recognize the chip type You may have to select the chip type manually if it is not recognizable from the data file In this case please select the chip type from the list of Supported chips Click Next The data files are imported into the project Click Finish The wizard now presents the New Analysis workflow In the first step you are asked to choose the appropriate statistical assay form see p 9 for explanations of the various Statistical assays possible Click Next 2011 Genomatix Software GmbH New Action Steps 1 Choose Action 2 Choose Files to Import 3 Microarray Data Import Genomatix New Action
37. er WiINdOW ssssssssssssseresssseserersssssrrrnsssssressssnrernnssssrerensssserreeesse 40 2011 Genomatix Software GmbH 2 GenomatiXx ChipInspector understanding gene regulation 9 5 THe Gene Information WINDOW occcciccceccdieceidsndoieacsnoinassnlbuaatsnsconadansdiaduaed eadeand cedenedicccenes 41 9 6 The Alternative Transcripts WiINdOW ccccccccssccccccesssssseeececsssseeeeeessessseeeeeeessssteeeeeeess 42 9 7 The Significance Curve WiINdOW ccccccesceccessstecceesseeccesseecceseeceseeeeeessenaeeeesssaaeeess 43 9 8 The Properties VWVINKOW vivcccisiscscavsinadsavotaxcansondenuscannsnantnusennotensinsheadesee od turdadreetaiaedins 43 9 9 The Probe Filters WINdOW vccascacosscsscacsadsecteds svctespnterdes aseetsenetsohsnashistansievcteniiciunonsiciseensiss 44 9 10 The Transcript Filters WiINdKOW cccccccccccccsssssseeeceeesessseeeeeeeessseeeeeesseessaeeeeeseseeaeeeeeees 45 9 11 The Unique Probe Statistics WINKOW cccccscccccecssssseeeeeesssseeececesessseeeeeesssseaeeeeeees 45 O12 The Memory MONIO tei setecctesetaccdesce sen lesctesceunnusnndunniencsunnsunidcanmmnciseailatddeuiteiaalercaaelaad lance 46 10 FPN IS CUS and ANIV EG eaten ssn sscsa rer saienosiancias TRE EEA ES EE EEEE 47 10 1 Creating a New Project cccccsssssccssssscccssssseccessseeccessensessessusecsseusecesseuseceesaseseeseaneeees 47 10 2 Opening an Existing Project seeeeese
38. et please click on Register EE Genomatix Login Page Mozilla M File Edit View Go Bookmarks Tools Window Help 7 2 x ki Ea 5 i e x gt a es amp http portall 0 genomatix de cgi bin sessions login pl s 789019cS_ s v 2 Back Reload A Print botadi v ii Fh Home W Bookmarks L mozilla org mozillaZine L mozdev org Genomatix GenomatixPortal Switch to encrypted login page Please log in Username Password Register 1 for a free of charge evaluation account if you haven t done so yet Lost your password Click here to retrieve your account info if you lost your username and or password For comments questions or bug reports please contact support genomatix de Genomatix Software GmbH 1998 2006 Al rights reserved License Agreement Fill in the form please enter your e mail correctly the GenomatixPortal incuces ElDorado Gene2Promoter a portal to explore various genomes BiblioSphere literature network mining GEMS Launcher Genomatix well known genome exploring and modeling software package MatInspector the de facto standard for transcription factor binding site search MatBase the transcription factor knowledge base ChipInspector microarray analysis based on Genomatix proprietary chip design optimization RegionMiner analysis of genomic regions from ChIP experiments LitInspector literature and pathway mining For evaluation accounts some limitations
39. finish the wizardnow a Click Next lt Back __Next gt _ Finish Cancel Help The time required for an x analysis depends on the Steps Run Analysis size of the microarray the 1 Choose Action Please wait while the analysis is performed This may take several 2 Name and Location minutes size of the data set and the 3 analysis Design hardware 4 Combinations and Store values Normalization Type BEES 30 f 5 Run Analysis The program gives an Performing analysis MyFirstAnalysis 1 1 overview of the progress ei 8 G e n oO m a t j X Analysis in progress Stop io t When the analysis is finished click Finish lt Back Next gt Finish Cancel Help 2011 Genomatix Software GmbH 34 Christian Zinser 15 7 08 16 47 Kommentar Anderer Screenshot Normalization type kann nicht mehr gew hlte werden Genomatix ChipInspector understanding gene regulation iD File Edit view Navigate Tools Window Help a a oa ee H Projects Ctrl 1 C h i p n S pe cto r Genome Browser Projects Q Gene Information lt gt tof d IS p ays a ta b le with Spee iy lestirclest Transcript View Number of Probes 237 rs MyFirstAnalysis oar th yal it ey e e ana ysis results H E Microarray Data G Properties 25 1 11 0 32 O NM_001037 0 73 a i 25 1 14 0 33 1 NM_052
40. g gene regulation 9 9 The Probe Filters Window Each single probe in the analysis has a x statistical score and a defined position in Probe Filters x the genome The list of probes can thus be restricted either by the significance score or by their proximity to other significant probes Significant Features fest 2656 FDR 0 2 Please refer to p 9 in this manual for an Median False Positives 4 51 explanation of the Delta value and False Discovery Rate The sliders in theProbe Filters window are used to vary this parameter in the analysis The Significant Feature number is an estimation at this Negative Delta 0 0 l 0 61 point and the numerical value may deviate in the resulting probe list Significant Features est 968 especially at low Delta settings FDR 0 3 Median False Positives 2 In general a more stringent statistic score Region a higher Delta results in less significant Size 3004 features with a higher confidence lower Minimum Probes 34 FDR Default To define regions of significant probes a sliding window approach is utilized the parameters here are the window size in basepairs and the number of required significant probes within this region 2011 Genomatix Software GmbH 44 Genomatix ChipInspector understanding gene regulation 9 10 The Transcript Filters Window Transcript Filters x Each transcript which is included in the micro
