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Agilent Genomic Workbench Lite Edition 6.5 Workflow User Guide

1. Workflow Progress Summary Console Abort Workflow 176 Close Tab Text Box Surenary Console Test R Vestn gt Workflow Progress Running lt Summary Console Abort Workflow tarting Workflow lorkflow Name K Test xperiment Nare ric Set Level Fiters NONE eeesese sereereseeserseesesessreeeesesseessesersereeeeee wecuting Feature Extraction Steps unning Feature Extraction xtraction started for US4510PP02_251729310003_S01 ISTEP Loading image and design There is no protocol attached with extraction FE is searching default protocol for extraction E automaticaly determined that required protocol is CGH_107_Sep09 id in use 017293_D_20070718 otocol in use CGH_107_Sepoo Initialization complete ocessing 4 arrays in single scan ISTEP Grid Placement rerforming BPS based GridPlacement Figure 58 Workflow progress tab The program creates a separate tab for each workflow that you run The name displayed is the name you specified as the Workflow Identifier when you started the workflow run Each workflow progress tab shows the analysis settings and a log of workflow activity Shows the status of the workflow either Running or Complete Opens the Summary Console tab where you see a list of all workflows and check the status of each See Summary Console tab on page 174 Appears only when a workflow is running and that workflow s progress tab is selected Click to a
2. Workflow User Guide Setting up Workflow Analysis Methods 3 Normalization method Description Instructions Inter array Median This kind of normalization corrects for variations between replicate arrays Agilent Genomic Workbench calculates and applies it separately for each channel It first calculates the median signal intensity over the common probes in each replicate array It then finds the average of these median intensities over all replicates of all arrays For each array it computes the ratio of its median signal intensity to the average of the median signal intensities of all arrays Finally it normalizes data by multiplying each signal intensity by the applicable ratio Inthe Analysis Method Navigator in Normalization select Inter array Median Dye bias intra array This kind of normalization corrects for dye bias within each array in an Median experiment and it normalizes the intensities of the IP channel to match the median of the WCE channel 1 Inthe Analysis Method Navigator in Normalization select Dye bias intra array Median 2 Inthe parameter panel in Normalize by select the way that the program compensates for dye bias By equalizing central tendencies of IP and WCE channels This method first calculates the ratio of the median IP signal intensity to the median WCE signal intensity Then it multiplies the signal intensities of the data probes by this ratio By normalizing central tendency of l
3. Select Imported Data ea Select Experiment Image Files Oy Extraction B Feature Extraction Report Type O Probe Based interval Based O Probe amp Interval Based Output Format complete Genome Switch Application Command Ribbon Select File Location F aGwResuts CGHI xls Browse overwrite if file exists Report Flat Intervals am Generate report per array A Probe Based Penetrance Sumr FF cyto Report FE CNR Report B SNP Genotype Report ml Aberration amp LOH Report Summary Console ole Workflow Progre3s R ature Level Filters NONI Wo rkfl ow Design Level Filters NONE Array Level Filters NONE Navigator SNP Copy Number OFF Summary Console Progress tabs LOH OFF ebkeo kk eco aaa ok Executing Step Importing GEML design file Summary Console Abort Workflow Figure 11 Workflow Main Window Figure 11 shows the Main Window when the Workflow tab is selected This window contains the elements listed in Table 10 below More detailed descriptions of each element appear later in this section 96 Workflow User Guide Workflow Reference 4 Table 10 Workflow Main Window Elements Element Purpose Tabs Used to change functional areas of Agilent Genomic Workbench The tab menu also contains the Switch Application command where you change to another licensed application CGH ChIP CH3 Command Ribbon When Workflow
4. Figure 12 Command ribbon for Workflow Workflow SE AEin Feature Preferences Run Import Export Apply password b ZS ma are o o A we B gt a A Op Extraction The commands in this ribbon are described in the following table Table 11 Commands for Workflow command ribbon Command Purpose Workflow Analysis Method Select Workflow New Delete Save Select to change the command ribbon for Workflow or Analysis Method Shows the existing workflows in the program To use an existing workflow select one from this list Opens the Create Run Workflow dialog box To create a new workflow type a name for the workflow in Enter Workflow Name then click OK Available only if a workflow is selected Opens a Confirm dialog box that asks if you want to delete the workflow To delete the current workflow from the program click Yes Available only if there are unsaved changes in the selected workflow Saves the changes in the selected workflow Note This does not save the workflow in a file To save a workflow in a file see To export a workflow on page 64 98 Workflow User Guide Workflow User Guide Workflow Reference 4 Command Purpose Save As Available only if workflow is selected Opens a Save As dialog box To save the selected workflow with a new name type the new name then click OK You must type a name that does not match an existing workflow or experiment Appl
5. Reports B CGH Aberration Report l B Probe Based Penetrance Sumr B Cyto Report E cnve Report i B SNP Genotype Report amp Aberration amp LOH Report Figure4 Workflow Navigator In DNA Analytics 4 0 workflow was the name for analysis method used in Agilent Genomic Workbench 5 0 and higher Workflow User Guide 15 1 1 16 Getting Started To change settings for CGH workflow analysis A workflow is helpful if you need to analyze multiple arrays and you know which algorithm settings you intend to use for the analysis First you set all parameters for an analysis in an analysis method When you run the workflow the program automatically runs the analysis method On 64 bit computers you can run up to three workflows simultaneously On 32 bit computers workflows are processed sequentially not simultaneously You create experiments for a workflow in one of two ways e You create and save a new experiment in interactive mode and then select the experiment for the workflow e Let the workflow create an experiment automatically to hold data from the selected input source The experiment is saved at the end of the workflow run and is available in the interactive mode You must create Cyto Report templates interactively before you can use them in an analysis method After you select the method settings and their options you set up and run the workflow After a workflow run is compl
6. Workflow User Guide Other User Guides This user guide which you are now reading supplies comprehensive help on all available Data Viewing tasks You can access it easily from anywhere within the program 1 In any tab of Agilent Genomic Workbench click the Help tab 2 On the Help Ribbon click Workflow The Workflow User Guide opens The Help tab in Agilent Genomic Workbench lets you view any of the available user guides that apply to the currently selected application type 1 Set the desired application type from the Switch Application menu 2 Inthe Agilent Genomic Workbench tab bar click Help The names of the available user guides appear in the command ribbon 3 Click the desired help guide The selected guide opens Workflow User Guide 33 1 Getting Started To contact Agilent Technical Support Technical support is available by phone and or e mail A variety of useful information is also available on the Agilent Technical Support Web site Resource To find technical support contact information Agilent Technical Support 1 Go to http chem agilent com Web site 2 Select a country or area 3 Under Quick Links select Technical Support 4 Select from the available links to display support information Contact Agilent Technical Telephone 800 227 9770 Support by telephone or E mail informatics_support agilent com e mail United States and Canada Contact Agilent Technical 1 Go to http chem a
7. New Condition Delete Condition Include Exclude matching values 130 New Update Reset Delete Rename e Operator How the filter uses the entry in Value to evaluate arrays For example the gt operator configures the filter to include or exclude arrays where the selected attribute is greater than or equal to the entry in Value e Value The value the filter uses as the basis to evaluate an attribute For example if you select the AmtCy3used ug attribute the operator and a value of 5 the filter includes or excludes an array if its AmtCy3used ug attribute is equal to 5 e Logical Operator Available only if you configure more than one condition The relationship between the condition and the next one in the list For example if you select AND in Logical Operator for the first condition the filter includes or excludes an array if it passes both the first condition and the next condition Adds a new blank condition row to the table Removes a condition from the list To remove a condition click anywhere within the condition row then click Delete Condition Select one of these options e Include matching values If an attribute passes the filter conditions the program includes the array in the analysis e Exclude matching values If an attribute passes the filter conditions the program excludes the array from the analysis Opens an Input dialog box where you can type a name for the new filter
8. Workflow Reference Agilent recommends that you use Sample Manager to set up your samples before you run a workflow After you organize your Array IDs and assign their attributes using Sample Manager you can use Workflow to automate Feature Extraction and analyze the data if you want In Workflow you select the image files to extract and then run Feature Extraction on the microarrays When you run the extraction using Workflow the following things happen e The extraction results are saved on your hard drive e The extracted arrays are available in the appropriate Data folder for workflow or interactive analysis and data display e The Array IDs in Sample Manager are updated For more information on using Sample Manager see the Sample Manager User Guide Workflow User Guide Setting Up and Running Workflows 2 B Agilent Genomic Workbench Lite Edition 6 5 CGH Home Sample Manager Workflow Preprocessing Analysis Discovery Reports View Iool Help Switch Application Y Create Edit Workflow Workflow QWorkFlow elect woridion Delete Save Save As ey Import Export ee C 9 SA T Besoa l a ae RE ae een Preferences Import FE Image Files Parameter Panel Description Import FE Files Import Feature Extraction images here and associate samples to them Import UDF Files Select Imported Data oe Select Experiment _ Image Name Global Display Name Array ID Ba ve Green Sample Red Sample Grid T
9. Workflow User Guide 155 156 Workflow Reference LOH Parameter Panel LOH Parameter Panel Description LOH Parameter Panel Description Figure 42 LOH Parameter Panel Purpose To set the threshold level for calculation of LOH Loss of Heterozygosity regions To open In the Analysis Method Navigator for CGH under SNP Algorithm select LOH Threshold Type the threshold to use for the LOH calculation For more information on this algorithm see the CGH Interactive Analysis User Guide Workflow User Guide Workflow Reference 4 Predefined peak shape detection v2 1 Pre defined Peak Shape detection v2 1 Description This peak detection algorithm slides a peak shape through the data searching for good fits Please refer to the user guide for a j detated description A The peak shape is computed from the estimated mean and standard deviation of DNA lengths of the shear distribution and the signficance of a potential fit is judged by comparing to fits on randomized data using a non parametric rank significance test For each peak that satisfies the non parametric test a score is computed by testing the quality of the fit under the assumption of ql an extrema valia distrib tinn of the mialitias nf the fits th randomized data The lt innificance derived From this hast ic conwerted to a Thresholds P value threshold maximum for non parametric test for reporting peaks Por Must be greater than I num
10. To accept the name click OK The program creates the filter and adds the new name to the Name list Saves any changes you make to the filter conditions without closing the dialog box Restores the values of the filter conditions to what they were before you made any changes to them Opens a Confirm dialog box that asks you if you want to delete the selected filter To delete the filter click Yes Opens an Input dialog box where you can type a new name for the filter To accept the name click OK Workflow User Guide Workflow Reference 4 Blank Subtraction Normalization Blank Subtraction Normalization Description Subtracts the median signal of negative control spats from all signals on the array Estimate central tendency of blank probes by median rey Figure 23 Blank Subtraction Normalization Purpose This parameter panel lets you select parameters for the Blank Subtraction normalization method for ChIP For more information see To configure normalization methods ChIP on page 84 To open This parameter panel appears when you click or select the check box beside Blank Subtraction under Normalization in the Analysis Method Navigator Estimate central Defines how the program calculates the central tendency of the negative tendency of blank control probes on an array The only option for this selection is Median probes by Workflow User Guide 131 4 Workflow Reference Centralization Parameter
11. Available only if you configure more than one condition The relationship between the condition and the next one in the list For example if you select AND in Logical Operator for the first condition the filter includes or excludes a probe if it passes both the first condition and the next condition Adds a new blank condition row to the table Removes a condition from the list To remove a specific condition click anywhere within the condition then click Delete Condition Select one of these options e Include matching values If a probe passes the filter condition the program includes it in the analysis e Exclude matching values If a probe passes the filter condition the program excludes it from the analysis Opens an Input dialog box where you can type a name for the new filter To accept the name click OK The program creates the filter and adds the new name to the Name list Saves any changes you make to the filter conditions without closing the dialog box Restores the values of the filter conditions to what they were before you made any changes to them Opens a Confirm dialog box that asks you if you want to delete the selected filter To delete the filter click Yes Opens an Input dialog box where you can type a new name for the filter To accept the name click OK Workflow User Guide Workflow Reference 4 Dye bias Intra Array Median Normalization Normalize by Workflow User Guide Dye
12. Figure 17 Central probe flanked by two neighbor probes The program accepts the probes as bound if the average p values for all three probes is less than a set cut off value and if either of the following is true Workflow User Guide Workflow Reference 4 e The p values for the central probe and at least one of its neighbors are less than set cut off values e The p value of one or optionally another number of the neighbors of the central probe is less than a set cut off value For a detailed description of the statistical calculations involved in event detection see the ChIP Interactive Analysis User Guide You can customize the settings of the model In the Analysis Method Navigator click Whitehead per array neighborhood model The parameters of the model appear in the parameter panel where you can edit them Predefined peak shape detection algorithm This peak detection algorithm slides a peak shape through the data searching for good fits The peak shape is computed from the estimated mean and standard deviation of DNA lengths of the shear distribution and the significance of a potential fit is judged by comparing it to fits on randomized data using a nonparametric rank significance test For each peak that satisfies the nonparametric test a score is computed by testing the quality of the fit under the assumption of an extreme value distribution of the qualities of the fits to randomized data
13. See To select to fuse designs CGH on page 73 To fuse designs select Fuse Design The Fuse Design Parameter Panel appears See Fuse Design Parameter Panel on page 143 Filter Before Analysis The options available in the Filter Before Analysis folder let you select filters that remove or include data based on specific criteria You can also create and edit these filters Select any of these options Option Description Design Level Filter Opens the Design Level Filter Parameter Panel Design level filters let you include or exclude probes based on criteria set in the filter For example a design filter can be used to filter out probes that fail the homology filter or have a low probe score See Design Level Filter Parameter Panel on page 137 Feature Level Filter Opens the Feature Level Filter Parameter Panel See Feature Level Filter Parameter Panel on page 141 Feature level filters let you include or exclude data from specific microarray features based on information from the imported Feature Extraction output files Array Level Filter Opens the Array Level Filter Parameter Panel See Array Level Filter Parameter Panel on page 129 Array level filters let you include or exclude arrays in the current experiment based on their attributes 111 4 112 Workflow Reference Combining Replicates For CGH arrays intra array replicates are features within the same array that contain the
14. The significance derived from this test is converted to a score by computing log10 significance for the peak fit e If a probe is not inside any of the peak objects then it is not bound Nothing special is drawn for this probe except a baseline at exactly a ratio of 1 log ratio of zero e If a probe is inside a peak then it gets the significance value and score value of the associated peak If it is inside two overlapping peaks it gets the values for the peak with the better score Peaks are drawn by computing ratios from the collection of detected peaks Output When you run a workflow the program creates a new Agilent Genomic Workbench experiment that you can display in the interactive tabs By default the program uses the name of the analysis method as the name of the experiment Workflow User Guide 121 4 Workflow Reference The program always creates an output experiment when it successfully runs a workflow To customize the name or description of the experiment click Experiment in the Analysis Method Navigator The Experiment Parameter Panel appears See Experiment Parameter Panel on page 140 122 Workflow User Guide Parameter P Aberrati Workflow Reference 4 anels As you select options for an analysis method in the Analysis Method Navigator the parameters available for each option appear in specific parameter panels If you do not need to set any parameters for a specific analysis method
15. where you can select a location and type a name for the Probe Report file See Select Report Folder on page 192 QC Report Settings QC Report Settings Description This will generate a QC report For peak detection results at the specified location IF experiment contains more than one array then reports would be created at the location in a folder with provided name To view the report open QCReport html file in a web browser Report Location fam Files Agilent DNA4 Analytics 0 68 QC_Report Browse Figure 46 QC Report Settings Purpose This parameter panel is used to select a location for QC reports generated in a ChIP workflow analysis To open This parameter panel appears when you click ChIP QC Report under Reports in the ChIP Workflow Navigator For information about the content and format of the QC report see the ChIP Interactive Analysis User Guide Report Location Shows the location of the QC report that the program creates during the workflow You can edit the location You can also click Browse to select a new location for the report The program saves the QC report as an HTML file Workflow User Guide 161 4 Workflow Reference Browse Opens the Select report folder dialog box where you can select a location and type a name for the QC report file See Select Report Folder on page 192 Run Analysis Application Panel Run Analysis Application Description Run a DNA Analysis Application Analy
16. 1 by default Click to add the images to the workflow Closes the dialog box No images are added Workflow User Guide Workflow Reference 4 Create Analysis Method Enter Analysis Method Name Apply Password OK Cancel Create Analysis Method Enter Analysis Method Name 5 Apply Password OK Cancel Figure 60 Create Analysis Method dialog box Purpose Used to give a name optionally apply a password and create an analysis method To open This dialog box appears when you click New on the workflow command ribbon Type the name for the analysis method you are creating Select this box if you want to protect the analysis method with a password When this box is selected the Set Workflow Password dialog box will appear when you click OK See Set Password on page 194 Click this button to create the new analysis method with the designated name Click to cancel the operation Workflow User Guide 179 4 Workflow Reference ate Workflow Create Workflow Create Workflow Enter Workflow Name workflow 5 Apply Password OK Figure 61 Create Workflow dialog box Purpose To name a new workflow and apply a password to the workflow To open From the Workflow command ribbon click New Enter Workflow Type the name of the new workflow here Name Apply Password Select the box to protect the workflow with a password If this check box is selected when you click OK the Set Password dialog b