41. he list of accepted chip types 2 The files should be stored locally or on a mounted drive Please be aware that depending on the file format and your network protocol remote storage could cause increased time demand 3 File extension ChipInspector analyzes files with the cel or CEL extensions in case of Affymetrix microarrays For other chip providers tab delimited files are expected and a data import interface is displayed We recommend a minimum of three replicates per experimental point It is possible to work with two replicates but the statistical evaluation should be considered with caution It is not possible to have less than two replicates per experimental point because this makes statistics non utilizable 5 Technical Requirements 5 1 Operating Systems The application has been tested on the following operating systems Windows Windows XP Windows Vista Macintosh At least MacOS X 10 4 Linux Unix systems SuSE Linux 8 0 or above or equivalent version of other distributors Minimum system requirements e 5 GB hard disc space e 1 GB RAM recommended 2GB e 1 GHZ processor speed If you do not have any of these operating systems or if you are not sure about your operating system please contact the Genomatix customer support support genomatix de 2011 Genomatix Software GmbH 14 Genomatix ChipInspector understanding gene regulation 5 2 Java Runtime Environment In order to run the ChipInspector
42. ificant evidence for both up and down regulation of a transcript two entries are listed for it displaying the according positive and negative fold changes The list can be sorted by any column by clicking on its header When a table cell is selected the corresponding information is updated correspondingly in the other windows 2011 Genomatix Software GmbH 38 Genomatix ChipInspector understanding gene regulation The list is filtered according to the filter settings in the Transcript Filters window Clicking the transcripts tag in the table header toggles the view to display the transcripts that are associated with the probes in the list t TreatmentControlAnalysis res Editor Oj x e TreatmentCortrolAnalysis res x e 4 xl o Probes Number of Transcripts 108 Gene ID Gene Symbol Accession Number 7 es mas laos O O oa sare lczas Jaos OO OOOO e e E E 1 nos sm woi O D SC SzST me pma pos OO O s 2598 a n mo n a 51 ss aca aga 4 2 Ce S e e T TE C a es aiam weee OO O a ass me wesss O O O on 23 Woo eos o o a aii me Wen S as ama eoa O ass kon Waos O a e9 ica aeos O O oa s e aa OO OO e a8 kesa Waas Oo O a ms ome aoso A519 SICA AK312157 ry 44 io Clicking the button ElDorado at the bottom of this window opens a browser with a view of the selected transcript in Genomatix annotation browser ElDorado coma var ie Vu d
43. ipt can be adapted by the user The restriction of the number of transcripts marked as significant by requiring a minimum number of significant probes to match this transcript is a form of biological quality checking More than one transcript can be annotated at a locus therefore many if not most probes are mapped to multiple transcripts 2011 Genomatix Software GmbH 10 Genomatix ChipInspector understanding gene regulation 3 Supported Microarray Platforms 3 1 Affymetrix Microarrays Array formats from Affymetrix Affymetrix Inc Santa Clara CA USA are recognized automatically when the corresponding raw data files CEL files are imported into a ChipInspector project 3 2 Agilent Microarrays The unique identifier for Agilent Agilent Technologies Inc Santa Clara CA USA probe sequences is the featureNum entity which is displayed in column 2 of the Agilent scanner output If the output comes from a GenePix scanner the corresponding unique identifier is the RefNumber in column 57 3 3 Illumina Microarrays The unique identifier for Illumina Illumina Inc San Diego CA USA probe sequences is the ArrayAddressID which is displayed in the Bead Studio output For array formats from Agilent and Illumina automatic recognition is not implemented During file import you are asked to identify the chip type and the column of your tab delimited raw data file where the unique probe identifier is ma
44. luation of Chromatin IP experiments In this case the workflow is amended Chromatin IP experiments are targeted towards sharply defined regions of the genome where the immunoreaction produces a signal It is therefore not advisable to use the transcript list view to see annotated genomic regions defined by single probes Instead use the Probe Filters section 8 9 to define the size of the significant regions in basepairs and the minimum number of probes that this region should contain Export the resulting significant regions as x Probe Piters a bed file File Export Use this bed Probe Fitters file as input in Genomatix RegionMiner a program found on our webserver RegionMiner provides information on Region Transcription Factor binding sites Size 30 independent of annotation and strand Minimum Probes 3 2 specificity and is thus optimally suited for L Chromatin IP evaluation Default 2011 Genomatix Software GmbH 35 Genomatix ChipInspector understanding gene regulation 9 The ChipInspector Workspace Area The Menu item Window shows a list of different data view options which can be turned on and off and are explained in detail in this section Each view presents various aspects of the data Projects projects and analyses which jls Window Help are currently open TE Projects Ctrl 1 Genome Browser probes graphically in the Genome Browser genomic environment Q Gere I