17. 158 Probe Based Penetrance Summary Report Parameter Panel 159 Probe Report Settings 160 QC Report Settings 161 Contents Run Analysis Application Panel 162 Select Experiment Parameter Panel 163 Select Imported Data Parameter Panel 164 Set Output Path for Feature Extraction Panel 165 SNP Copy Number Parameter Panel 166 SNP Genotype Report Parameter Panel 167 Variance Stabilization 168 Whitehead Error Model 169 Whitehead Per Array Neighbourhood Model 170 Whitehead Per Array Neighbourhood Model Modified 172 Z Score Parameter Panel 172 Summary Console Progress Tabs 174 Summary Console tab 174 Workflow progress tabs 176 Dialog Boxes 178 Add Image Pack Information for FE Extraction 178 Create Analysis Method 179 Create Workflow 180 Export 181 Export Analysis Method s 182 Export Workflow s 183 Feature Extraction Preferences 184 Feature Extraction Properties 185 Import 186 Import Analysis Method s 187 Import Workflow s 188 Open 189 Provide Workflow Identifier 190 Save As 191 Select Report Folder 192 Select Report Name 193 Set Password 194 10 Workflow User Guide Agilent Genomic Workbench Lite Edition 6 5 Workflow User Guide ee 1 Getting Started Starting the Workflow Program 12 Setting Up and Running Workflows for Extraction and or Analysis 15 Getting Help 33 In Agilent Genomic Workbench Workflow is used to automate feature extraction and or analysis of CGH and ChIP data This chapter gives an overview
18. Analysis Method When Analysis Method is selected the command ribbon displays commands related to the Analysis Method On the Workflow command ribbon under Create Edit Analysis Method click A A list of analysis methods appears From the list select the analysis method to change On the Workflow command ribbon click Edit The Analysis Method window appears Change the parameters of the analysis method For specific instructions see the topics in the sections entitled Setting up CGH Analysis Methods on page 73 or Setting up ChIP Analysis Methods on page 83 When you are finished do one of the following e To save the existing analysis method with the changes click Save e To save the analysis method with changes as a new analysis method click Save As In the dialog box that appears type a name for the new analysis method then click OK By default the program also gives this name to the experiment it creates when you run the workflow See Save As on page 191 To save an analysis method You must save an analysis method before you can use it in a workflow e In the Analysis Method window do one of the following To save the analysis method with the same name click Save Workflow User Guide Setting up Workflow Analysis Methods 3 To save the analysis method with a different name click Save As In the dialog box that appears type a name for the new analysis method then click OK By default
19. Array List Although the program displays the arrays from one design at a time you can add arrays from more than one design to the Selected Array List Select Genome If the design you select in Select Design has arrays from more than one Build genome build select the desired genome build Although the program displays the arrays from one genome build at a time you can add arrays from more than one genome build to the Selected Array List Array List Displays the arrays for the selected design and genome build Selected Array Displays the arrays the program uses as input for the workflow List 164 Workflow User Guide Workflow Reference 4 Moves selected arrays from the Array List to the Selected Array List To select an array click its name To select additional arrays hold down the Ctrl key and click command click on a Mac their names To select a block of arrays click the name of the first array in the block then hold down the Shift key and click the last one lt Moves selected arrays from the Selected Array List to the Array List gt gt Moves all of the arrays in the Array List to the Selected Array List lt lt Clears all of the arrays from the Selected Array List and restores them to their original location s Set Output Path for Feature Extraction Panel Output path for FE File output Description Set the FE extraction file output path here Output path for Feature Extracted files Piiacw Da
20. Click to accept the workflow identifier and start the workflow Cancel Click to cancel the operation without running the workflow 190 Workflow User Guide Workflow Reference 4 Save As Enter Analysis Method Name l Apply Password OK Cancel Figure 72 Save As dialog box Purpose To type a name apply a password optional and save an analysis method or a workflow To open Click Save As from the workflow command ribbon Enter Workflow Type the name for the analysis method or workflow to save Name Enter Analysis Method Name Apply Password Click this box to protect the analysis method or workflow with a password OK Click to accept the password and close the dialog box If the Apply Password box is selected the Set Analysis Method Password dialog box opens See Set Password on page 194 Cancel Click to cancel the operation Workflow User Guide 191 4 192 Workflow Reference Select Report Folder File name Files of type Save Cancel Select report folder E Genomic Data Analysis Methods Image Files Reports File name JE Documents and Settings All Users Documents Genomic Data Files of type Cancel Figure 73 Select report folder dialog box Purpose Used to select the folder location and name to store a CGH or ChIP report To open From any of the CGH or ChIP Reports parameter panels click Browse Click the arrow and select the folder or browse to the locat
21. Files Parameter Panel Purpose This parameter panel lets you configure the workflow to import one or more Universal Data Files UDFs as input for the analysis To open The Import UDF Files Parameter Panel appears when you select Import UDF Files in Input in the Workflow Navigator Most of the parameters help to identify the array and its contents UDFs are tab delimited txt files Use the arrow buttons at the top of the table to map the column to a program parameter A UDF file must have these columns in any order Column Description ProbeName The name Probe ID of the probe ChrName The name of the chromosome to which the probe binds Start The first base pair on the chromosome to which the probe binds Workflow User Guide 151 4 Workflow Reference Add Main data table File Name Design ID Design type Data type Select Species Select Genome Build Select Mapping 152 Column Description Stop The last base pair on the chromosome to which the probe binds Description Additional probe annotation such as gene names LogRatio Log ratio data for the probe The file can contain more than one column of log ratio data Click this button to open an Open dialog box where you can select a UDF file for the workflow to import The first row of the main data table contains the first line of the UDF file Although the first line of the UDF file must contain column headings the workflow does not
22. ID Workflow Import FE Image Files Parameter Panel Add Purpose This panel lets you import or remove FE image files to extract using a workflow For each image sample parameters are displayed Buttons at the bottom of the tab are used to add or remove images from the list See To import an image file on page 41 and To remove an image from the list on page 43 To open This parameter panel is shown when you select Image Files under Input in the Workflow Navigator For additional information on associating samples to images see the Sample Manager User Guide Read only The file name of the image The Global Display Name for the array To change the Global Display Name double click the name and type the new name Read only The unique identifier for the microarray Workflow User Guide 149 150 Workflow Reference Barcode Sample ID lt Red Green ArraylD gt Grid Template Protocol Add Remove Read only This is the barcode identifier for the slide A microarray slide contains one array or for multiplex slides multiple arrays on one slide If the Red Green attributes for the array are assigned in Sample Manager they appear here Otherwise a appears in the field By default the Feature Extraction program automatically determines the Grid Template for the microarray Or you can select a Grid Template from the list or select a grid file grd from your hard disk By de
23. Panel Centralization Parameter Panel Description Centralization Threshold E o Centralization Bin Size fio Figure 24 Centralization Parameter Panel Purpose This parameter panel lets you set up centralization parameters Centralization recenters log ratio values It finds a constant value to subtract from or add to all values and makes sure that the zero point reflects the most common ploidy state For a description of the centralization algorithm see the CGH Interactive Analysis User Guide To open The Centralization Parameter Panel appears when you select Centralization in Normalization in the CGH Analysis Method Navigator Centralization This value is the ADM 1 threshold used to call aberrations for the Threshold centralization algorithm Ideally set this value to the ADM 1 threshold defined in the current analysis However because the centralization value is fairly robust over a wide range of threshold values Agilent does not recommend that you change the default settings for this algorithm 132 Workflow User Guide Centralization Bin Size Workflow Reference 4 Type a whole number To improve performance the algorithm finds the average of groups or bins of contiguous probes and runs the centralization process on these averages rather than on individual probes The centralization bin size is the number of probes that the program groups together for each of these averages The default value is 10 prob
24. Reference Table 14 Input for ChIP 4 Option Description Select Experiment Image Files Configures the workflow to use ChIP microarray data from an existing Agilent Genomic Workbench experiment When you select this option the Select Experiment Parameter Panel appears in the parameter panel Use this tab to select the desired experiment See Select Experiment Parameter Panel on page 163 When you run the workflow the program creates a new experiment with the same name as the workflow The original experiment is unchanged To customize the name of the new experiment click Experiment in the Output folder of the Workflow Navigator If you select this option you must also select Feature Extraction This option lets you select the image files to extract during the Feature Extraction workflow See Import FE Image Files Parameter Panel on page 149 105 4 106 Workflow Reference Feature Extraction You can use the Workflow application to automatically perform Feature Extraction on a series of microarray images In order to run a Feature Extraction workflow you must have both a Feature Extraction license and either a CGH or ChIP license You run Feature Extraction workflows from either the CGH or the ChIP Workflow Navigator For Feature Extraction the only workflow selections required are Input gt Image Files and Extraction gt Feature Extraction If Image Files is selected as the Input for the wor
25. Running Workflows for Extraction and or Analysis 15 To change settings for CGH workflow analysis 16 Quick start instructions for analyzing CGH data ina workflow 18 To change settings for ChIP Workflow Analysis 31 Getting Help 33 To get help within Agilent Genomic Workbench 33 To contact Agilent Technical Support 34 To learn about Agilent products and services 34 2 Setting Up and Running Workflows 35 Creating and Managing Workflows 36 To create a new workflow 36 To edit an existing workflow 36 To save a workflow 37 To save a workflow to a new name 37 To delete a workflow 37 To set a password for an existing workflow 37 Setting up a Workflow for Feature Extraction 38 To use eArray to update design template files 39 To set the location of the Feature Extraction software 41 To import an image file 41 To remove an image from the list 43 To correlate sample attributes with workflow microarrays 43 To set the output path for Feature Extraction results 44 To display or change the default FE parameters 44 Workflow User Guide 5 Contents Setting up an Analysis Workflow 45 To setup the Run Analysis 46 To change the Application Type 47 To select workflow input 47 To select FE data files for the workflow to import To select UDF data files for the workflow to import 49 50 To use previously imported data as the workflow input To use an experiment s arrays as the workflow input To select data files for the workflow to import To
26. Selects the interarray median normalization method for ChIP No parameters are required To open This parameter panel appears when you click Inter Array Median under Normalization in the Analysis Method Navigator See To configure normalization methods ChIP on page 84 Agilent Genomic Workbench calculates the central tendency of the common probes on replicate arrays using the median of the probe signal intensities No further parameters are necessary in this panel 154 Workflow User Guide Workflow Reference 4 Intra Array Lowess Intensity Dependent Normalization Intra Array Lowess Intensity dependent Normalization Description Normalizes the median signal across arrays in an array set in both channels Regression curve is fitted to all data probes a Figure 41 Intra Array Lowess Intensity dependent Normalization Purpose This parameter panel is used to configure the intra array Lowess normalization method for ChIP workflow analysis To open This parameter panel is displayed when you select Intra Array Lowess Intensity Dependent under Normalization in the ChIP Analysis Method Navigator Regression curve Select the data to use for the regression curve is fitted to e All data probes Includes all of the data probes in the regression curve e All common probes Includes probes whose names start with LACC e Gene desert probes Includes data for probes whose names start with LACC GD
27. This normalization is useful for data that is either blank subtracted or spatially detrended but it may have utility for data processed by other means as well Variance stabilization is an alternative to Lowess normalization that fits a regression curve to signal intensities after applying an arcsinh x transform to each channel This approach uses a two parameter error model to compress the reported ratios of probes with weak signals after blank subtraction After the transform is applied the variance of the reported log ratios should be independent of the signal strength Note If you are using feature extraction data that have been normalized by the Lowess approach you do not need to apply Variance Stabilization here 1 Inthe Analysis Method Navigator in Normalization select Variance Stabilization 2 Inthe parameter panel select how to fit the regression curve Note If Variance Stabilization normalization is selected Dye bias intra array Median normalization and Intra array Lowess normalization are not available 86 Workflow User Guide Setting up Workflow Analysis Methods 3 To configure error model for analysis method ChIP Workflow User Guide The goal of ChIP data analysis is to find the chromosomal locations where protein binding or other events occur The error model calculates the likelihood that probes represent binding events and assigns p values to probes A p value close to 1 indicates th
28. bottom of the window in the Summary Console Progress tabs pane displays the status of the workflow run s See Summary Console tab on page 174 A workflow progress tab is created in the Summary Console Progress tabs pane for the named workflow and displays the progress of the run and any errors that occur A not yet completed run has running displayed in its progress tab A completed run has completed displayed in its progress tab See Workflow progress tabs on page 176 Workflow User Guide 59 2 60 Setting Up and Running Workflows The Summary Console and workflow progress tabs include workflows for CGH ChIP and SureSelect Target Enrichment For information on SureSelect Target Enrichment workflows see the SureSelect Target Enrichment User Guide To run a series of workflows Once a workflow run has started you can set up and run additional workflows which are run in the order in which you started them Each workflow has its own progress tab On 64 bit computers you can run up to three workflows simultaneously On 32 bit computers workflows are processed sequentially not simultaneously To run a series with different workflows 1 Run the first workflow Follow the instructions in To run a workflow on page 59 Create a new workflow or select an existing one from the list See To create a new workflow on page 36 or To edit an existing workflow on page 36 3 In Output click E
29. box click to select the workflow s or analysis method s you wish to export See Export Analysis Method s on page 182 for more information The Export dialog box appears See Export on page 181 5 In the Export dialog box go to the location where you want to save the file and type the file name 6 Click Export To import an analysis method If you have exported one or more analysis methods and you want to import them into the program 1 On the workflow command ribbon under Create Edit Analysis Method click Analysis Method On the command ribbon click Import Workflow s The Import Analysis Method s dialog box appears See Import Analysis Method s on page 187 Browse to the location where the file is located click to select it and then click Import The Import dialog box appears See Import on page 186 4 Click to select the analysis method s to import 5 Click OK to import the analysis method s An import status box appears Click OK Workflow User Guide Setting up Workflow Analysis Methods 3 Setting up CGH Analysis Methods This section describes how to set up analysis methods to run a workflow for CGH analysis To select to fuse designs CGH If you have two arrays that use different design files you can combine fuse them into one larger virtual array You do this to increase the coverage of the genome in your design This can make it easier to work with multiple
30. file s when you select the files for the workflow run The workflow will automatically import them during the workflow e For information on how to add designs grid templates to the Feature Extraction database see the Feature Extraction User Guide For information on how to add data files and designs to Agilent Genomic Workbench see the CGH Interactive Analysis User Guide To set the output folder for Feature Extraction see To set the output path for Feature Extraction results on page 44 Workflow User Guide 19 1 Getting Started Quick start instructions for analyzing CGH data in a workflow Table 1 Steps for setting up and running a CGH workflow continued To do this Follow these instructions Comments Create or selecta workflow 1 Click the Workflow tab You see the Workflow Navigator 2 Onthe command ribbon under Select Workflow select an existing workflow to run OR On the command ribbon click New Type a name for the workflow and then click OK Using a workflow you can run Feature Extraction with image files before the CGH analysis if you have an Agilent Feature Extraction 10 7 or higher license Import UDF Files Select Imported Data 3 Select Experiment B CGH Aberration Report B Probe Based Penetrance Sumr B Cyto Report E cre Report B SNP Genotype Report B Aberration amp LOH Report 20 Workflow User Guide Table 1 Step
31. for SNP Copy Number and LOH analyses All of the aberration algorithms can use the Centralization calculation used to normalize data Centralization is recommended for SNP Copy Number and LOH analyses Workflow User Guide 23 1 Getting Started Quick start instructions for analyzing CGH data in a workflow Table 1 Steps for setting up and running a CGH workflow continued To do this Follow these instructions Comments 1 2 Set up the aberration algorithms Select one of the aberration algorithms Type or change parameters To remove long low aberrations from the ADM1 or ADN2 results select the Fuzzy Zero check box To filter out nested aberrations select the Apply Nesting Filter and type a value for the filter If you use HMM Hidden Markov Model you must also select the State Parameters ADM1 and 2 can use Fuzzy Zero to make the results more reliable by taking into account the global error across the chromosomes If the Nesting Filter value is set to zero you will filter out all nested aberrations By default no filter is applied See the Statistical Algorithms chapter of the CGH Interactive Analysis User Guide for information on the aberration algorithm calculations and suggested thresholds Set up the SNP algorithms Select one or both of the SNP calculations for analysis of CGH SNP data Type or change the parameters SNP Copy Number detec