45. lues are rire z plotted against the expected ratio 3 values z 05 28054 The color intensity of the curve j ia Ba 3 mirrors the number of features 20 with the corresponding 25 Observed Expected coordinates 3 0 scaled in the bar on the right E Most of the features will be 0 75 0 50 0 25 0 00 0 25 0 50 0 75 grouped around the center of the Expected Ratio graph near Zero By right clicking the graph you have access to zooming functions 9 8 The Properties Window A right click on any object in the program opens ChiP on chipAnalysis Properties a Properties window Here the parameters Properties Pgs Name ChiP on chipAnalysis description texts and file locations for the ee MERIDAN selected object are displayed core Mane Ae Nase Grackanex First 9 Analysis Design In the case of an analysis this includes e g the Analysis Type chip type the file combination and the ElDorado ee database version on which this analysis is Pararots based Combination Type ElDorado Version ChIP on chipAnalys is This analysis examines 3 replicates of E2F4 Ab pulldown against 3 replicates of Input DNA The files are combined exhaustively The experiment is based on datafrom the NCBI s Gene Omnibus Data Set GSE7516 ChIP chip analysis of human DREAM complex subunits in GO arrested and S phase synchronized cells Homo sapiens 2011 Genomatix Software GmbH 43 Genomatix ChipInspector understandin
46. me AND password are case sensitive You will find the GenomatixSuite at http portall O genomatix de cgi bin eldorado main pl You can change your password using the change password link at the top of the program page or on your Personal profile page You can also subscribe to unlimited MatInspector access on your Personal profile page If you should run into trouble logging onto our server please first read http portall O genomatix de online help help techfaq html After that do not hesitate to contact us in case of any problems or questions Best regards Your Genomatix Support tearm PE RRR ne For comments or suggestions please contact Genomatix Software at support genomatix de DE DE DE DE DE DE DE DE DE DE DE DE DE DE DE DE DE DE DE ne ae a a DE DE DE DE DE DE DE DE DE ae ae a ai a ei ee i ei Oi DE DE ee ei en on ai a ee i i i ei er en ena The login and password is not only valid for Chiplnspector but for all Genomatix products 2011 Genomatix Software GmbH 21 Genomatix ChipInspector understanding gene regulation 6 2 2 Change Password Open your internet Browser and switch to www genomatix de Click on Login on the webpage see above Enter your login and password which was sent to you via e mail E Genomatix Login Page Mozilla File Edit wiew Go Bookmarks Tools Window Help so ee FA KI TIES v a wi A http portal1 0 genomatix de cgi bin sessions k is z re Reload Print
47. n Steps Analysis Design 1 Choose Action Presence Absence is 2 Name and Location Microarray Data Sample oe a ee gt Eire CENENE eae E g only nog Momalzaton Type E TisMap_Kidney_03_v1_WTGene1 cf EE ro ron osv were l isplayed which accepts all the files for the current analysis Genomatix Click Next ee lt Back _net gt Finish Cancel Help 2011 Genomatix Software Gmb Genomatix ChipInspector understanding gene regulation Choose the file xi combinations fo r the Steps Combinations and Normalization Type icti j 1 Choose Action Statistical analysis see p 9 5 Meese Amon o cman for an expla nation of the ss ede a Created Combinations i GsM51674_24_ 24 GSM51676_24_0 j C GSM51677_13_24 G5M51676_24_0 possible variants Normalization Type GSMS1680_17_24 G5M51676_24_0 5 Run Analysis GSM51674_24 24 G5M51679_13_0 GSM51677_13_2 4 GSM51679_13_0 j i G5M51680_17_24 G5M51679_13_0 The program will display Genomatix G5M51674_24_24 G5M51682_17_0 the combination of the files aii Sa accordingly when the o Som bi n ati an type Is nr Normalization Total Intensity Normalization bd selected A In the next step the analysis will be started IF you want to run the analysis later a N for instance with a batch job you can finish the wizard now Click Next lt Back ne gt Finish Cancel Help If you select Finish
48. n s Proxy Settings No Proxy system administrator Use System Proxy Settings C Manual Proxy Settings HTTP Proxy Port OK Cancel Help 2011 Genomatix Software GmbH 26 Genomatix ChipInspector understanding gene regulation 6 4 2 Configuring automatic updates Periodically Genomatix provides important ChipInspector updates The Genomatix Update Service helps you to keep your application current Pee ele Yelle tae and select Plugins File Edit wiew Navigate Tools window Help Oaa ka Options Projects 41 newproject kj ChipInspector Analysis Microarray Data In the Settings tab xl set the inte rval of Updates Available Plugins 6 Downloaded Installed 24 Settings Ht Configuration of Update Centers ANOMANG UPEAIG Active fame l i checks 3 iv Ai Genomatix Update Center Genomatix Update Center Edt Remove Last Check 7 11 08 2 22 PM URL http release genomatix de updates chipinspector updates xml If you select Never then the application will only update itself add if O u m a n u a st a rt Automatically Check For Updates y y Check Interval Every Week v Proxy Settings this process Advanced J Force install irpstbeulbimmudeacus Close Help 2011 Genomatix Software GmbH 27 Genomatix understanding gene regulation When an updated ChipInspector 2 0 File Edit View Navigate Tools
49. nformation Transcript View Gene Information known annotation of a selected gene Significance Curve Properties Transcript View alternative transcripts for Probe Fiters the selected locus Transcript Filters Significance result curve of the statistical Close Window Curve calculation for the Maximize Window experiment Undock Window Alt Shift D Properties properties of the selected Docur project or analysis Close All Documents Probe Filters parameters used for selectina subsets of the Documents Transcript Filters Reset Windows Size and screen position of all windows are manageable 2011 Genomatix Software GmbH 36 Genomatix ChipInspector understanding gene regulation 9 1 The Projects Window Projects 4 x Se ely FirstProject k Brain_ s_Heart Brain_vs_Heart res j Spleen_vs_Brain The project management panel shows the currently open projects and analyses in a tree structure Right clicking on an item in the tree opens a context menu for performing actions on the respective object such as data import or starting a new analysis Spleen_vs_Brain res Var Microarray Data EA TisMap_Brain_01_v1_WTGene1 cif EA TisMap_Brain_02_v1_WTGene1 cif EA TisMap_Brain_03_v1_WTGene1 cif EA TisMap_Heart_01_v1_WTGene1 cif E TisMap_Heart_02_v1_wTGene1 cif EA TisMap_Heart_03_v1_WTGene1 cif EA TisMap_Spleen_O1_v1_WTGenel1 cif EA TisMap_Spleen_O2_ 1_WTGenel1 cif EA TisM
50. nomatix Software GmbH 12 Genomatix ChipInspector understanding gene regulation Affymetrix expression arrays Number of Perfect match Transcripts Species cont d columns probes Genomatix rows Genomatix annotated optimized Rat Expression Set 230 A 144141 18676 Rat Genome 230 2 0 284875 26353 R norvegicus Other Array providers Number of Perfect match Transcripts Species identifiers probes Genomatix Genomatix annotated optimized Human Genome 12391 41675 29678 106485 G4112A Human Genome 14850 43376 1379 82289 H sapene G4112F a Le SurePrint G3 Human 8x60k 62972 Po Human Promoter 474411 473123 47281 014706 014707 G4489A more a S Human RefSeq 8 Version 22185 21448 78853 2 0 Human RefSeq 8 Version 24526 23750 81826 3 0 Version 2 0 Version 3 0 Human HT 12 0 Version 3 48803 45775 135472 Mouse RefSeq 8 Version 25697 24972 90332 2 0 Mouse Whole Genome 6 45281 43331 116468 MAUSE S Version 2 0 Rat RefSeq 12 22523 20968 39569 2011 Genomatix Software GmbH 13 GenomatiXx ChipInspector understanding gene regulation 4 File Requirements ChipInspector has a number of requirements for the data files The files as they are produced in the experiment usually meet all of them but if the files cannot be analyzed it might be advisable to check the following list 1 The chip type given in the data file must be compliant with the currently 69 chips supported cf t
51. nomatix de What should Mozilla do with this File O Open it with choose Save it to disk C Always perform this action when handling files of this type Choose the option save to disk and click ok 2011 Genomatix Software GmbH 17 ChipInspector A window will show up where you can choose a folder to save the file Choose the folder where you would like to save the installer and press ok Mac users will find a folder named GenomatixApplications on their desktop or in their designated download folder It contains an installer package a ReadMe and the license file Double clicking the GenomatixApplications installer package will start the installation of the software EE a EL ns _ GenomatixApplications C7 GenomatixApplications 2011 Genomatix Software GmbH 18 ChipInspector Genomatix understanding gene regulation 6 2 Get Login and Password To apply the ChipInspector application you require a Genomatix user account with login and password Registration for a two week trial account is free of charge An e mail with your personal username and password will be sent to you immediately 6 2 1 Registration Open your internet browser and navigate to www genomatix de Click on Login in the left frame of the webpage Genomatix understanding gene regulation Home Sitemap Contact DOTT Software Training Software To help you get the m
52. ost from our products Genomatix offe rious training options n ep tutorials and online rom your Collaborative Research Training algorithms Customers About us Downloads News amp Events Careers ulation regulatory path microarra s Learn more about our recent collaborations fram our project list or contact us at research genomatix de Genomatix and Molecular Connections Ltd Training Resources Genomatix releases new versions of GEMS account Buy a license online Search our site Cj Launcher ElDorado Gene2Promoter and MatBase NEW Genomatix has released EiDorado Gene2Promoter 4 4 GEMS Launcher 4 5 and MatBase 6 3 The new features include a new Java Graphics component new and updated genomes CAGE tag annotations user defined data display in ElDorado and many more Read more in the product news form strategic partnership Read more in our press release Genomatix wins BioChance grant for 1 6 Mio research project into RNAi mediated regulatory networks Read more in our press release 2011 Genomatix Software GmbH 19 e read manuals or tutorials e book a training seminar courses on promoter and microarray analysis To learn more about their contents have a look at the overview page or contact us at seminars genomatix de ChipInspector understanding gene regulation If you do not have an account y