32. is selected contains the commands used to create save delete and run a workflow and to set locations for Feature Extraction When Analysis Method is selected contains the commands to create save edit and delete an analysis method Workflow Navigator Displays current workflow and options As you select each option the relevant parameters appear in the parameter panel Parameter Panel Displays parameters for the selection in the Workflow Navigator Summary Console For Workflow this view contains the following tabs Progress View Summary Console tab that displays the application type workflow name experiment name status and current step for each workflow completed or in process Workflow Progress tab for each named workflow that displays the status of the workflow progress including any errors that might have occurred In Workflow the program only lets you set up workflow procedural steps and not explore the data You must switch to one of the other interactive tabs to review data and perform further analysis Workflow User Guide 97 4 Workflow Reference Workflow Command Ribbons The Workflow commands are divided into two groups located at the top of the window directly below the tabs The Create Edit Workflow commands on this ribbon change when you select Workflow or Analysis Method Command ribbon for Workflow Create Edit Workflow workFlow Select Workflow O Analysis Method Heli oo New Delete
33. option No input parameters required appears This section describes the parameter panels that appear in Workflow mode organized alphabetically by name on amp LOH Report Parameter Panel Aberration amp LOH Report Parameter Panel Description Report will be created at the specified location Select File Location Browse overwrite if file exists Figure 18 Aberration amp LOH Report Parameter Panel Select File Location Workflow User Guide Purpose To set location for Aberration amp LOH Report created by Workflow To open In the Analysis Method Navigator for CGH under Reports select Aberration amp LOH Report Displays the location where the workflow saves the files To select a location for the report click Browse An Open dialog box appears Type a name and select a location for the report then click Open 123 4 Workflow Reference Overwrite if file exists If you select this option the workflow deletes an existing file if it has the same name and location as a generated report Aberration Filter Parameter Panel Aberration Filter Parameter Panel Description You can select an already existing filter from the drop down below or can create a new filter Name New 124 Edit Aberration Filters Name DefaultAberrationFilter lca Minimum number of probes in region Minimum absolute average log ratio for region po Maximum number of aberrant regions Delete Percent pen
34. overrides a mark in Show probe information If you select this check box the resulting gene report contains additional information about the probes in the array Shows the location where the program saves the gene report Type a new location in the text box or click Browse to select a location Opens the Select report folder dialog box where you can select a location for the gene report See Select Report Folder on page 192 145 4 Workflow Reference HMM Parameter Panel Number of states FDRO value 146 HMM Parameter Panel Description HMM Aberration Detection Algorithm Number of States FDRG Value ps Figure 35 HMM Parameter Panel Purpose This parameter panel lets you set up the parameters required for applying the Hidden Markov Model HMM algorithm See the CGH Interactive Analysis User Guide for more information To open The HMM Parameter Panel appears when you select HMM under Aberration in the Analysis Method Navigator Select the number of states This is the number of distinct aberration states into which the observed data is to be partitioned For example if the number of states equals 3 then this would correspond to amplification deletion and no change aberration status The number of states can be set to either 3 5 or 7 Type the FDRQ value This is a False Discovery Rate threshold used in the calculation of the Discrete Haar Wavelet Transform and is used to keep only h
35. probes from the analysis based on selected design attributes To open In the Analysis Method Navigator under Filter Before Analysis select the box next to Design Level Filter Name Select the name of the design filter you want to edit To create a new filter and add its name to the list click New Filter Conditions For each condition row select options from the list or type a value then table press Enter To add another row to the table click New Condition Each condition has these elements e Attribute The design attribute evaluated by the filter e Operator How the filter uses the entry in Value to evaluate data For example the gt operator configures the filter to include or exclude probes where the selected attribute is greater than or equal to the entry in Value Workflow User Guide 137 4 Workflow Reference New Condition Delete Condition In m 138 clude Exclude atching values New Update Reset Delete Rename e Value The value the filter uses as the basis to evaluate an attribute You either select a value or for some attributes type a value For example if you select the Homology attribute with an operator of the filter will include or exclude probes that exhibit homology if the value is set to 1 The homology attribute is assigned to probes that have more than one mapping in the genome or probes that have secondary hits that are not perfect matches e Logical Operator
36. program To open On the command ribbon click Analysis Method and then click Import gt Analysis Method s Click the arrow and select the folder from which you want to import a file Type the name of the file you wish to import Or click to select the file from the displayed files Shows the type of files displayed in the window Click the arrow to change the type of files displayed Click to import the currently selected analysis method file into the program 187 4 Workflow Reference Cancel Click to cancel the operation Import Workflow s Look in File name Files of type Import Cancel 188 Import workflow s Lookin C My Documents My Music My Pictures File name Files of type xml Import Cancel Figure 69 Import Workflow s dialog box Purpose Used to select an workflow file to be imported into the program To open Click Import gt Workflow s from the command ribbon Click the arrow and select the folder from which you want to import a file Type the name of the file you wish to import Or click to select the file from the displayed files Shows the type of files displayed in the window Click the arrow to change the type of files displayed Click to import the currently selected workflow file into the program Click to cancel the operation Workflow User Guide Open Look in File name Files of type Open Workflow User Guide Workflow Ref
37. select reports CGH 53 To select and configure reports ChIP 57 Running Workflows 59 To run a workflow 59 To run a series of workflows 60 To run a series with different workflows 60 To run a series with the same workflow 60 To monitor workflow runs 61 To abort a single workflow 62 To abort all workflows 62 To run an existing workflow on a new set of data To display the results of a workflow 63 To export a workflow 64 To import a workflow 65 Setting up Workflow Analysis Methods 67 Setting up an Analysis Method 68 To create a new analysis method 69 To select an existing analysis method 69 To edit an existing analysis method 70 To save an analysis method 70 To delete an analysis method 71 To export an analysis method 71 62 51 Workflow User Guide Contents To import an analysis method 72 Setting up CGH Analysis Methods 73 To select to fuse designs CGH 73 To filter the data before analysis CGH 74 To configure the analysis method to apply a design level filter 74 To configure the analysis method to apply a feature level filter 75 To configure the analysis method to apply an array level filter 75 To create or modify a feature level filter 75 To create or modify an array level filter 76 To combine replicates CGH 76 To configure the analysis method to combine intra array replicates 76 To configure the analysis method to combine interarray replicates 77 To select to correct for GC content 77 To select to Centralize t
38. setting up and running a CGH workflow continued To do this Follow these instructions Comments Set up a workflow and select input files 1 To display the Workflow Navigator to set up and run a workflow on the Workflow command ribbon click Workflow 2 Inthe Workflow Navigator under Input click one of these option buttons Import FE Files Select these files to analyze Feature Extraction log ratio data Import UDF Files Select these text delimited files to analyze non Agilent data Select Imported Data Select to analyze data that appears in the Navigator for Interactive Mode Select Experiment Select an existing experiment to analyze data in the experiment Image Files Select to run Feature Extraction e The program creates a new experiment when you run a workflow If you select Image Files the Feature Extraction check box must be selected Image files are the only input allowed for Feature Extraction If you select image files you must have a Feature Extraction license If you selected Image Files as the input you can run a CGH analysis in addition to Feature Extraction If you selected any other input type you can only run the analysis not Feature Extraction You may want to use the imported data from the design file 014698 that comes with the program to run a workflow 26 Workflow User Guide Getting Started 1 Table 1 Steps for setting up and running a CGH
39. the Agilent Feature Extraction program Select this model if you have tried both models and know this one provides a better match to biological truths and or positive controls you have available for your experiment If you select this model the edit button becomes unavailable you do not need to set any additional parameters For more information see the Agilent Feature Extraction Software Reference Guide Whitehead Error The program uses this error model by default Select this model unless Model you have tried both models and know the FE Error Model provides a better match to biological truths and or positive controls you have available for your experiment If you choose this model the edit button becomes available and you can set additional advanced parameters For a description of the statistical algorithm used in this model see the ChIP Interactive Analysis User Guide Peak Detection and Evaluation The ChIP program uses two versions of the Whitehead per array neighbourhood model to make binding calls These models consider the p values of both the probe in question and its neighbors You can customize the parameters of the models including the maximum distance between neighbor probes and the stringency of the detection process The models consider probes in groups of three shown in Figure 17 In this figure two neighbor probes blue flank a central probe red Binding Signal P amp P Xb Genomic Location bp
40. the parameter panel See Probe Based Penetrance Summary Report Parameter Panel on page 159 2 Under Output Format select if the report file will contain output from the Complete Genome or if individual files will be generated Per Chromosome 3 Under Select File Location click Browse The Select report folder dialog box appears 4 Select a location for the report and if necessary change the File name 5 Click Open The location of the Text Penetrance Summary Report appears in the parameter panel in Report Location 6 Under Select File Location select Overwrite if file exists to overwrite a previous report saved as the same filename and location 54 Workflow User Guide Workflow User Guide Setting Up and Running Workflows 2 Table5 CGH module reports continued Report Description Instructions Cyto Report To set parameters for the report 1 Inthe Workflow Navigator in Reports select Cyto Report Three settings appear in the parameter panel See Cyto Report Parameter Panel on page 136 2 In Select Report select an existing Cyto Report 3 Under Select File Location click Browse The Select report folder dialog box appears 4 Select a location for the report and if necessary change the File name 5 Click Open The location of the Cyto Report appears in the parameter panel in Report Location 6 Under Select File Location select Overwrite if file exists to overwrite a previous report saved as the same filena
41. the program also gives this name to the experiment it creates when you run the workflow See Save As on page 191 When you use the Save or Save as command to save an analysis method it is not saved in an external file on your hard disk To save an analysis method to a location on your hard disk use the Export Workflow s gt Analysis Method command To delete an analysis method When you delete an analysis method the program removes the name parameters and settings of the analysis method from the program The program does not delete any microarray data files 1 On the Workflow command ribbon click Analysis Method When Analysis Method is selected the command ribbon displays commands related to the Analysis Method 2 On the Workflow command ribbon under Create Edit Analysis Method select the analysis method to delete 3 Click Delete A Confirm dialog box appears 4 Click Yes To export an analysis method To save an analysis method in a file on your hard disk you will need to export it To export an analysis method you must have one or more analysis methods saved in the program 1 On the command ribbon under Create Edit Analysis Method click Analysis Method 2 On the command ribbon click Export Workflow s A selection menu appears Workflow User Guide 71 3 72 Setting up Workflow Analysis Methods 3 To export an analysis method select Analysis Method s 4 In the Export Analysis Method s dialog
42. to the ADM 1 algorithm except that it is more accurate and also takes into account probe quality It is especially useful for the detection of small aberrant intervals When you select this option the ADM 2 Parameter Panel appears where you can set the parameters of the algorithm See ADM 2 Parameter Panel on page 127 For a discussion of the ADM 2 algorithm see the CGH Interactive Analysis User Guide Workflow User Guide 113 4 114 Workflow Reference Option Description CBS HMM The CBS algorithm partitions probes into subsets that share the same copy number to identify copy number change points It is useful for putative aberration characterization copy number estimates and downstream analysis You do not set any parameters for this algorithm For a discussion of the CBS algorithm see the CGH Interactive Analysis User Guide The Hidden Markov Model HMM algorithm identifies all aberrant intervals in a given sample based upon the individual likelihood of such signals in a genomic context A Hidden Markov Model is a method to partition a large number of observations into a smaller number of hidden states The HMM algorithm differs from other detection algorithms in that it identifies local probabilities in the data See HMM Parameter Panel on page 146 For more information on the HMM algorithm see the CGH Interactive Analysis User Guide SNP Algorithm These options let you set up the workf
43. type Type a value The filter excludes putative aberrant regions if the average log2 ratio within the region is less than the value you type Type a whole number For each microarray the filter includes up to this number of aberrant regions that have the highest statistical significance Type a value The filter excludes putative aberrations that have less than the specified minimum percent penetrance across the set of selected arrays 125 4 126 Workflow Reference ADM 1 Parameter Panel Threshold Nesting Level ADM 1 Parameter Panel Description ADM1 Aberration detection method By default Nesting Level is set to maximum integer value 2147483647 which means no nesting level filter will be applied You can set it to any desired integer value between 1 and 2147483647 Threshold Nesting Level Eo Wi Fuzzy Zero 01 s E 147483647 B Figure 20 ADM 1 Parameter Panel Purpose This panel lets you set the parameters for the ADM 1 aberration detection algorithm For more information on ADM 1 see the CGH Interactive Analysis User Guide To open The ADM 1 Parameter Panel appears when you select ADM 1 under Aberration in the Analysis Method Navigator Type a numerical value from 0 1 to 50 or use the slider to set a value The threshold is the minimum ADM 1 score for the detection algorithm to consider a given genomic interval significant In general increase this value to make the detect
44. you want to open Click Open The Add image pack information for FE Extraction dialog box appears See Add Image Pack Information for FE Extraction on page 178 For each image file select the Number of packs for the image file Select 1 if your image is a 1x1M or 1x244K slide select 2 if your image is a 2x400K or 2x105K slide select 4 if your image is a 4x180K or 4x44K slide or select 8 if your image is an 8x60K or 8x15K image Click Add Images The sample images from the file are displayed in the image list Workflow User Guide Setting Up and Running Workflows 2 To remove an image from the list 1 In the Import FE Image Files Parameter Panel click an image to highlight it See Import FE Image Files Parameter Panel on page 149 2 Click Remove The selected image is removed from the list To correlate sample attributes with workflow microarrays Workflow User Guide To correlate sample attributes for image files to use for Workflow input import an attribute file with the Array ID in Sample Manager or add the Array ID before you add the FE image file in Workflow The successful correlation is indicated by lt red attribute gt lt green attribute gt array ID displayed in the Sample ID lt Red Green Array ID gt field of the Import FE Image Files Parameter Panel If the association of sample attributes has not been successful a appears in the Sample ID lt Red Green Array ID gt field of the Impo
45. 08328 45666 chr4 12740 P 4 115743403 137912518 22 1 Mb 4 2 2 3 14 15 137 Mb 130 Mb 123 Mb115 Mb ple ES FIER 3 38 fat 433 a Workflow User Guide 29 1 Getting Started Table 1 Steps for setting up and running a CGH workflow continued To do this Follow these instructions Comments See the CGH Interactive Analysis At this point you can add tracks with With these tracks you can see if User Guide for instructions on how other gene information to the Gene View the aberrations correlate with copy to 1 Inthe My Entity List pane double click number variant regions You can Create and use gene lists the Tracks folder import other tracks as well Create and use tracks 2 Right click one of the tracks in the list Customize the appearance ofthe 3 Select the Show in UI check box display See Jo set up the Run A My Entity List Analysis on page 46 for E Gene List instructions on how to change the HE RECN OE analysis method Hs_hg17_CpGlsland_20080404 See the CGH Interactive Analysis a HEL PAR Z0OOMto User Guide for instructions on how bai Hs _hg18_CpGIsland_200804 A show In UI to use postprocessing Discovery S Kalmana zos a i nepot statistics on the results p Mm_mm7 _CpGIsland_20080 Mm_20080510 Mm_mms_Cp a Genomic Boundaries Mm_mm9_CpGlIsland_200805 Show In UCSC Mrn_rmm9_miRNA_20080510 Rn_rn3_CpGlIsland_2008051 Rn_rn4_CpGlIsland_2008051 Ren
46. 1 E Output L 9 Experiment Figure 16 The Analysis Method Navigator for ChIP To select any analysis method option click the option button or select the check box next to its name To edit the parameters for an option without changing its selection status click the name of the option Workflow User Guide Workflow Reference 4 This section describes the options for ChIP analysis methods in the order in which they appear in the Analysis Method Navigator Combining Replicates In the ChIP application replicate probes are probes that have the same probe name When you combine replicates you define how the program handles replicate probes The program can combine multiple biological and technical replicates within and among arrays See the ChIP Interactive Analysis User Guide for a discussion of the statistical algorithm the program uses to combine replicate probe data Select any of these options Option Description Intra Array Replicates Combines replicate probes within each array If you select this option you do not need to set any additional parameters for it Inter Array Replicates Combines replicate probes within designated groups of arrays When you select this option the Inter Array Replicates Parameter Panel appears in the parameter panel Use this tab to select the array attribute the program uses to group arrays when it combines interarray replicates See Inter Array Replicates Parameter Panel on p
47. 1_CGH v4_95_Feb07_1_1 2 U522502705_251469814934_501_CGH v4_95_Feb07_1_2 3 U522502705_251469814935_501_CGH v4_95_FebO7_1_1 4 U522502705_251469814935_501_CGH v4_95_Feb07_1_2 Aberration Filters minProbes 3 AND minAvgAbsLogRatio 0 0 AND maxAberrations 100 AND percentPeneti Feature Level Filters glIsSaturated true OR rIsSaturated true OR glsFeatNonUnifOL true OR rIsFeatNont kokk The actions are listed in the named workflow tab next to the Summary Console tab during the run The named workflow tabs show the progress of each workflow run and its completion status The new workflow experiment appears in the Experiment pane The experiment is marked with a W while the workflow is running When you select the new experiment tabular data from the experiment appear in the Tab View and the aberration results appear in Genome Chromosome and Gene views 28 Workflow User Guide Getting Started 1 Quick start instructions for analyzing CGH data in a workflow Table 1 Steps for setting up and running a CGH workflow continued To do this Follow these instructions Comments 4 5 6 7 8 9 10 q4 12 16 17 18 FeatureNum Description Name of Gene Accession u52250270 i u522 L osassa 0 12479165 0 13345937 Mossan 127025509 __ 15335 3 127083931 127083990 23461 5 jiaviaaass 127148514 104344 3 127207960 127208019 70039 K j127316179 127316238 42541 chr4 12731 _16_PO10 1274