53. our former logins Our commitment is to offer the best quality and most accurate data based on state of the art science The new GenomatixPortal is the result of more than a year of intensive research and development Improvements and scientific progress have consequences for ElDorado genome annotation Homology groups Affymetrix probe set annotation transcription factor matrix database more than 200 matrices of our matrix library have been improved promoter module library For more information see the Release Notes For consistency and a smooth transition for the next few months you will be able to choose between GenomatixPortal Genomatix Suite Current Release December 05 Previous Release April 05 Account upgrades If you have purchased an account upgrade in our online shop and received a voucher code or if you received a voucher during a conference you can enter the voucher code here including the dashes Click Continue and a confirmation page will open stating that your account has been upgraded Users will not receive a voucher code if their account has already been upgraded during the online shop purchase or if the Genomatix staff did the upgrade manually For comments questions or bug reports please contact support qenomatix de Genomatix Software GmbH 1998 2006 AH rights reserved License Agreement te GL a Es COCCCCCCCCC a Fill in the form and click on Chang
54. output comes from a GenePix scanner the corresponding unique identifier is the RefNumber in column 57 The unique identifier for Illumina probe sequences is the ArrayAddressID which is displayed in the Bead Studio output This not the Illumina ProbelD starting with ILMN_ but rather the strictly numeric ArrayAddressID The number of columns that can hold expression data from the experiment is not restricted The data files are x i m po rted i n to th e Steps Microarray Data Import program 1 Choose Action Please wait while the data is imported to your project data folder This may take a 2 Choose Files to Import few minutes 3 Microarray Data Import Importing GSM51676_24_0 CEL to GSM51676_24 O cif a 17 G e n O ma ti AN Importing microarray data Files Import in progress Stop Click Finish lt Back Next gt Finish Cancel Help 2011 Genomatix Software GmbH 51 Genomatix ChipInspector understanding gene regulation 10 6 Editing a Project Right click on the project node in the Projects Window to perform a number of tasks on the project Projects 41 x G ExampleProject New b Treatment Control Pairing eat or FF Cel File Import Set as Main Project Clase a Other Action Rename Move Copy Delete Properties e New o Importing additional data files for analysis o Starting a new analysis on imported data file
55. ple of an expression array analysis If you wish to go to greater depths in understanding the inner workings of the program read Chapter 2 of this manual for theoretical considerations about the statistics and the scientific rationale behind ChipInspector Chapter 6 gives information about the installation and configuration of the program and points to other resources for additional technical information The FAQ item in the programs Help menu takes you to our website where some of the most commonly appearing issues are posted This is continuously updated It will also refer you to a download area where you can obtain a pre calculated and configured example project 2011 Genomatix Software GmbH 4 Genomatix ChipInspector understanding gene regulation 2 Theoretical Background 2 1 Rationale of the Single Probe Approach The definition of probe sets greatly eases the handling of the large data amounts that are produced by microarray experiments Considerable thought has gone into the design of the probes regarding comparable hybridization temperatures GC content and genomic position to create sets of single probes for one transcript Gene expression levels are calculated as cumulated signals from several probe expression values The algorithms used for calculating this cumulated signal assume that all probes in a set represent only one specific target sequence i e one transcript In many cases this sequence information i
56. port for other platforms Click Next Use the upper Browse button to navigate to the folder where the data files are located Choose the appropriate data files in the File Chooser dialog and click Open gt New Action Steps 1 Choose Action H ChipInspector Onew action x Steps Choose Action 1 Choose Action Project MyTestprojet o YS 2 Categories Actions IS EJ Cel File Import EA Tabular File Import Genomatix cs Description CEL files From Affymetrix microarray experiments are imported converted and associated with the project lt Back ne gt Finish Cancel Help 2 Choose Files to Import 3 Microarray Data Import Choose Files to Import O sess Microarray Data Browse P Genomatix unknown Click Protocol Files k Browse al Created Files Zi Please select some data files lt Back Next gt Finish Cancel Help t Select Microarray Data Files x Look in lo ALLexample A Fe ii My Recent Documents Eigene Dateien 2011 Genomatix Software GmbH File name eg GaMs1676 24 0 CEL a G5MS1686_32_24 CEL eg GSM51697_38_ E Gsm51677_13_24 CEL fg csms1683_32_0 ceL fj csms1693_40_ E GSMS1679_13_0 cEL fj csm51689_33_24 cEL fg Gsms51700_40_ E Gsm51680_17_24 CEL fg c5m51691_33_0 ceL fj csms51701_43_ E Gsm51682_17_0 cEL E c5m51692_37_24 cEL fj Gsm51703_43_ fag Gsm5168