48. 2 1 94 Workflow User Guide Agilent Genomic Workbench Lite Edition 6 5 Workflow User Guide 4 Workflow Reference Main Window 96 Workflow Command Ribbons 98 Workflow Navigators 102 Analysis Method Navigator 110 Parameter Panels 123 Summary Console Progress Tabs 174 Dialog Boxes 178 This chapter describes the commands tabs views and parameter panels specific to Agilent Genomic Workbench Workflow A workflow is a sequence setup or roadmap to automatically run an analysis with selected data input and output An analysis method is a method you set up to run in the workflow The analysis method contains parameters you select to prepare the data for event detection detect events and generate reports using the CGH or ChIP licensed applications If you also have an Agilent Feature Extraction license you can set up to use image files in the workflow which can be extracted and then analyzed when the workflow is run all without intervention ot Agilent Technologies 95 4 Workflow Reference Main Window Z Agilent Genomic Workbench Lite Edition 65 L CGH Home Sample Manager Workflow Preprocessing Analysis Discovery Create Edit Workflow QworkFiow Select Workflow Balt Reports Save As Q analysis Method con Pi N we E Apply password lope CGH Aberration Report eter Panel Description Import FE Files Report will be created at the specified location P arameter P ane l import UDF Files
49. 4 Type the name of the workflow and click OK To delete a workflow 1 Next to the Select Workflow list click the right arrow 2 Select a workflow name from the list 3 Click Delete To set a password for an existing workflow 1 Next to the Select Workflow list click the right arrow 2 Select a workflow name from the list 3 If you intend to restrict access to this workflow select Apply Password The Set Password dialog box appears See Set Password on page 194 4 Type a password Type the password again to confirm it and click OK Workflow User Guide 37 2 Setting Up and Running Workflows Setting up a Workflow for Feature Extraction 38 This section provides how to help for the Feature Extraction tasks available in the Workflow tab of Agilent Genomic Workbench If you have installed a license for the Agilent Feature Extraction software you can automatically perform Feature Extraction on image files without exiting Agilent Genomic Workbench whether or not you have any licenses installed for analysis applications such as CGH or ChIP If you have one or more licenses installed for analysis applications you can also set up workflows to automatically perform feature extraction and analysis For more information on setting up Workflows for CGH or ChIP analysis see Setting up an Analysis Workflow on page 45 For a detailed description of all of the parameter panel and dialog boxes that appear see Chapter 4
50. 59 4 Workflow Reference Select File Location Overwrite if file exists Displays the location where the workflow saves the files To select a location for the report click Browse An Open dialog box appears Type a name and select a location for the report then click Open If you select this option the workflow deletes an existing file if it has the same name and location as a generated report Probe Report Settings Report Location 160 Probe Report Settings Description This will generate a Probe report for peak detection results at the specified location If experiment contains more than one array then reports would be created at the location in a Folder with provided name Report Location j Browse Figure 45 Probe Report Settings Purpose This parameter panel lets you select the location for Probe Reports generated during ChIP workflow analysis To open This parameter panel appears when you click Probe Report under Reports in the ChIP Workflow Navigator For information about the content and format of the Probe Report see the ChIP Interactive Analysis User Guide Shows the location of the Probe Report that the program creates during the workflow You can edit the location You can also click Browse to select a new location for the report The program saves the Probe Report as a tab separated value tsv file Workflow User Guide Workflow Reference 4 Browse Opens the Select report folder dialog box
51. Agilent Genomic Workbench Lite Edition 6 5 Workflow User Guide ote Agilent Technologies Notices Agilent Technologies Inc 2010 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 63800 90033 Edition Revision A0 December 2010 Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Trademarks Microsoft is a registered trademark of Microsoft Corporation in the United States and other countries Adobe Adobe Acrobat and Adobe Reader are either registered trade marks or trademarks of Adobe Systems Incorporated in the United States and or other countries Software Revision This guide is valid for the Agilent Genomic Workbench Lite Edition 6 5 software and later revisions until superseded Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness
52. Refresh Status Abort Workflows amp Clear Table Figure 3 Workflow User Guide Workflow Main Window The selected DNA Analytics application appears at the top of the window in brackets You must change applications to set up a workflow for a different application CGH or CHIP To change the application type click Switch Application at the top right corner of the Agilent Genomic Workbench tab bar and click the application type 13 14 Getting Started To start the Workflow program For more information on the contents of the main window of Agilent Genomic Workbench see Main Window on page 96 Workflow User Guide Getting Started Setting Up and Running Workflows for Extraction and or Analysis Agilent Genomic Workbench Workflow lets you set up and run automatic feature extraction and analysis for multiple samples You use a workflow to e Run the workflow to extract image files with Agilent Feature Extraction software FE and produce a QC report that contains sample ID information from the Sample Manager table or e Run the workflow to analyze CGH or ChIP not CH3 data using Agilent Genomic Workbench and create reports or Run the workflow to extract image files and then analyze the extracted results to create both sets of reports O Import FE Files Import UDF Files Select Imported Data O Select Experiment i Image Files E Extraction amp Feature Extraction Er Analysis
53. Reports 5 CGH Aberration Report Probe Based Penetrance Sumr 5 Cyto Report E CNR Report i 5 SNP Genotype Report L F Aberration amp LOH Report CGH Import Data Files O Select Imported Data Select Experiment Image Files H Extraction i am Feature Extraction Ey Analysis i Run Analysis io E Reports B Probe Report B Gene Report m QC Report Figure 14 Workflow Navigator for CGH and ChIP 102 Workflow User Guide Workflow Reference 4 To select any workflow option click the option button or select the check box next to its name To display the parameters for a workflow option without changing its selection status click the name of the option Input In Input you select the source of data for the workflow See To select workflow input on page 47 Select one of the input options Table 13 Input for CGH Option Description Import FE Files Configures the workflow to import Agilent Feature Extraction microarray data files When you select this option the Import Data Files Parameter Panel appears Use this pane to select data files for import See Import Data Files Parameter Panel on page 147 To use this option you must have already imported the representative design files into the program See the CGH Interactive Analysis User Guide for more information In Workflow mode the CGH application supports the import of Agilent and UDF microarray data files only To use Axon files i
54. TION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met Workflow User Guide In This Guide Workflow User Guide This guide describes how to use the Workflow utility of Agilent Genomic Workbench Lite Edition 6 5 to extract image files with Agilent Feature Extraction software and or analyze data using CGH and ChIP analysis software Getting Started This chapter gives an overview of Workflow and how it is used in Agilent Genomic Workbench Lite Edition It also provides flow charts for setting up and running CGH and ChIP analysis workflows Setting Up and Running Workflows This chapter describes how to set up and run Feature Extraction and analysis workflows It includes instructions for creating new workflows Setting up Workflow Analysis Methods This chapter describes how to set up an analysis method for a CGH or ChIP analysis workflow Workflow Reference This chapter describes the main window parameter panels and the dialog boxes for Workflow Workflow User Guide Contents Contents 1 Getting Started 11 Starting the Workflow Program 12 To start the Workflow program 12 Setting Up and
55. age 153 Normalization Normalization corrects data for known factors that cause measured values to deviate from their true values For ChIP analysis select any of these options Workflow User Guide 117 4 Workflow Reference Option Description FE Output Select FE Output to use the processed feature intensity values in the output files of the Agilent Feature Extraction program The ChIP program uses these values instead of applying its own normalization methods Normally the program uses the raw unprocessed feature intensities If you use processed FE output instead of applying normalization within the ChIP program you do not need to set any additional normalization parameters Blank Subtraction This kind of normalization corrects for nonspecific binding It first calculates the central tendency of the negative controls on the array for both the immunoprecipitated IP and whole cell extract WCE channels It then subtracts these central tendencies from the raw signal intensities of each feature on the array As with all of the normalization methods if the method causes a probe to have a negative value for intensity it flags the probe as excluded See the ChIP Interactive Analysis User Guide for more information If you select this type of normalization central tendencies are calculated using the median of the negative control probes as a baseline See Blank Subtraction Normalization on page 131 Dye b
56. ame Rn_rn4_miRNA_200805 Delete View Details 4 Toclear the tracks right click the Gene Select the Show in Report check View and click Preferences boxes to add track information in 5 Click Tracks and clear the Show in UI the report check boxes and click OK e Select Genomic Boundaries to limit the analysis within the boundaries defined in the tracks 30 Workflow User Guide Getting Started 1 To change settings for ChIP Workflow Analysis A ChIP workflow is helpful if you need to analyze multiple arrays and you know which algorithm settings you intend to use for the analysis First you set all parameters for an analysis in an analysis method When you run the workflow the program automatically runs the analysis method On 64 bit computers you can run up to three workflows simultaneously On 32 bit computers workflows are processed sequentially not simultaneously With a workflow you can use already imported FE data or existing experiments as the source of data for your analysis Or you can import data and create experiments automatically as you run the workflow Workflows also let you analyze different data sets with the same analysis method and multiple data sets with multiple analysis methods After completion of the workflow run you use the Navigator to select the workflow experiment to display the results in the Genome Chromosome and Gene Views To learn how to set up an analysis method and workflow and r
57. ame US23502418_252152910035 Array ID 252152910035_1_1 Barcode 252152910035 Sample ID lt Red Green 4rra Grid Template lt Automatically Determine gt Protocol lt Automatically Determine gt 4 US23502418_252152910035 US23502418_252152910035 252152910035_1_2 252152910035 lt Automatically Determine gt lt Automatically Determine gt US23502418_252152910035 US23502418_252152910035 252152910035_1_3 252152910035 lt Automatically Determine gt lt Automatically Determine gt US23502418_252152910035 US23502418_252152910037 US23502418_252152910035 us23502418_252152910037 252152910035_1_4 252152910037_1_1 252152910035 252152910037 lt Automatically Determine gt lt Automatically Determine gt lt Automatically Determine gt lt Automatically Determine gt US23502418_252152910037 US23502418_252152910037 252152910037_1_2 252152910037 lt Automatically Determine gt lt Automatically Determine gt US23502416_252152910037 US23502418_252152910037 252152910037_1_3 252152910037 lt Automatically Determine gt lt Automatically Determine gt US23502418_252152910037 US23502418_252152910037 252152910037_1_4 252152910037 lt Automatically Determine gt lt Automatically Determine gt Image Name Global Display Name Array
58. ame probe To configure the analysis method to combine intra array replicates e In the Analysis Method Navigator in Combining Replicates select Intra Array Replicates The Intra Array Replicates Parameter Panel appears No parameters are required for this option Workflow User Guide Setting up Workflow Analysis Methods 3 To configure the analysis method to combine interarray replicates 1 In the Analysis Method Navigator in Combining Replicates select Inter Array Replicates The Inter Array Replicates Parameter Panel appears See Inter Array Replicates Parameter Panel on page 153 2 In Group By select an array attribute When you run the workflow the program combines replicates from arrays with matching values for the selected attribute To select to correct for GC content Workflow User Guide When you select to correct for GC content the algorithm corrects for wave artifacts by performing a robust regression fit to GC content in a specified region flanking the probes and then corrects for it The correction is done for both CGH and SNP probes GC Content correction is recommended for calculation of SNP Copy Number and LOH 1 In the Analysis Method Navigator in Normalization select GC Correction The GC Correction parameter panel appears See GC Correction Parameter Panel on page 144 2 Click to select the Window Size to use for the GC correction For more information on how this algori
59. an or equal to probe spacing on the array Selection increases accuracy but it doubles the runtime Workflow User Guide Workflow Reference 4 Use errors estimated by Error model Select to use the estimated error for each probe to weight its contribution to the peak fit measurement Probe Based Penetrance Summary Report Parameter Panel Probe Based Penetrance Summary Report Parameter Panel Description Report will be created at the specified location Output Format _ _ Select File Location Browse complete Genome O Per Chromosome overwrite if file exists Figure 44 Probe Based Penetrance Summary Report Parameter Panel Purpose This parameter panel lets you configure the Probe Based Penetrance Summary Report and select a location for it This report displays each probe that shows a significant aberration and gives the percentage of selected arrays that show a significant deletion or amplification in the region for each probe The workflow creates one or more xls files that you can work with in Microsoft Excel To open The Probe Based Penetrance Summary Report Parameter Panel appears when you select Probe Based Penetrance Summary Report in Reports in the Workflow Navigator for CGH Output Format Select one of these options e Complete Genome Creates a single report file e Per Chromosome Creates a separate report file for each chromosome Workflow User Guide 1
60. analysis method To delete the selected analysis method click Yes Edit Opens the Analysis Method window Select the analysis method to edit and change the parameters Click Save to save the changes in the selected analysis method Click Save As to save the changes with a new analysis method name Apply password Lets you type your password for a password protected analysis method Import Lets you select to import workflow s or analysis method s Export Lets you select to export workflow s or analysis method s Feature Extraction Lets you set the path for the installation folder of your Feature Preferences Extraction Software Workflow User Guide 101 4 Workflow Reference Workflow Navigators The Workflow application has two Navigators the Run Workflow Navigator and the Analysis Method Navigator The Run Workflow Navigator is always displayed at the left side of the main Workflow window The Analysis Method Navigator is displayed in the Analysis Method window when you create or edit an analysis method The Navigators contain different information depending on whether you select the CGH or ChIP application Workflow Navigators for CGH and ChIP When CGH or ChIP is selected as the analysis application the Workflow Navigator changes to look like Figure 14 3 Import FE Files Import UDF Files Select Imported Data Select Experiment 3 Image Files E Extraction t 5 Feature Extraction Oy Analysis L 5 Run Analysis
61. arameter Panel Inter Array Replicates Parameter Panel Description Tf this is checked the data from arrays which have the same value for selected attribute will be combined and treated as a single array in analysis Group By Group By Select Attribute a Figure 39 Inter Array Replicates Parameter Panel Purpose This parameter panel lets you configure how the workflow combines replicate probes across multiple arrays To open The Inter Array Replicates Parameter Panel appears when you select Inter Array Replicates under Combining Replicates in the Analysis Method Navigator In Select Attribute select an array attribute The program uses the selected array attribute to group arrays when it combines replicate probes For example if you have some arrays where the Sample Name attribute is set to C4 and other arrays with the same array attribute set to D95 the program combines the corresponding probes for the C44 arrays to make a virtual array C44 It combines the probes with the same names for the Workflow User Guide 153 4 Workflow Reference D95 arrays to make another virtual array To edit the attributes of an array see the CGH Interactive Analysis User Guide or the ChIP Interactive Analysis User Guide Inter Array Median Normalization Inter Array Median Normalization Description Normalizes the median signal across arrays in an array set in both channels Figure 40 Inter Array Median Normalization Purpose
62. arrays that are part of an array set For information about fusing designs see the CGH Interactive Analysis User Guide Agilent Genomic Workbench cannot combine arrays from more than two different design files at a time Arrays to be fused must have the same value for the ArraySet attribute This is set interactively 1 In the Analysis Method Navigator in Fuse click the Fuse Design option button The Fuse Design Parameter Panel appears See Fuse Design Parameter Panel on page 143 Click Select Normalization and select None or Centralization Select Remove arrays from experiment after fuse if you wish to use only fused arrays in the experiment rather than fused and individual arrays This deletes the initial unfused arrays from the experiment and reduces the duplication of data within the experiment If the fused arrays have common probes these probes are treated as replicates in the fused array You can combine these replicates See To configure the analysis method to combine intra array replicates on page 83 Workflow User Guide 73 3 74 Setting up Workflow Analysis Methods To filter the data before analysis CGH Some features on an array can lead to erroneous results for example saturation in either channel attenuates the true log ratio With a feature level filter the workflow can remove these data before analysis Also measurable log ratios can be established with a minimum log ratio value The Defau
63. ase the value to find more peaks Score threshold Minimum threshold for extreme value distribution EVD based score Decrease value to find more peaks Workflow User Guide 157 4 Workflow Reference Estimated mean shear length distribution of sample DNA Estimated standard deviation of the shear length distribution of sample DNA Precision of peak placement on the chromosome in base pairs Number of randomizations for determining peak significance via non parametric test and score Window size in bp for computing local baseline Desired spacing of interpolated datapoints between probe Automatically re run calculation after learning peak shape 158 Peak Shape Parameters Type a mean to be used in the gamma distribution calculation for approximation of the distribution of sheared DNA fragments Type a standard deviation to be used in the gamma distribution calculation of the distribution of sheared DNA fragments Other Algorithmic Parameters This is the window within which the algorithm searches for potential positions for the peak center Decreasing this window increases the time it takes for the algorithm to run The program computes p value and peak score through a number of random samplings Increasing the number of samples increases the accuracy of the prediction however it also increases the time to do the calculation Use smaller number for smaller genomes Must be less th
64. at a probe is unlikely to represent a significant binding event A very small p value for example lt 0 001 indicates that the probe is very likely to represent one Later the program combines p values from groups of probes to make binding calls 1 In the Analysis Method Navigator in Error Model click the option button next to the desired error model Option Description FE error model Uses the error model from the Agilent Feature Extraction program Select this model if you have tried both models and know this one provides a better match to biological truths and or positive controls you have available for your experiment If you select this model the edit button becomes unavailable you do not need to set any additional parameters Whitehead Error Model The program uses this error model by default Select this model unless you have tried both models and know the other one provides a better match to biological truths and or positive controls you have available for your experiment If you choose this model the edit button becomes available and you can set additional advanced parameters For a description of the statistical algorithm used in this model see the ChIP Interactive Analysis User Guide 2 If you selected the Whitehead Error Model you can set several advanced parameters that are optional You can set them to optimize the statistical calculations of the error model with training data specific to your particular a
65. ber of randomization runs Increase to find more peaks Score threshold minumum for EvD based score Decrease to find more peaks po Peak Shape Parameters Estimated mean shear length distribution of sample DNA Estimated standard deviation of the shear length distribution of sample DNA Other Algorithmic Parameters Precision of peak placement on the chromosome in base pairs Number of randomizations for determining peak significance via non parametric test and score Window size in bp for computing local baseline Use smaller number for smaller genomes Desired spacing of interpolated datapoints between probe must be less than or equal to probe spacing on the array 8 Automatically re run calculation after learning peak shape Doubles runtime but increases accuracy Aa Use errors estimated by Error model If selected the estimated error for each probe is used to weight its contribution to the peak fit measurement Figure 43 Pre defined peak shape detection v2 1 Purpose This parameter panel is used to configure the predefined peak shape detection v2 1 algorithm for peak detection in ChIP analysis To open This parameter panel appears when you select Pre defined Peak Shape detection v2 1 under Peak Detection and Evaluation in the ChIP Analysis Method Navigator Thresholds P value threshold Maximum threshold for the nonparametric test for reporting peaks Value must be greater than 1 number of randomized runs Incre
66. bias intra array Median Normalization Description Normalize channels within each array so that medians are equivalent Normalize by Figure 29 Dye bias intra array Median Normalization Purpose This parameter panel lets you configure the Dye Bias intra array Median Normalization method for ChIP To open These parameters appear in the parameter panel when you click Dye bias intra array Median under Normalization in the Analysis Method Navigator See To configure normalization methods ChIP on page 84 Defines how the program computes the dye bias when it applies this kind of normalization Select one of these options e By equalizing central tendencies of IP and WCE channels This method first calculates the ratio of the median IP signal intensity to the median WCE signal intensity It then multiplies the signal intensities of the data probes by this ratio e By normalizing central tendency of log ratios to 1 This method multiplies the signal intensities of all data probes on the array by a correction factor This correction factor adjusts the central tendency of the log ratios of data probes on the array to 1 139 Workflow Reference Experiment Parameter Panel Experiment Parameter Panel Description When workflow run is complete an experiment with specified name will be created and can be further analyzed from interactive mode Experiment Name Be uto created Experiment From workflow E
67. bine replicates see the ChIP Interactive Analysis User Guide Intra array replicates are features within the same array that contain the same probe Interarray replicates are features on different arrays that contain the same probe When you combine interarray replicates you select an array attribute Agilent Genomic Workbench combines replicates from arrays with matching values for the selected attribute To configure the analysis method to combine intra array replicates e In the Analysis Method Navigator in Combining Replicates select Intra Array Replicates To configure the analysis method to combine interarray replicates Workflow User Guide 1 In the Analysis Method Navigator in Combining Replicates select Inter Array Replicates The Inter Array Replicates Parameter Panel appears in the parameter panel See Inter Array Replicates Parameter Panel on page 153 2 In Group By select an array attribute When you run the workflow the program combines replicates from arrays with values that are the same as the selected attribute 83 3 84 Setting up Workflow Analysis Methods To configure normalization methods ChIP Normalization corrects microarray data for known factors that cause the reported signal intensities to be different from the actual signal For a discussion of the statistical algorithms the program uses to normalize data see the ChIP Interactive Analysis User Guide e In the Analysis Meth
68. bort the running workflow and remove its tab from the Summary Console Progress tabs pane Removes the current tab and removes its row in the Summary Console Tab Displays analysis settings and a running log of workflow activity Workflow User Guide Workflow Reference 4 KI Available if tabs are hidden past the left edge of the pane Shifts the display of tabs to the left to reveal hidden tabs gt Available if tabs are hidden past the right edge of the pane Shifts the display of tabs to the right to reveal hidden tabs Workflow User Guide 177 4 Workflow Reference Dialog Boxes 178 Add Image Pack Information for FE Extraction Add Images Cancel Add image pack information for FE Extraction Please select the number of packs for each image No File Name Number of Packs 1 Hu22K_GE2_251209710036 tif ES 2 Hu244K_CGH_251469312458 tif 3 Hudx44k_GE1_251485034336_H tif Add Images Cancel Figure 59 Add image pack information for FE Extraction dialog box Purpose To select the number of image packs to open for multi pack image files to be opened To open This dialog box appears when you click Open after you select an image file from the Open dialog box For each image file click the Number of Packs and select the number of packs included in the image file to be imported For example for a 2 pack array select 2 for Number of Packs The Number of Packs is set to
69. cence intensity between some red and green dyes The Lowess normalization algorithm normalizes the channels within each array using a nonlinear polynomial fit to the data and effectively normalizes by probes and by arrays See Intra Array Lowess Intensity Dependent Normalization on page 155 Variance This normalization is useful for data that is either blank subtracted or Stabilization spatially detrended but it may have utility for data processed by other means as well Variance stabilization is an alternative to Lowess normalization that fits a regression curve to signal intensities after applying an asinh x transform to each channel This approach uses a two parameter error model to compress the reported ratios of probes with weak signals after blank subtraction After the transform is applied the variance of the reported log ratios should be independent of the signal strength See Variance Stabilization on page 168 Error Model The error model calculates the likelihood that probes represent binding events and assigns I values to probes A p value close to 1 indicates that a probe is unlikely to represent a significant binding event A very small p value for example p lt 0 001 indicates that the probe is very likely to represent a significant binding event Select one of these error models 119 4 120 Workflow Reference Option Description FE Error Model Uses the error model from
70. ch for the correct folder The default location for the Feature Extraction software is C Program Files Agilent MicroArray Feature Extraction Click on the folder and then click Open The path will appear in the FE Location box To import an image file As part of the Workflow to automate extraction you must import an image file that contains one or more scanned microarrays along with the Array ID that identifies the microarray For example this can be a tif image file generated by an Agilent scanner The only input allowed for Feature Extraction workflows are image files If any other input is selected then Feature Extraction cannot be selected 1 From the Workflow tab in the command ribbon select Workflow The Workflow Navigator appears in the Navigator Pane 2 The DNA folder should be open by default If not double click the DNA folder to open the folder Input Extraction and Analysis folders are displayed Workflow User Guide 41 2 42 Setting Up and Running Workflows Click the Input folder to open it 4 Click Image Files The Import FE Image Files Parameter Panel is displayed See Import FE Image Files Parameter Panel on page 149 Click Add The Open dialog box appears Click to highlight the image file you wish to open Or click the Look in arrow and search for the desired folder Then click to highlight the image file To select more than one image file hold down the Ctrl key and click the files
71. ck Select Imported Data The Select Imported Data Parameter Panel appears See Select Imported Data Parameter Panel on page 164 2 In the parameter panel in Select Design select an array design In the parameter panel in Select Genome Build select an array design build The arrays associated with the design and genome build appear under Array List 4 In Array List click the name of an array to include in the workflow To select additional arrays hold down the Ctrl key and click their names To select a contiguous block of arrays click the name of the first one in the block then hold down the Shift key and click the name of the last one 5 Click The program moves the selected arrays to the Selected Array List You can also use the other buttons in the dialog box to change the array lists For more information see Select Imported Data Parameter Panel on page 164 Workflow User Guide 51 2 52 Setting Up and Running Workflows To use an experiment s arrays as the workflow input You can use the arrays selected in an existing CGH or ChIP experiment as the input for a workflow The program only uses the arrays linked to the experiment and does not overwrite the original experiment or use any of its settings 1 In the Workflow Navigator under Input click Select Experiment The Select Experiment Parameter Panel appears See Select Experiment Parameter Panel on page 163 2 In Select E
72. create a new filter Feature level filters include or exclude data based on selected criteria To open The Feature Filter Parameter Panel appears when you select Feature Level Filter under Filter Before Analysis in the Analysis Method Navigator Name Select the name of the feature filter you want to edit To create a new filter and add its name to the list click New Filter Conditions Below the Name is a list that displays the conditions defined for the table selected feature level filter For each criterion row select options from the list or type a value then press Enter To add another row to the table click New Condition Each condition has these elements e Attribute The feature attribute evaluated by the filter e Operator How the filter uses the entry in Value to evaluate arrays For example the gt operator configures the filter to include or exclude features where the selected attribute is greater than or equal to the entry in Value Workflow User Guide 141 4 Workflow Reference New Condition Delete Condition Include Exclude matching values New Update Reset Delete Rename 142 e Value The value the filter uses as the basis to evaluate a feature For example if you select the gIsSaturated attribute the operator and a value of true the filter includes or excludes a feature if its gIsSaturated attribute is true e Logical Operator Available only if you configure more than one con
73. ction and one of the analysis applications To select workflow input When you set up a workflow you must select its source of input data e In the Workflow Navigator under Input click the option button next to the source of microarray data See the tables below for a description of the available options Table 2 Workflow Input for Feature Extraction Option Description Image Files Opens the Import FE Image Files Parameter Panel where you can add image files to be extracted during a workflow See Import FE Image Files Parameter Panel on page 149 This is the only input option allowed for running a Feature Extraction in a workflow Workflow User Guide 47 2 Setting Up and Running Workflows Table 3 Workflow Input for CGH Analysis Option Description Import FE Files Import UDF Files Select Imported Data Select Experiment Extracted microarray data that you have not yet imported into Agilent Genomic Workbench For instructions on how to set the parameters for this option see To select FE data files for the workflow to import on page 49 Tab delimited Universal Data Files created by non Agilent programs For instructions on how to set the parameters for this option see To select UDF data files for the workflow to import on page 50 CGH microarray data that you have previously imported into Agilent Genomic Workbench For instructions on how to set the parameters for this
74. dition The relationship between the condition and the next one in the list For example if you select AND in Logical Operator for the first condition the filter includes or excludes a feature if it passes both the first condition and the next condition Adds a new blank condition row to the table Removes a condition from the list To remove a specific condition click anywhere within the condition row then click Delete Condition Select one of these options e Include matching values If a feature passes the filter condition the program includes it in the analysis e Exclude matching values If a feature passes the filter condition the program excludes it from the analysis Opens an Input dialog box where you can type a name for the new filter To accept the name click OK The program creates the filter and adds the new name to the Name list Saves any changes you make to the filter criteria without closing the dialog box Restores the values of the filter conditions to what they were before you made any changes to them Opens a Confirm dialog box that asks you if you want to delete the currently selected filter To delete the filter click Yes Opens an Input dialog box where you can type a new name for the filter To accept the name click OK Workflow User Guide Workflow Reference 4 Fuse Design Parameter Panel Fuse Design Parameter Panel Description You can fuse arrays from 2 different designs The
75. e Workflow Reference 4 Read only The name of the workflow described in the row Read only The name of the analysis method used in the workflow Read only The name of the output experiment that the workflow generates By default the program gives the experiment the same name as the Analysis Method However you can change the experiment name before you run the workflow You look at the experiment including its results in the interactive tabs Read only Indicates whether the workflow is Running or Complete Read only For a workflow that is running shows the progress through the steps of the workflow In this column in the row of the desired workflow click _ 8__ to open the tab for the selected workflow This lets you review information about the workflow Updates the status of all workflows The program also updates the status of the workflows in the workflow list in real time Opens a Confirm dialog box that asks if you are sure you want to abort all workflows If you click Yes the program stops the execution of all workflows and removes all workflows from the list It also removes all named workflow tabs Available if tabs are hidden past the left edge of the pane Shifts the display of tabs to the left to reveal hidden tabs Available if tabs are hidden past the right edge of the pane Shifts the display of tabs to the right to reveal hidden tabs 175 4 Workflow Reference Workflow progress tabs
76. e a file from the list in the parameter panel click its name then click Remove 4 In Dye Flip select either Normal or Flipped for each array Select Normal if e The test samples were labeled with cyanine 5 red e The control samples were labeled with cyanine 3 green e The imported ratio test control should be reported directly Select Flipped if e The test samples were labeled with cyanine 3 green e The control samples were labeled with cyanine 5 red e The imported ratio control test should be reported with the ratio inverted test control Agilent Feature Extraction txt array files must use GEML xml design files Axon gpr array files must use Axon gal design files This helps Agilent Genomic Workbench to match data and design files correctly To select reports CGH Workflow User Guide When you run a workflow the program can produce four different reports that present the analysis method s experimental results See Table 5 Reports are files that contain output from the CGH module that you can open with other programs e In the Workflow Navigator in Reports select the reports you want the analysis method to produce In the parameter panel set the parameters for each report See Table 5 for a description of the available reports and instructions on how to set the specific parameter s for each 53 2 Setting Up and Running Workflows Table5 CGH module reports Report Description Ins
77. e a minimum percent penetrance across samples 1 In the Analysis Method Navigator in Filter After Analysis select Aberration Filter The Aberration Filter Parameter Panel appears See Aberration Filter Parameter Panel on page 124 In Select Filter choose either the DefaultAberrationFilter or any filter predefined in interactive mode See the CGH Interactive Analysis User Guide for details Or click New to open the Input dialog box to type a name for the new filter Complete the parameter panel for the selected edited or new filter Workflow User Guide Setting up Workflow Analysis Methods 3 To select to calculate SNP Copy Number This selection causes the program to calculate allele specific copy number for SNP probes In order to select this option you must first select an aberration algorithm except z score or HMM GC Correction and Centralization are recommended In the Analysis Method Navigator in SNP Algorithm select SNP Copy Number The SNP Copy Number Parameter Panel appears See SNP Copy Number Parameter Panel on page 166 In SNP Conf Level type a value to use for the confidence level in the calculation For more information see the CGH Interactive Analysis User Guide To select to calculate LOH Use this selection to identify regions of LOH oss or lack of heterozygosity for microarrays with SNP content Workflow User Guide In order to select this option you must first select SNP Co
78. e design files into the program before you run a workflow that imports FE files Global Display Lists the global display names of array data files selected for import To Name add files click Add To edit the global display name of a file double click the name type the name then press Enter Dye Flip Lets you annotate an array as a dye flip array an array in which the Cy3 and Cy5 fluorochrome labeled samples have been reversed If you select Flipped the program inverts ratios so you can make proper comparisons Also the program does not combine dye flip pairs Select one of these options for each array Workflow User Guide 147 4 Workflow Reference Design Name Design Build Design Status Overwrite arrays with duplicate names Remove Add Arrays 148 Select this option If Normal The test samples were labeled with cyanine 5 red The control samples were labeled with cyanine 3 green The imported ratio test control should be reported directly Flipped The test samples were labeled with cyanine 3 green The control samples were labeled with cyanine 5 red The imported ratio control test should be reported with the ratio inverted test control The design name for the array is displayed in this field The design build for the array is displayed in this field If this field is blank you need to import the design file from the Home tab or add the design before you can run a wor
79. e parameters for the ADM 2 aberration detection algorithm For more information on ADM 2 see the CGH Interactive Analysis User Guide To open The ADM 2 Parameter Panel appears when you select ADM 2 under Aberration in the Analysis Method Navigator Workflow User Guide 127 4 Workflow Reference Threshold Type a numerical value from 0 1 to 50 or use the slider to set a value The threshold is the minimum ADM 2 score for the detection algorithm to consider a given genomic interval significant In general increase this value to make the detection process more stringent Nesting Level To apply a nesting level filter select Apply Nesting Filter then type a whole number from 0 to 2147483647 in the box The ADM 2 aberration detection algorithm reports nested aberrations that is aberrations within other aberrations If you set the nesting level to 0 the program reports only the parent aberration without any child nested aberrations If you set it to 1 the programs reports the first level of child aberrations By default the program sets the nesting level filter to its maximum value which applies no filter To make the filter more stringent decrease the value Fuzzy Zero Select Fuzzy Zero to apply Fuzzy Zero correction to detected aberrant intervals This correction applies a global error model to the intervals and can result in fewer aberration call errors For more information on the fuzzy zero algorithm see the CGH Interac