57. py the ChipInspector application to your Applications folder Mac Linux Unix type java jar InstallGenomatixApplications jar Please note that the Genomatix licensing model for Chiplnspector is a single user floating license This means that you may install the program on any number of machines however not run several instances of the program at the same time If a second instance of ChipInspector is started while another instance is running the user is given the choice of ending the concurring session This can lead to data loss on the first instance if the analysis results have not been stored yet The above said is true also for parallel instances of ChipInspector 2 and older versions The programs may be run alternately but not at the same time 2011 Genomatix Software GmbH 24 Genomatix ChipInspector understanding gene regulation If you run a windows system the following screen will pop up Genomatix ChipInspector 2 0 Setup Welcome to the Genomatix ChipInspector 2 0 Setup Wizard This wizard will guide you through the installation of Genomatix ChipInspector 2 0 It is recommended that you close all other applications before starting Setup This will make it possible to update relevant system files without having to reboot your computer Click Next to continue cme Click Next gt and follow the instructions After Chiplnspector is installed successfully you can start
58. rked In addition these array formats do not use the probe set approach The coverage should therefore be set to 1 cf chapter 9 10 Genomatix calculates a proprietary annotation for the database ElDorado ChipInspector data is based on this For the currently supported chips more than 85 of the perfect match probes are used to calculate the statistics The following tables show the data for each chip 2011 Genomatix Software GmbH 11 GenomatiXx ChipInspector understanding gene regulation 3 4 Platforms and Probe Numbers Affymetrix exon arrays Number of Perfect match Species columns rows probes Genomatix optimized H sapiens M musculus Affymetrix tiling arrays Number of Perfect match Species columns rows probes Genomatix optimized 2888550 2726143 Affymetrix expression arrays Number of Perfect match Transcripts Species columns probes Genomatix rows Genomatix annotated optimized 16861 20501 C 39164 15996 Arabidopsis ATH1 Genome 220039 29840 A thaliana i B Human Genome U133Plus2 0 1164 525438 61158 Human Genome U133A 207689 39876 Human Genome U133A 2 0 207689 39876 H sapiens elegans 22693 24755 15267 143190 12329 10303 64195 62161 34676 89895 M musculus Mouse Genome 430A 2 0 207750 62161 Murine Genome U74v2 A 141087 37949 Chimpanzee on HG 1164 398160 16364 P toclodvies U133Plus2 0 abated Poplar Genome 1162 355285 27395 P trichocarpa 2011 Ge
59. rojects Browse Project Folder ahn GenomatixProjects MyTestProject Steps Name and Location Genomatix Enter a name for the new project and click Finish ext gt __ Finish Cancel Help 2011 Genomatix Software GmbH 30 Genomatix ChipInspector understanding gene regulation In the next step the raw etry x data files are imported The wizard opens this dialog automatically Depending on the type of data choose CEL File Import for Affymetrix or Tabular File Import for other platforms Click Next Use the upper Browse button to navigate to the folder where the data files are located Choose the appropriate data files in the FileChooser dialog and click Open Steps Choose Action 1 Choose Action Project MyTestprojet o YS 2 Categories Actions A EJ Cel File Import EJ Tabular File Import Genomatix Description CEL files from Affymetrix microarray experiments are imported converted and associated with the project Back _ ne gt Finish Cancel Help t New Action x Steps Choose Files to Import 1 Choose Action 2 Choose Files to Import Microarray Data Browse 3 Microarray Data Import Genomatix TR facom bs Cli ck Protocol Files Browse A Created Files Sea z Please select some data files lt Back Next gt Finish Cancel Help t S
60. s e Renaming e Deleting e Copying to another directory i e for sharing the result with collaborators e Closing e Moving to another location on your computer e Set As Main Project this project will be suggested as the default project when a new analysis is started e Editing the project Properties i e adding a description text 2011 Genomatix Software GmbH 52 Genomatix ChipInspector understanding gene regulation 10 7 Creating a New Analysis To create a new x analysis right click Steps Choose Action on a project node E eee Project exampleProect o and select New Categories Actions Other Action n breyr s Treatment Control Pairing B Time Course Titration Experiment a Presence Absence Calling LS Analysis Batch Job Description i files from Affymetrix microarray experiments are imported converted and associated with the project e cancel Heo Choose an analysis type from the selection please refer to page 9 for a more in depth explanation Available types are Treatment Control Pairing Use this option if you want to perform a one class analysis which compares a treated sample to a control A single sided permutation T test analysis is performed Time Course Titration Experiment Select this option if you want to compare a set of data points in a multi class analysis e g for subsequent cluster analysis of the results in other programs In this case a multi class