80. emplate Protocol us23502418_25 U523502418_25 252152910035 ITa lt Automatically lt Automatically Feature Extraction Oy Analysis TFA Run Analysis Parameter panel for selected item Oy Reports fA CGH Aberration Report fil Probe Based Penetrance Sumr amp Cyto Report E we Report E SNP Genotype Report Summary Console A Summary Console Close Tab Workflow 7 Navigator Run workflow tab s Running Analysis AppMation istae Analysis Settings Aberration algorithm aom MSA LLULL GIA A ULES ot G11 Threshold 6 0 IGC Correction OFF Centralization OFF Figure 7 Workflow Main Window for Feature Extraction To use eArray to update design template files To run a Feature Extraction workflow the design template file s for the files you want to extract must be present in the Feature Extraction Grid Template Browser or in the workflow FE output folder and also in the Agilent Genomic Workbench database Workflow User Guide 39 2 40 Setting Up and Running Workflows When you run a workflow the program will check the eArray Web site for design templates and add them to the Feature Extraction database automatically if e The eArray Login Settings dialog box in Feature Extraction has a valid Username and Password AND e You selected Use eArray server during extraction and Check for updates of grid template in the Advanced Options of the eArray Logi
81. en this file in Microsoft Excel To open The CNVR Report Parameter Panel appears when you select CNVR Report in Reports in the CGH Workflow Navigator Type a name If you select CNVR Report in the Workflow Navigator the results folder of the output experiment of the workflow will contain a CNVR node with the name that you typed The location where the workflow saves the generated CNVR report file Click Browse The Select report folder dialog box opens where you can type a name and select a location for the CNVR report file If you select this option the workflow deletes an existing file if it has the same name and location as the generated CNVR report 135 4 Workflow Reference Cyto Report Parameter Panel Cyto Report Parameter Panel Description You have to create a template from the interactive mode which will be available from the drop down below Only Formatting settings will be used From the selected Cyto Report template Analysis settings will be used from the selected Analysis Method Select Report Select File Location Browse Select Report ica _ overwrite if File exists Figure 27 Cyto Report Parameter Panel Purpose This panel lets you select the Cyto Report template to use and a location for the report Cyto Reports summarize analysis settings and detected aberrations by array The workflow creates a separate PDF file for each array To open The Cyto Report Parameter Pane
82. ence level to use in the SNP copy number calculation 166 Workflow User Guide Workflow Reference 4 SNP Genotype Report Parameter Panel SNP Genotype Report Parameter Panel Description Report will be created at the specified location Output Format Select File Location Browse complete Genome O Per Chromosome overwrite if File exists Figure 52 SNP Genotype Report Parameter Panel Purpose To set the format and storage location for SNP Genotype Reports generated in the Workflow To open In the Analysis Method Navigator for CGH under Reports select SNP Genotype Report Output Format Select one of these options e Complete Genome Creates a single report file e Per Chromosome Creates a separate report file for each chromosome Select File Displays the location where the workflow saves the files To select a Location location for the report click Browse An Open dialog box appears Type a name and select a location for the report then click Open Overwrite if file If you select this option the workflow deletes an existing file if it has the exists same name and location as a generated report Workflow User Guide 167 4 Workflow Reference Variance Stabilization Variance Stabilization Description Variance Stabilization is an alternative to Lowess normalization that fits a regression curve to signal intensities after applying an asinh x transform to each channel This approach uses a two
83. entralization algorithm see the CGH Interactive Analysis User Guide All of the aberration algorithms can use the Centralization calculation used to normalize data Centralization is recommended for SNP Copy Number and LOH analyses Workflow User Guide Workflow Reference 4 Aberration The options in the Aberration folder let you select the aberration detection algorithm for the workflow For a detailed discussion of all aberration detection algorithms see the CGH Interactive Analysis User Guide Select one of these options Option Description Z Score The Z Score algorithm is a quick method of detecting aberrant regions It assesses intervals using a sliding window of fixed size and is especially useful in the exploratory phase of CGH data analysis When you select this option the Z Score Parameter Panel appears where you can set the parameters of the algorithm See Z Score Parameter Panel on page 172 For a discussion of the Z Score algorithm see the CGH Interactive Analysis User Guide ADM 1 The ADM 1 algorithm searches for intervals in which the average log ratio of the sample and reference channels exceed a threshold that you specify When you select this option the ADM 1 Parameter Panel appears where you can set the parameters of the algorithm See ADM 1 Parameter Panel on page 126 For a discussion of the ADM 1 algorithm see the CGH Interactive Analysis User Guide ADM 2 The ADM 2 algorithm is similar
84. ere you configure the report and type a name and select a location for it Probe Based Penetrance Summary Report Parameter Panel on page 159 Cyto reports summarize analysis settings and detected aberrations by array The workflow creates a separate PDF file for each array When you select this option the Cyto Report Parameter Panel appears where you select the desired cyto report template to use anda location for the report See Cyto Report Parameter Panel on page 136 Note You create Cyto Report templates in the Reports tab See the CGH Interactive Analysis User Guide Also when you generate a Cyto Report in a workflow the program uses the data analysis settings from the workflow not from the selected Cyto Report template The program uses only the format settings from the template The CNVR report contains a list of the copy number variant regions CNVRs detected by the analysis saved as an xls file The report also contains a list of the parameters of the analysis You can open this file in Microsoft Excel When you select this option the CNVR Report Parameter Panel appears where you can select a location for the report You also provide the name for the CNVR node that appears in the results folder of the output experiment generated by the workflow See CNVR Report Parameter Panel on page 135 Workflow User Guide 107 4 108 Workflow Reference Table 15 Report selections for CGH Repo
85. erence 4 Lookin MicroArray a FeatureExtraction File name E Program Files Agilent Micro4rray Files of type Feature Extraction Output Folder Cancel Figure 70 Open dialog box Purpose To select the files you want to open or to identify the location where you want files to be stored To open This dialog box appears when you click Add at the bottom of the Image Files parameters panel This also appears when you click Browse in a parameter panel or dialog box Select folder where the files are located To browse for a location click the arrow and browse to the desired folder Type the name for the file you want to open or click the file to select it To select multiple files to open hold down the Ctrl key and click the files to open Expected file type is displayed Click to change displayed file types Click open the open the Add Image Pack Information dialog box See Import FE Image Files Parameter Panel on page 149 189 4 Workflow Reference Cancel Click this to cancel the operation Provide Workflow Identifier Provide Workflow Identifier Please provide name to identify this workflow Cancel Figure 71 Provide Workflow Identifier dialog box Purpose To designate a name used in the Summary Console to identify a workflow to be run To open This dialog box appears when you click Run to run a workflow Provideanameto Type a name for the workflow progress tab identify this workflow OK
86. es Centralization is recommended for SNP Copy Number and LOH analyses CGH Aberration Summary Report Parameter Panel Report Type Workflow User Guide Figure 25 CGH Aberration Report Parameter Panel Purpose This parameter panel lets you configure the CGH Aberration Summary Report for CGH and select a location for it This report describes regions that have detected aberrations You can report these regions by genomic interval by probe or both The program reports aberrations separately for each array in the workflow and creates one or more xls files that you can work with in Microsoft Excel To open The CGH Aberration Report Parameter Panel appears when you select CGH Aberration Report in Reports in the CGH Workflow Navigator Configures the organization of reported aberrations Select one of these options Option Description Probe Based Creates a report that contains one line for each probe showing an aberration Interval Based Creates a report that contains one line for each aberrant genomic interval Probe amp Interval Creates both a probe based report and an interval based report Based 133 4 Workflow Reference Output Format Select one of these options Option Description Complete Genome Creates a single report file for each requested report type Per Chromosome Available only for probe based reports Creates a separate report file for each chromosome Select File Displays the loca
87. es displayed in the window Click the arrow to change the type of files displayed Open Click to save the report file Cancel Click to cancel the operation Workflow User Guide 193 4 194 Workflow Reference Set Password Password Confirm Password OK Cancel Set Password x Password I Confirm Password OK Cancel Figure 75 Set Password dialog box Purpose Used to create a password for a newly created analysis method or workflow or to type the password when trying to open a password protected analysis method or workflow To open This dialog box appears when you create a password protected analysis method or workflow or when you select a password protected analysis method or workflow Type the password you wish to use Retype the password to confirm it Click this button to accept the password and close the dialog box Click this to cancel the operation Workflow User Guide Workflow Reference 4 Set Password Workflow User Guide 195 www agilent com In this book This guide describes how to use the Workflow utility of Agilent Genomic Workbench Lite Edition 6 5 to extract image files with Agilent Feature Extraction software and or analyze data using CGH and ChIP analysis software Agilent Technologies Inc 2010 Revision A0 December 2010 G3800 90033 Ag Agilent Technologies
88. eted you return to the Home tab or one of the interactive analysis tabs to select the workflow experiment and display the results in the Genome Chromosome and Gene Views In the procedure described in this section you configure an analysis method to analyze CGH microarray data You set up and run the workflow Then you use the Genomic Viewer to review the result files and data generated from the workflow run See Quick start instructions for analyzing CGH data in a workflow on page 18 On the next page is a typical CGH workflow analysis For more information see Chapter 3 Setting up Workflow Analysis Methods Workflow User Guide Import or download design files Create or select a workflow Create report templates Home Reports tabs Switch to workflow view set up and run workflow Select input data Select Feature Monitor progress Extraction or not of workflow run s Select analysis Select reports Click Run Display results under newly created Click Home tab experiment Navigator Genomic Viewer Figure 5 Typical CGH Workflow analysis pathway Create analysis method Configure analysis method Fuse designs Select filters before analysis Combine replicates Select Normalization Configure CGH algorithm Configure SNP algorithm Select filter after analysis Select experiment for output Save analysis method 1 Getting Started Quick start instructions for analyzing CGH data in a work
89. etrance per Feature Rename Figure 19 Aberration Filter Parameter Panel Purpose This parameter panel is used to create or edit aberration filters Aberration filters exclude detected aberrations from the output of the workflow based on selected criteria To open The Aberration Filter Parameter Panel appears when you select Aberration Filter under Filter After Analysis in the Analysis Method Navigator Select the name of the filter you want to use To create a new aberration filter click New Opens an Input dialog box where you can type a name for the new aberration filter To accept the name click OK The program creates the filter and adds the new name to the Name list Workflow User Guide Update Reset Delete Rename Minimum number of probes in region Minimum absolute average log ratio for region Maximum number of aberrant regions Percent penetrance per feature Workflow User Guide Workflow Reference 4 Saves any changes you make to the filter criteria Restores the values of the filter criteria to what they were before you made any changes to them Opens a Confirm dialog box that asks you if you want to delete the currently selected filter To delete the filter click Yes Opens an Input dialog box where you can type a new name for the filter To accept the name click OK Type a whole number The filter excludes putative aberrant regions that contain fewer probes than the number you
90. fault the Feature Extraction program automatically determines the Protocol for the microarray Or you can select a Protocol from the list Click this button to open the Open dialog box where you can select the image file s to be added See Open on page 189 Highlight one or more microarrays and then click this button to remove them from the list To select more than one microarray hold down the Ctrl key and then click the microarrays to remove To select a contiguous series of microarrays hold down the Shift key and click the first microarray and then click the last microarray Workflow User Guide Workflow Reference 4 Import UDF Files Parameter Panel Import UDF Files Parameter Panel Description 4 tab delimited CGH data can be imported as a new custom formatted array data file The order of tabular data must follow the column headers in the Universal Data Importer Map column headers dialog box File Name udf_sample_file Design type cgh I Select species H sapiens a Select Mapping customs Design Id Custom Datatype ratio Select Genome Build hg18 1 Save Mapping As ProbeName ChrName Start LogRatio Sample2 l descrip Select ES Select ES Select ES Select rey Select xs Select re Select rey 1oiize 1000 0 0023 0 0044 p102121 i 1100 p 0022 0 1123 jai03z22 1200 0 0033 p 121 ja104343 1300 b 1125 p 123 h105122 1400 b 0123 b o112 Figure 38 Import UDF
91. fault the program also gives this name to the experiment it creates when you run the workflow 4 Click OK The program creates the new analysis method and opens the Analysis Method window The name of the new analysis method appears in the toolbar under Select Analysis Method You can now set the parameters of the new analysis method You can also create a new analysis method from the Run Analysis application pane See Run Analysis Application Panel on page 162 for more information To select an existing analysis method Workflow User Guide The current analysis method appears in the toolbar under Create Edit Analysis Method To select a different analysis method follow these steps 1 On the Workflow command ribbon click Analysis Method When Analysis Method is selected the command ribbon displays commands related to the Analysis Method 2 On the command ribbon click the arrows next to Select Analysis Method A list of existing analysis methods appears If no analysis methods appear you must create a new one See To create a new analysis method on page 69 69 3 70 Setting up Workflow Analysis Methods 3 Select the analysis method from the list You can now set the parameters for the selected analysis method To edit an existing analysis method You can edit an existing analysis method or use an existing analysis method as the basis for a new one 1 On the Workflow command ribbon click
92. flow These instructions apply when you have started the program with the CGH license installed See the CGH Interactive Analysis User Guide for information on how to install the license 18 Workflow User Guide Table 1 Steps for setting up and running a CGH workflow Getting Started 1 To do this Follow these instructions Comments Import designs and array data In order to perform Feature Extraction ina workflow e The design s for the data you want to extract must be in the Feature Extraction database To automatically add designs during workflow your eArray settings for Feature Extraction must contain a valid Username and Password and you must select Use eArray server during extraction and Check for updates of grid template in the Advanced Options of the eArray Login Settings dialog box OR Manually add the designs to the Feature Extraction program e The design s for the data you want to extract must be in the Agilent Genomic Workbench database Manually import the design s from the Home tab of Agilent Genomic Workbench OR Make sure the design file is in the workflow output path for Feature Extraction In order to perform an analysis workflow no Feature Extraction the design s for the extractions you want to analyze must be in the Agilent Genomic Workbench database Manually import the design s from the Home tab of Agilent Genomic Workbench OR Select the location path of the design
93. for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAU
94. ghbor probes blue flank a central probe red The program considers a probe bound if the average p values for all three probes is less than a set cut off value and if either of the following is true Workflow User Guide Workflow Reference 4 e The p values for the central probe and at least one of its neighbors are less than set cut off values e The p value of one or optionally another number of the neighbors of the central probe is less than a set cut off value To configure the model you set the cut off values of this significance heuristic The ChIP program lets you set the following parameters of the Whitehead per array neighbourhood model Parameter Comments Maximum distance in bp for two probes to be considered as neighbors P Xpar lt Central probe has P X lt At least one neighboring probe has P X lt At least n of the neighbors has P X lt The program only considers probes to be neighbors if their genomic locations are within this threshold distance The default value for this parameter is 1000 base pairs This parameter refers to the average p value for the central probe and its neighbors The default cut off value is 0 001 Decreasing the cut off value makes the selection more stringent The central probe is the red probe in Figure 55 The default cut off value is 0 001 e Decreasing the cut off value makes the selection more stringent e Neighbo
95. gilent com Support by telephone or 2 Select Contact Us e mail for your country 3 Under Worldwide Sales and Support Phone Assistance click to select a country and then click Go Complete e mail and telephone contact information for your country is displayed To learn about Agilent products and services To view information about the Life Sciences and Chemical Analysis products and services that are available from Agilent go to www chem agilent com 34 Workflow User Guide Agilent Genomic Workbench Lite Edition 6 5 Workflow User Guide 2 Setting Up and Running Workflows Creating and Managing Workflows 36 Setting up a Workflow for Feature Extraction 38 Setting up an Analysis Workflow 45 Running Workflows 59 This chapter gives instructions on how to set up and run workflows The first section explains how to set up a workflow using Feature Extraction software for automatic feature extraction of microarray images The second section describes how to set up a workflow for automatic analysis of data using the Agilent Genomic Workbench CGH or ChIP analysis packages The third section explains how to run a workflow and review results phe Agilent Technologies 35 2 Setting Up and Running Workflows Creating and Managing Workflows 36 In this section you learn to create edit save and delete workflows For a detailed description of the window of the Workflow tab see Main Window on page 96 and f