61. s outdated today as genomic annotations are becoming more and more complete Today it can be seen from the latest genomic annotations that most genes are flexible entities which are translated into different mRNAs alternative transcripts These variants were often not or only partially known at the time of the array design thus leading to ambiguous probe design i e probes cannot be assigned uniquely to different gene variants Therefore the probes in a probe set are often distributed among different alternative transcripts of the same gene locus As a consequence the calculated signals do not reflect the expression value of the gene properly 15235 bp LOC146443 GXT_2776032 NC_000016 15235 bp LOC146443 GXT_2781581 NC_000016 noexonin noexonin transcript2 transcript2 ee Se 6 Oe eS ee Situation 1 transcript 1 present Situation 2 transcript 2 present Detected by probe set approach Detected by probe set approach D4 Detected by Chiplnspector Detected by Chip nspector Figure 1 Assay sensitivity is increased by a single probe approach 2011 Genomatix Software GmbH 5 Genomatix ChipInspector understanding gene regulation With increasing computing power available to desktop benchtop computers the advantages of the single probe approach can be accessed The annotation is always up to date even if some of the probes which were originally included in a probe set are now annotated differently The pooling of th
62. seesesessseseeesserrsssereerssreerssttsssretsssereesssreessereeesssreee 48 10 3 Saving the Project Tie S asiriar n an eA N Eaa NENE RANS 48 10A DSE a TO E aa E A 48 10 5 Importing Data Files into a Project sccssssssecessssssctscecsesssssseesesesseesecesssssneesseeesees 48 10 6 Editing PPO CCU acc cteaieaistire sae iernii ra a ENEA ATTE 52 10 7 Creating a New Analysis ccccssscccssssecssssseccssseseccssseneccssssusecessusessesnseceesusecsessaneeses 53 10 8 Committing an Analysis for a Batch Job ssessssssssssssesessssrerrssrresssrsssereessereesssrrerssseeese 57 109 Expornimng RESURS xi soasiici Soctccscioien ienie E R 58 109 Data EXPO enera ea A E EIE OETA E EA OO AS 58 1092 TS eS TS OI oiran aa ENON ONTE E E O 58 10 9 3 Transonpt List EXPO sensorien krsni E nEn EEE EEEE E EEEE R RARA 59 2011 Genomatix Software GmbH 3 Genomatix ChipInspector understanding gene regulation 1 Introduction to ChipInspector 1 1 What is Genomatix ChipInspector ChipInspector analyzes raw data files from microarray experiments It selects those probes from the chip where the signal is significantly different from the background and puts them in their biological context ChipInspector extracts significant information from the expression level of single probes of Affymetrix GeneChip microarrays Agilent DNA microarrays and Illumina whole genome gene expression arrays The program uses the world s largest database of alternative
63. sence Calling to Analysis Batch Job Categories A ChipInspector Description Creates a batch job which processes a list of analyses that have already been created but not executed yet lt Back _ n gt Finish Cancel Help GLET Action Steps Select those analyses 1 choose action E 2 Analyses Selection which you want to run iN 3 Runanalyss one continuous process e g over night Genomatix Click Next Analyses Selection Analyses for Batch Job 2 Available Analyses Is Analysis1 Tq Analysis2 Analysis3 Select at least one analysis for batch job lt Back Finish Cancel Help 2011 Genomatix Software GmbH 57 Genomatix ChipInspector understanding gene regulation 10 9 Exporting Results You can export the results in different formats for further analysis To do so click on File Export in the menu and choose from the available export options 10 9 1 Data Export Both the transcript list and the probe list can be exported as a table for MS Excel or other spreadsheet programs This table will contain the following data e Chromosome e Position e Length e Strand e Significance Score e Region e Transcript e Fold Change log2 10 9 2 Bed file Export The probe ist TT x can be veered dar Save in Desktop pi d m E bed file for D Eigene Dateien further A O arbeitsplatz downstre
64. sociate them with the Control list Steps 1 Choose Action 2 Name and Location 3 Analysis Design 4 G Combinations and ChipInspector Name and Location Destination Folder Analysis Description tix S Rin Analysis 9 Project Created Folder tig ie I TE f EE i Steps Choose Action Name and Location Analysis Design Combinations and Normalization Type Run Analysis Pratt sae a Genomatix 3 w G Steps Choose Action Name and Location Analysis Design Combinations and Normalization Type Run Analysis dit Jas ee Genomatix 2011 Genomatix Software GmbH x MyFirstAnalysis MyTestproject hents and Settings hahn Genomatix lt Back net gt Finish Cancel Help Analysis Design Treatment A el gt gt Microarray Data EJ csms1674_24 24 EJ csms1676_24_0 Q Please add at least 2 treatments Analysis Design Treatment lt Back Next gt Microarray Data g EA G5M51674_24_24 G5M51676_24_0 E gt EJ comsi677_13_24 amp G5M51679_13_0 Please add at least 2 treatments 33 lt Back E E G5m51680_17_24 Control EJ csms1677_13_24 EJ csmsi679_13_0 E csms1680_17_24 EJ csm51682_17_0 gt Finish Cancel Help Control x Finish Cancel Help Christian Zinser 15 7 08 16 36 Kommentar Screenshot pa