96. he button under Display Tab in the Summary Console row to go to the progress tab for the selected run or in the progress tabs click a named workflow tab A progress tab displays the workflow progress and any errors that occur A not yet completed workflow run has running displayed in its progress tab A completed workflow run has completed displayed in is progress tab See Workflow progress tabs on page 176 Click Close Tab in a progress tab to stop that run and remove the workflow from the list Click lt Summary Console to return to a summary table of each workflow and experiment Click Abort Workflows amp Clear Table in the Summary Console tab to stop all workflow runs and remove workflows and workflow experiments from the run list Workflow User Guide 61 2 62 Setting Up and Running Workflows To abort a single workflow 1 In the Summary Console Progress tabs pane click the workflow progress tab for the workflow to abort Click Abort Workflow You are asked to confirm that you want to abort the workflow Note that the Abort Workflow button will not appear unless the workflow is currently running The workflow is aborted and the workflow progress tab and workflow status in the Summary Console tab are removed To abort all workflows 1 In the Summary Console Progress tabs pane click the Summary Console tab Click Abort Workflows amp Clear Table You are asked to confirm that you want
97. he data CGH 78 To select aberration algorithms CGH 78 To configure the analysis method to apply the Z score algorithms 79 To configure the analysis method to apply the ADM ADM 1 or ADM 2 algorithms 79 To configure the analysis method to apply the CBS algorithm 80 To configure the analysis method to apply the HMM algorithm 80 To select to filter the results after analysis CGH 80 To select to calculate SNP Copy Number 81 To select to calculate LOH 81 To define an output experiment 82 Setting up ChIP Analysis Methods 83 To combine replicates ChIP 83 To configure the analysis method to combine intra array replicates 83 To configure the analysis method to combine interarray replicates 83 To configure normalization methods ChIP 84 To configure error model for analysis method ChIP 87 To configure peak detection and evaluation ChIP 89 To set the parameters for the Whitehead per array neighbourhood model 90 Workflow User Guide 7 Contents To set the parameters of the modified Whitehead per array neighbourhood model 91 To set the parameters for the predefined peak shape detection algorithm v2 1 92 4 Workflow Reference 95 Main Window 96 Workflow Command Ribbons 98 Command ribbon for Workflow 98 Command ribbon for Analysis Method 100 Workflow Navigators 102 Workflow Navigators for CGH and ChIP 102 Input 103 Feature Extraction 106 Reports 106 Analysis Method Navigator 110 Analysis Method Navigator for CGH 110 Fu
98. ias intra array This kind of normalization corrects for dye bias within each array in the Median workflow and it normalizes only the intensities of the IP channel See the ChIP Interactive Analysis User Guide for more information If you select this type of normalization the parameters for it appear in the parameter panel Use this tab to select the way the program calculates the dye bias and also how the program calculates central tendencies See Dye bias Intra Array Median Normalization on page 139 Inter Array Median This kind of normalization corrects for variations from one replicate array to another The program calculates and applies the normalization separately for each channel It first calculates the median signal intensity over the common probes in each replicate array It then finds the average of these median intensities over all replicates of all arrays For each array it computes the ratio of its median signal intensity to the average of the median signal intensities of all arrays Finally it normalizes data by multiplying each signal intensity by the applicable ratio See Inter Array Median Normalization on page 154 118 Workflow User Guide Workflow User Guide Workflow Reference 4 Option Description Intra Array Lowess Intra array normalization attempts to correct for artifacts caused by Intensity Dependent nonlinear rates of dye incorporation as well as inconsistencies in the relative fluores
99. ick Fuse Design Select whether you want to normalize the data Select if you want to remove the arrays from the experiment after the designs are combined When you set up a workflow and click Select Imported Data remember to select the designs to be fused In this step you combine two design files into a larger file when the same sample has been hybridized to multiple designs Arrays from the same design and already fused designs cannot be fused Select to use filters before analysis 1 In the Analysis Method Navigator under Filter Before Analysis select one or more of the check boxes Design Level Filter Feature Level Filter or Array Level Filter Select DefaultFeatureFilter or another one from the list or create a new one Select a filter from the Array Filter list or create a new one When you apply a Design Level probe filter you include or exclude probes based on design filter conditions When you apply an Array filter microarrays that fail the filter criteria are not included in the evaluation When you apply a Feature Level filter features from the array that fail the criteria are not included in the evaluation To create a new filter see instructions in the Workflow User Guide 22 Workflow User Guide Table 1 Getting Started 1 Quick start instructions for analyzing CGH data in a workflow Steps for setting up and running a CGH workflow continued To do this Foll
100. if file exists to overwrite a previous report saved as the same filename and location Aberration amp LOH To set parameters for the report Report 1 Inthe Workflow Navigator in Reports select Aberration amp LOH Report Three settings appear in the parameter panel See Aberration amp LOH Report Parameter Panel on page 123 2 Under Select File Location click Browse The Select report folder dialog box appears Select a location for the report and if necessary change the File name 4 Click Open The location of the Aberration amp LOH Report appears in the parameter panel in Report Location 5 Under Select File Location select Overwrite if file exists to overwrite a previous report saved as the same filename and location ow 56 Workflow User Guide Setting Up and Running Workflows 2 To select and configure reports ChIP When you run a workflow the program can produce three different reports that present the analysis method s experimental results See Table 6 Reports are files that contain results from the ChIP module that you can open with other programs e In the Analysis Method Navigator in Reports select the reports to produce In the parameter panel set the parameters for each report See Table 6 for a description of the available reports and instructions on how to set the parameter s for each Table6 ChIP module reports Report Description Instructions Probe Report This report contains inf
101. igh amplitude coefficients which mark occurrences of true breakpoints rather than noise in the data Workflow User Guide Workflow Reference 4 Import Data Files Parameter Panel Import Data Files Parameter Panel Description If the Reference Sample is in Red channel for a CGH SNP array please select flipped from the Dye Flip drop down during import Please specify the reference sample in Red Channel using Sample Manager or Microarray Properties Genomic Workbench will create a new array node in the data section of the navigator in interactive mode The new node will have the name of the imported file However you can use this dialog to edit the file name s Additionally you can specify if an array is dye flipped In this case the ratios will be inverted but dye flip pairs will not be automatically combined Global Display Name Dye Flip Green Sample Red Sample Design Name Design Build Design Status 15235024 18_251470410096_501_CGH v4_91 Normal ppis704 fois Already Present 522502637 _251713010006_S01_H_CGH_105_ Normal prizo I E iJ Not Found overwrite arrays with duplicate names Remove f Add Designs Figure 36 Import Data Files Parameter Panel Purpose This panel lets you configure the workflow to import Agilent Feature Extraction FE array data files To open The Import Data Files Parameter Panel appears when you select Import FE Files in Input in the Workflow Navigator You must import representativ
102. ighbourhood model 1 In the Analysis Method Navigator in Peak Detection and Evaluation click Whitehead Per Array Neighbourhood Model Modified Parameters for the model appear in the parameter panel Set any of the parameters in the parameter panel Default values appear for each parameter but you can change them Refer to Table 8 for descriptions of each parameter Table 8 Parameters for the Whitehead per array neighbourhood model modified Parameter Comments Maximum distance in bp for two probes to be considered as neighbors A probe is considered bound if P Xbar lt Central probe has P X lt At least one neighboring probe has P X lt At least n of the neighbors has P X lt The program only considers probes to be neighbors if their genomic locations are within this threshold distance The default value for this parameter is 1000 base pairs To change the value delete the old value and type a new one in the box This parameter refers to the p value of the average error corrected ratio X of the central probe and its left and right neighbors The bar indicates the average The default value is 0 001 To make detection more stringent decrease the value The central probe is the red probe in Figure 10 The default value is 0 001 To make detection more stringent decrease the value Neighboring probes are probes to either side of the central probe The blue probes in Fig
103. ill be created and can be further analyzed from interactive Analysis Method ears Analysis method selection i B Fuse Design E Filter Before Analysis i 5 Design Level Filter i B Feature Level Filter t B Array Level Filter Experiment Name KF1 Combining Replicates B Intra Array Replicates uto created Experiment From workflow B Inter Array Replicates Normalization Experiment Description B GC Correction B Centralization Aberration O z Score ADM 1 aDmM 2 ces i O HMM 24 SNP Algorithm z F F SNP Copy Number Analysis Method Navigator Parameter panel for selected item MoH 9 Filter After Analysis 5 Aberration Filter 2g Output Experiment Figure 9 Analysis Method window for CGH Workflow User Guide Setting up Workflow Analysis Methods 3 To create a new analysis method An analysis method is a group of settings that the program uses to run an analysis in Workflow Before you run an analysis method you must create a new one or select an existing one see To select an existing analysis method on page 69 1 On the Workflow command ribbon click Analysis Method When Analysis Method is selected the command ribbon displays commands related to the Analysis Method 2 On the Analysis Method command ribbon click New The Create Analysis Method dialog box appears See Create Analysis Method on page 179 3 Type a name for the new analysis method By de
104. import status box will appear Click OK 65 2 Setting Up and Running Workflows To import a workflow 66 Workflow User Guide Agilent Genomic Workbench Lite Edition 6 5 Workflow User Guide eece 3 Setting up Workflow Analysis Methods Setting up an Analysis Method 68 Setting up CGH Analysis Methods 73 Setting up ChIP Analysis Methods 83 This chapter describes how to create analysis methods to use in the workflow An analysis method is a list of actions and parameters to use for an analysis workflow The analysis method was called the workflow in DNA Analytics 4 0 You can save analysis methods and apply the same method to many different sets of data Use workflows to analyze data and generate results and then review the results in Genomic Viewer After you set up and run your workflow you see the results graphically with chromosomal aberrations or binding events correlated with genes and chromosomal locations The workflow also produces report files that you can analyze further with a spreadsheet or other analysis program a Agilent Technologies 67 3 Setting up Workflow Analysis Methods Setting up an Analysis Method 68 Analysis methods are created edited and saved from the Analysis Method window This window appears when you create or edit an analysis method Analysis Method Experiment Parameter Panel Description When workflow run is complete an experiment with specified name w
105. ings parameter panel click Browse A Select report folder dialog box appears Select a location for the report and if desired change the File name 4 Click Open The location of the QC Report appears in Report Location ow Workflow User Guide Setting Up and Running Workflows 2 Running Workflows When you run a workflow the selected analysis Feature Extraction CGH ChIP application analysis or both is run on the selected input files To run a workflow 1 Create a new workflow or select one from the list See To create a new workflow on page 36 or To edit an existing workflow on page 36 On the Workflow ribbon click Run 3 At the prompt type a name into the Provide Workflow Identifier dialog box and then click OK This name becomes the name of the progress tab in the Summary Console Progress tabs pane The default name is the name of the workflow Typing a new name does not change the name of the experiment or the workflow See Provide Workflow Identifier on page 190 An experiment is created in the Experiments pane of the Home tab Navigator The experiment folder lists the Experiment Name you specified when you set up the analysis method Otherwise the experiment has the name of the Analysis Method used for the workflow Feature Extraction only workflows do not create experiments The experiment folder is marked with a W while the workflow is running The Summary Console tab at the
106. interpret them Instead you select the most appropriate label for each column from the lists The rest of the main data table contains the first few data lines of the file to help you identify the columns properly You must use all of the labels exactly once except LogRatio which you can use more than once Read only The name of the UDF file to be imported Read only A unique identifier for the design If Custom appears here the workflow creates the necessary design ID s after it imports the files Select the application type that best represents the data either cgh or expression Select the mathematical relationship that best exemplifies the data The following options appear in the list e ratio linear ratio e logs ratio binary log ratio logio ratio common log ratio e In ratio natural log ratio base e Select the species appropriate to data in the UDF file Select the genome build appropriate to the data in the UDF file The choices available depend on the species you select in Select Species Select a saved column mapping if desired To create a new mapping select CUSTOM Workflow User Guide Save Mapping As Workflow Reference 4 Opens an Input dialog box where you can type a name for the current column map This lets you save the current column map for future use This is especially useful if you want to import many UDF files that have columns arranged in the same manner Inter Array Replicates P
107. ion process more stringent To apply a nesting level filter select Apply Nesting Filter then type a whole number from 0 to 2147483647 in the box The ADM 1 aberration detection algorithm reports nested aberrations that is aberrations within other aberrations If you set the nesting level to 0 the program reports only the parent aberration without any child nested aberrations If you set it to 1 the programs reports the first level of child Workflow User Guide Workflow Reference 4 aberrations By default the program sets the nesting level filter to its maximum value which essentially applies no filter To make the filter more stringent decrease the value Fuzzy Zero Select Fuzzy Zero to apply Fuzzy Zero correction to detected aberrant intervals This correction applies a global error model to the intervals and can result in fewer errors in aberration calls For more information on the fuzzy zero algorithm see the CGH Interactive Analysis User Guide ADM 2 Parameter Panel ADM 2 Parameter Panel Description ADM2 Aberration detection method By default Nesting Level is set to maximum integer value 2147483647 which means no nesting level filter will be applied You can set it to any desired integer value between 1 and 2147483647 Threshold Nesting Level F o Wipe ng Filter s Fuzzy Zero 0 1 fds E 147483647 Figure 21 ADM 2 Parameter Panel Purpose This parameter panel lets you set th
108. ion where you want to save the report file Type the name of the file to save Or click to select the file from the displayed files Shows the type of files displayed in the window Click the arrow to change the type of files displayed Click to save the report file Click to cancel the operation Workflow User Guide Workflow Reference 4 Select Report Name Select report name Lookin Agilent_sample_mamt_demo QCReport_Graphs a US22502637_251713010C 251713010006_501_H_GE2_105_Dec08_1_1 pdf US22502637_251713010006_S01_H_GE2_105_DecO8_1_2 pdF a US22502637_251713010006_S01_H_GE2_105_DecO8_1_3 pdf a US22502637_251713010006_S01_H_GE2_105_DecO8_1_4 pdf a US22502637_251713010006_S01_H_GE2_105_DecO8_2_1 pdf a US22502637_251713010006_S01_H_GE2_105_Dec08_2_2 pdf a US22502637_251713010006_S01_H_GE2_105_Dec08_2_3 pdf Filename 0522502637_251713010006_S01_H_GE2_105_DecO8_1_1 pdF Files of type PDF File a Cancel Figure 74 Select report name dialog box Purpose Used to select the folder location and name to store a CGH report To open From any of the Cyto CNVR SNP Genotype or Aberration amp LOH Reports parameter panels click Browse Click the arrow and select the folder or browse to the location where you want to save the report file Filename Type the name of the file you wish to save Or click to select the file from the displayed files Files of type Shows the type of fil
109. ith signal intensity Normally it is computed for you but you can also provide it here instead 5 Custom defined f value Figure 54 Whitehead Error Model Purpose This parameter panel lets you customize the advanced parameters of the Whitehead error model Set parameters to optimize the statistical calculations of the error model using training data specific to your particular assay To open This parameter panel appears when you select Whitehead Error Model under Error Model in the ChIP Analysis Method Navigator See To configure error model for analysis method ChIP on page 87 Source of additive The choices for this parameter change the additive intensity dependent intensity depen component of the estimate of the error in IP WCE Select one of these dent errorin sources each channel is e Standard deviation of background pixels e Additive error as computed by Agilent Feature Extractor e Observed spread of negative controls Custom defined Select this check box to define a custom f value In the box to the right f value type an f value The f value of one replicate of an array is the rate at which the multiplicative error increases with signal intensity By default the ChIP program calculates f values automatically Workflow User Guide 169 4 170 Workflow Reference Whitehead Per Array Neighbourhood Model Whitehead Per Array Neighbourhood Model Description eto ar gt a Genomic Locatio
110. kflow you must also select Feature Extraction whether or not you choose to do a CGH or ChIP analysis The options in the Reports folder let you select the reports that are created by the workflow The reports contain one or more files that you can open in other programs such as Microsoft Excel or Adobe Reader Select any of these options Workflow User Guide Workflow Reference 4 Table 15 Report selections for CGH Report CGH Aberration Report Probe Based Penetrance Summary Report Cyto Report CNVR Report Description The CGH Aberration Report describes regions that have detected aberrations You can report these regions by genomic interval by probe or both The program reports aberrations separately for each array in the workflow and creates one or more xls files that you can work with in Microsoft Excel When you select this option the CGH Aberration Report Parameter Panel appears where you can configure the report and type a name and select a location for it See Variance Stabilization on page 168 The Probe Based Penetrance Summary Report lists each probe with a significant aberration and gives the percentage of selected arrays that show a significant deletion or amplification in the region for each probe The workflow creates one or more xls files that you can work with in Microsoft Excel When you select this option the Probe Based Penetrance Summary Report Parameter Panel appears wh
111. kflow at a time with a Type a name for the workflow 32 bit computer but they will run Click OK to start the workflow one right after the other not simultaneously Workflow User Guide 27 1 Getting Started Quick start instructions for analyzing CGH data in a workflow Table 1 Steps for setting up and running a CGH workflow continued To do this Follow these instructions Comments f Summary Console My CGH Exp WorkFlaw st gt Workflow Progress Running combine Repiicates inter Array Monitor progress of workflow run Array Level Filters NONE Executing Step Creating Experiment Running Analysis on following samples Setting recite The Summary Console tab shows alist of all the runs you have started and their status 1 Click the Home tab E In the Navigator double click the newly created workflow experiment and click Yes when asked if you want to select this experiment s Expand the Results folder under the experiment Note that the WF result label is also in blue to indicate this set of results is active 4 Select a chromosome in Genome View that appears to have a significant number of aberrations Chr 4 was selected in figure below 5 Move the blue cursor in Chromosome View to a region of interest for display in Gene View 6 Inthe Home command ribbon click Save Experiment Result and click Yes then OK Review and save results 3 1 U522502705_251469814934_50