65. t nicht das sind Treatment Files Christian Zinser 15 7 08 15 48 Genomatix ChipInspector understanding gene regulation Select the files that x represent the experimental Steps agu 1 Choose Action condition and associate RE Treatment Microarray Data Control j p 3 Analysis Design BB esmsi674_24_24 ci ej them with the Treatment 4 Combinations and E GsM51677_13_24 cil E G5M51680_17_24 cll m Normalization Type amp list 5 Run Analysis Genomatix lt Back _ net gt Finish Cancel Help Click Next EE x Steps Combinations and Normalization Type Choose the file 1 Choose Action i i i inati i xhaustive mes 2 com bi n atio ns fo r th e 2 Name and Location Combination Type hausti Tim So Analys Doss Created Combinations f1 G5M51674_24_24 G5M51676_24_0 statistical analysis see p 9 ss ee ie GSM51677_13_24 GSMS1676_24_0 i orna K an ype GSM51680_17_24 GSM51676_24_0 for an explanation of the RunApaysj G5MS1674_24_24 GSM51679_13_0 GSM51677_13_24 GSM51679_13_0 possible va riants i GSM51680_17_24 GSM51679_13_0 m GSM51674_24 24 GSM51682_17_0 Gen omatix GSMS51677_13_24 GSM51682_17_0 GSMS1680_17_24 GSM51682_17_0 Normalization Total Intensity Normalization v a 2 A In the next step the analysis will be started IF you want to run the analysis later for instance with a batch job you can
66. that is selected by clicking and drawing the mouse can be displayed in the genome annotation browser ElDorado by clicking the corresponding button A comprehensive overview over all the transcription factor binding sites that are located in the selected region can be obtained by clicking the Matlinspector button A browser window is opened in both cases taking you directly to the corresponding Genomatix webpage Please note that this feature depends on synchronized genome versions in ChipInspector and ElDorado and is therefore disabled when an analysis is based on an older version of the genome 9 5 The Gene Information Window Gene Information x A digest of the comprehensive annotation pacer a r aeons nen from ElDorado is presented The link behind fous Jeans SSS the GenelD leads to a complete overview of name kinesin family member 5A the information in the various databases on preten TTT iaaii a eri Gch Mad cai spastic paraplegia 10 autosomal dominant the Genomatix servers while the button Symbol KIFSA ElDorado leads directly to the More Gene Synonyms 01251869 KIFSA MYOSO NKHC SPG10 Info page of ElDorado Organism Homo sapiens human Chromosomal 1213 13 Location GeneID 3798 Unigene Cluster Hs 151219 ElDorado 2011 Genomatix Software GmbH 41 Genomatix ChipInspector understanding gene regulation 9 6 The Alternative Transcripts Window The Transcript
67. the application either from the program group Genomatix with the executable Chiplnspector by double clicking the Chiplnspector icon on your desktop 2011 Genomatix Software GmbH 25 Genomatix ChipInspector understanding gene regulation 6 4 Configuration of ChipInspector Before you start working with Chiplnspector please take a minute to configure the application concerning the proxy configuration for internet access and the application update behavior to get the latest program version automatically 6 4 1 Proxy Configuration Many companies and institutions use proxies and firewalls for secure and fast access to the Web ChipInspector will try to learn the appropriate settings from your preferred browser application InternetExplorer or Firefox but it may be necessary to configure these settings to allow the application to pass your local proxy or firewall In ChipInspector go to the Tools Chipinspector 2 0 menu and select Options File Edit view Navigate Tools Window Help Lo a ab FH a e A 1 a Projects 40 newproject Is ChipInspector Analysis Microarray Data In the General tab tell the CM x application whether to use By J Pe OS aa ne the proxy known to your General Keymap Miscellaneous computer or to use a manual setting which you will be Web Browser lt Default System Browser gt Edit able to receive from your institutio
68. ting a Data Server If data downloads are slow a different data server may be closer to your geographical location Configure the data server which you want to use by going to the Tools menu and selecting Options In the Download Options tab choose the server for data download Gz Asay General Data Download Server lo EE Keymap Download Miscellaneous Choose automatically Munich Germany Tokyo Japan C Seattle USA 2011 Genomatix Software GmbH 28 OK Cancel Help Genomatix ChipInspector understanding gene regulation 6 4 5 Configuring the Java memory settings Every Java program such as ChipInspector runs within a defined environment the Java Virtual Machine JVM When a Java program is started the computer allocates a portion of its main memory its RAM to the JVM When ChipInspector is first installed the amount of allocatable RAM is calculated However this calculation may be off or new hardware RAM may have been added subsequently It is possible to increase the portion of RAM for the JVM thus giving ChipInspector more memory Step 1 Find out how much RAM your computer has Under Windows this parameter can e g be found in the System Properties Control Panel Here you can also find out whether you have a x64 operating system Step 2 Find out which Java machine is installed See page 15 for details Step 3 Cal

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