112. kflow that imports data files This field displays the status of the design file e Not Found The design file is not currently in the program or a path has not been selected e Already Present The design file was imported from the Home tab of Agilent Genomic Workbench e Path Provided The design file was not imported but a location was selected for the file If you select this option the workflow deletes an existing array if it has the same name as one you import For a complete description of what happens when you import FE data files see the Sample Manager User Guide Removes files from the list of files to be imported To remove a file from the list click its name then click Remove Opens an Open dialog box where you can select a data file to import The file you select appears in the list of arrays to import Workflow User Guide Figure 37 Add Designs Workflow Reference This option is enabled after you add an array to the list that does not have an associated design present in the program When you select this an Open dialog box appears where you can select a design file for the arrays in the list After you select the design Path Provided appears under Design Status for the array Import FE Image Files Parameter Panel Import FE Image Files Parameter Panel Description Import Feature Extraction images here and associate samples to them Image Name US23502418_252152910035 Global Display N
113. l appears when you select Cyto Report in Reports in the CGH Workflow Navigator Select Report Select the desired Cyto Report template You create and edit Cyto Report templates in the Reports tab See the CGH Interactive Analysis User Guide for more information Also when you generate a Cyto Report in a workflow the program uses the data analysis settings from the workflow not from the selected Cyto Report template The program uses only the format settings from the template Select File Click Browse The Select report folder dialog box appears where you can Location type a name and select a location for the Cyto Report file s The workflow creates a new folder that contains all of the files Overwrite if file If you select this option the workflow deletes an existing file if it has the 136 exists same name and location as the generated Cyto Report Workflow User Guide Workflow Reference 4 Design Level Filter Parameter Panel Design Level Filter Parameter Panel Description You can select an already existing filter From the drop down below or can create a new filter Edit Design Level Filters Name New laa Attribute Operator Logical Ope New Condition Homology Delete Condition include matching values Ob xclude matching values Update Reset Figure 28 Design Level Filter Parameter Panel Purpose This parameter panel is used to create or edit design filters that let you include or exclude
114. le 7 Parameters for the Whitehead per array neighbourhood model Parameter Comments Maximum distance in bp for two probes to be considered as neighbors P Xbar lt Central probe has P X lt At least one neighboring probe has P X lt At least n of the neighbors has P X lt The program only considers probes to be neighbors if their genomic locations are within this threshold distance The default value for this parameter is 1000 base pairs To change the value delete the old value and type a new one in the box This parameter refers to the p value of the average error corrected ratio X of the central probe and its left and right neighbors The bar indicates the average The default value is 0 001 To make detection more stringent decrease the value The central probe is the red probe in Figure 10 The default value is 0 001 To make detection more stringent decrease the value Neighboring probes are probes to either side of the central probe The blue probes in Figure 10 are the neighbors of the central red probe The default value is 0 1 To make detection more stringent decrease the value The default value for nis 1 The default cut off value for P X is 0 005 To make detection more stringent decrease the value 90 Workflow User Guide Setting up Workflow Analysis Methods 3 To set the parameters of the modified Whitehead per array ne
115. lect UDF data files for the workflow to import You can configure a workflow to import custom tab delimited UDF data files and to use them as the workflow input This input option is only available when CGH is selected as the application 1 In the Workflow Navigator in Input click Import UDF Files The Import UDF Data Files Parameter Panel appears See Import UDF Files Parameter Panel on page 151 2 In the parameter panel click Add An Open dialog box appears 3 Select the file s to import then click Open 4 Agilent Genomic Workbench attempts to use information in the UDF file to set the data and design type parameters For more information see the CGH Interactive Analysis User Guide Make any necessary changes to these parameters Workflow User Guide Setting Up and Running Workflows 2 5 If necessary select the proper column correlation from the drop down lists in the data display view Alternatively select a predefined correlation using the Select Mapping drop down box 6 Optionally click Save Mapping As to save the correlated column fields in Agilent Genomic Workbench for future import of UDF data files To use previously imported data as the workflow input When you configure a workflow you can use array data that you have previously imported into Agilent Genomic Workbench as the source of data for the workflow This applies to both CGH and ChIP analysis types 1 In the Workflow Navigator under Input cli
116. location where your Feature Extraction software is installed To open On the Workflow Command Ribbon click Feature Extraction Preferences FE location Enter the path where the Feature Extraction software is installed Browse Click to browse for the folder where the Feature Extraction software is installed OK Click to accept the displayed location and close the dialog box Cancel Click to cancel the operation Apply Click to apply the displayed location but keep the dialog box open 184 Workflow User Guide Workflow Reference 4 Feature Extraction Properties Feature Extraction Properties on Propertie FE Property Default Value Output JPEG File Output Grid File Output Visual Results Tab Text output Type Compact Project Default Protocol Output QC Report File Overwrite Previous Results MAGE output Type Save Figure 66 Feature Extraction Properties dialog box Purpose To use Workflow to display and change the parameters for Feature Extraction To open In the Run Workflow Navigator make sure image files are already open click Feature Extraction and then in the Output path for FE output parameter panel click FE default parameters being used This dialog box displays the current FE parameters To change a parameter click the field next to the parameter and select the new value Click Save to save any changes and close the dialog box Workflow Use
117. low to calculate results for CGH arrays that contain SNP probes Select one or both of the following options Option Description SNP Copy Number LOH For each SNP site that is represented on the array SNP Copy Number ASCN algorithm calculates an expectation value for the copy number of the uncut SNP allele See SNP Copy Number Parameter Panel on page 166 For more information on the SNP Copy Number algorithm see the CGH Interactive Analysis User Guide Note In order to select SNP Copy Number you must first select an Aberration except z score or HMM LOH algorithm identifies copy neutral genomic regions with a statistically significant scarcity of heterozygous SNP calls The algorithm reports the regions where the LOH score exceeds a definable threshold See LOH Parameter Panel on page 156 For more information on the LOH algorithm see the CGH Interactive Analysis User Guide Note In order to select LOH you must first select SNP Copy Number Workflow User Guide Workflow User Guide Workflow Reference 4 Filter After Analysis Aberration filters exclude certain detected aberrations from the output of the workflow based on specific criteria To apply an aberration filter to the results of the workflow select Aberration Filter The Aberration Filter Parameter Panel appears where you can select a filter See Aberration Filter Parameter Panel on page 124 You create and edit aberration fil
118. lt FeatureFilter removes saturated and nonuniform features See the CGH Interactive Analysis User Guide for more information Array level filters include or exclude arrays from an experiment based on the values of specific array attributes It is sometimes useful to filter arrays based on the parameters used in array preparation For example if you set a minimum degree of labeling in either channel this can help produce better results Agilent Genomic Workbench allows feature and array attribute criteria to be applied during analysis See the CGH Interactive Analysis User Guide for more information By default all probes are present in an experiment unique and non unique The design level filter lets you filter out probes based on specified design filters such as homology or probe score See the CGH Interactive Analysis User Guide for more information To configure the analysis method to apply a design level filter 1 In the Analysis Method Navigator in Filter Before Analysis select Design Level Filter The Design Level Filter Parameter Panel appears See Design Level Filter Parameter Panel on page 137 2 Do one of the following e To create a new filter click New In the dialog box that appears type a name for your new filter then click OK e To edit an existing filter select its name The name of the filter and its conditions appear in the dialog box 3 Change the conditions of the filter To restore changed values to
119. me and location CNVR Report To set parameters for the report 1 Inthe Workflow Navigator in Reports select CNVR Report Three settings appear in the parameter panel See CNVR Report Parameter Panel on page 135 2 In CNVR Node Name type a node name under which to access the CNVR report in interactive mode 3 Under Select File Location click Browse The Select report folder dialog box appears 4 Select a location for the report and if necessary change the File name 5 Click Open The location of the CNVR Report appears in the parameter panel in Report Location 6 Under Select File Location select Overwrite if file exists to overwrite a previous report saved as the same filename and location 55 2 Setting Up and Running Workflows Table5 CGH module reports continued Report Description Instructions SNP Genotype To set parameters for the report Report 1 Inthe Workflow Navigator in Reports select SNP Genotype Report Three settings appear in the parameter panel See SNP Genotype Report Parameter Panel on page 167 2 In Output Format select to format the report on a Full Genome or Per Chromosome basis 3 Under Select File Location click Browse The Select report folder dialog box appears 4 Select a location for the report and if necessary change the File name 5 Click Open The location of the SNP Genotype Report appears in the parameter panel in Report Location 6 Under Select File Location select Overwrite
120. ms are available to provide detection of contiguous aberrant regions See the CGH Interactive Analysis User Guide for more information To select an aberration algorithm e In the Analysis Method Navigator in Aberration select the algorithm to use for the analysis Workflow User Guide Setting up Workflow Analysis Methods 3 To configure the analysis method to apply the Z score algorithms 1 In the Analysis Method Navigator in Aberration select Z score The Z score Parameter Panel appears See Z Score Parameter Panel on page 172 In Window select the size of the sliding window from the drop down list The probe abundance is calculated within the selected size for the genomic region Note that this is a fixed value in the Z score algorithm For more information see the CGH Interactive Analysis User Guide In Threshold type or select with the green slider the parameter used to calculate whether a region is statistically different from a log ratio value of 0 To configure the analysis method to apply the ADM ADM 1 or ADM 2 algorithms Workflow User Guide 1 In the Analysis Method Navigator in Aberration select either ADM 1 or ADM 2 See the CGH Interactive Analysis User Guide for more information The ADM 1 or ADM 2 Parameter Panel appears See ADM 1 Parameter Panel on page 126 and ADM 2 Parameter Panel on page 127 In Threshold type or select with the green slider the parameter used to calculate whe
121. n Settings dialog box You cannot do automatic design template upload from a workflow for CGH SNP custom designs You must first import these designs with the Home gt Import gt Design Files gt GEML file command You must import or download the design template manually in Feature Extraction before you run the workflow if e The eArray settings in Feature Extraction are blank or e If you entered an eArray Username and Password but did not select Use eArray server during extraction and Check for updates of grid template in the Advanced Options of the eArray Login Settings dialog box e If you use a design that is not in eArray For information on the eArray Login Settings in Feature Extraction see the Feature Extraction User Guide Workflow User Guide Setting Up and Running Workflows 2 To set the location of the Feature Extraction software In order for Workflow to automate Feature Extraction you need to tell it where your Feature Extraction software is installed Feature Extraction software must be version 10 7 or higher to work with Agilent Genomic Workbench Lite Edition 1 From the Genomic Workbench menu bar click Workflow On the command ribbon click Feature Extraction Preferences The Feature Extraction Preferences dialog box opens See Feature Extraction Preferences on page 184 2 Type the location where your Feature Extraction software is installed To browse for the location click Browse and sear
122. n bp The values x and Xbar are computed from spot intensities and error estimates In order for a genomic region to be identified as a peak these values must satisfy the thresholds specified here Xbar represents the average x value over three consecutive probes close to each other Maximum distance in bp For two probes to be considered as neighbors fi ooo 4 probe is considered bound if P X lt p 001 AND EITHER Central probe red probe above has P X lt 001 At least one neighboring probe blue has P x lt pa OR At least fi of the neighbors blue has P x lt p 005 Figure 55 Whitehead Per Array Neighbourhood Model Purpose This parameter panel is used to configure the Whitehead per array neighbourhood model for peak detection in ChIP workflow analysis To open This parameter panel is displayed when you click Whitehead Per Array Neighbourhood Model under Peak Detection and Evaluation in the ChIP Analysis Method Navigator See To configure peak detection and evaluation ChIP on page 89 The ChIP program uses the Whitehead per array neighbourhood model to make binding calls This model considers the p values of both the probe in question and its neighbors You can customize the parameters of the model including the maximum distance between neighbor probes and the stringency of the detection process The model considers probes in groups of three as shown at the bottom of Figure 55 Two nei
123. n Select Experiment Experiment Select an experiment from the list The program uses the arrays from the selected experiment as input for the workflow The program creates a new experiment during the workflow and does not change the selected experiment You create experiments in the Genomic Viewer See the Data Viewing Guide for information on how to use the Genomic Viewer Workflow User Guide 163 4 Workflow Reference Select Imported Data Parameter Panel Select Imported Data Parameter Panel Description Select what data you would like to include in the analysis by highlighting arrays in the left column and clicking gt to move them into the right column A new experiment will be created with the selected arrays Select Design Select Genome Build 014698 hg18 Array List Selected Array List 0522502705 _251469814934_501 _CGH v4_95_Feb07_1_1 U522502705_251469814934_501_CGH v4_95 _Feb07_1_2 lu522502705_251469814935_S01_CGH v4_95_Feb07_1_1 lS22502705_251469814935_S01_CGH v4_95_Feb07_1_2 Figure 49 Select Imported Data Parameter Panel Purpose This parameter panel lets you select previously imported arrays as input for the workflow To open The Select Imported Data Parameter Panel appears when you select Select Imported Data under Input in the Workflow Navigator Select Design Displays the array designs available in Agilent Genomic Workbench Select the design from the list The arrays for the design appear in the
124. n a workflow first import them in Interactive mode For more information see the CGH User Guide Then in Workflow mode use the Select Imported Data option in the Workflow Navigator to select them Import UDF Files Configures the workflow to import UDF microarray data files When you select this option the Import UDF Data Files Parameter Panel appears Use this pane to select UDF data files for import and to identify their columns See Import UDF Files Parameter Panel on page 151 Select Imported Configures the workflow to use microarray data that you have previously Data imported into the CGH application When you select this option the Select Imported Data Parameter Panel appears Use this tab to select previously imported arrays for the workflow See Select Imported Data Parameter Panel on page 164 Workflow User Guide 103 4 104 Workflow Reference Table 13 Input for CGH Option Description Select Experiment Image Files Configures the workflow to use microarray data from an existing CGH experiment When you select this option the Select Experiment Parameter Panel appears in the Parameters tab Use this tab to select the desired experiment See Experiment Parameter Panel on page 140 When you run the workflow the program creates a new experiment with the same name as the workflow The original experiment is unchanged To customize the name of the new experiment click Experiment in the Ou
125. n algorithm for CGH The Z Score algorithm is a quick method of detecting aberrant regions It calculates intervals using a sliding window of fixed size and is especially useful in the exploratory phase of CGH data analysis For more information on the Z Score algorithm see the CGH Interactive Analysis User Guide To open The Z Score Parameter Panel appears when you Select Z Score under Aberration in the Analysis Method Navigator The size of the sliding window You can type a window size in Kb or Mb for example 2 Mb You can also select a value from the list Alternatively you can specify the window size in terms of data points pt This sets the window size to the specified number of contiguous data points Type the desired number of data points for example 30 pt or select a value from the list The minimum Z Score for the Z Score algorithm to consider a region aberrant Type a value from 0 1 to 50 or use the slider to set the value In general to increase the stringency of aberration detection increase the threshold value 173 4 Workflow Reference Summary Console Progress Tabs In Workflow mode the Summary Console and Workflow Progress Tabs provide information on the status of each workflow you run The Summary Console tab displays the workflows you run and the status of each Named workflow progress tabs show information about each single workflow run including the analysis settings used and a running log of progres
126. ng filter select its name The name of the filter and its conditions appear in the dialog box 3 Change the conditions of the filter To restore changed values to their original values click Reset 4 Click Update to save the filter Workflow User Guide 75 3 76 Setting up Workflow Analysis Methods To create or modify an array level filter 1 In the Analysis Method Navigator in Filter Before Analysis select Array Level Filter The Array Level Filter Parameter Panel appears See Array Level Filter Parameter Panel on page 129 2 Do one of the following e To create a new filter click New In the dialog box that appears type a name for your new filter then click OK e To edit an existing filter select its name The name of the filter and its conditions appear in the dialog box 3 Change the conditions of the filter as desired To restore changed values to their original values click Reset 4 Click Update to save the filter To combine replicates CGH You can configure an analysis method to combine intra array or interarray replicate probes or both This increases the statistical power of your analysis For a discussion of the statistical model Agilent Genomic Workbench uses to combine replicates see the CGH Interactive Analysis User Guide Intra array replicates are features within the same array that contain the same probe Interarray replicates are features on different arrays that contain the s
127. nt Name See Experiment Parameter Panel on page 140 5 In Experiment Name type a new name for the experiment 6 In the command ribbon click Save 7 In the command ribbon click Run gt The program runs the workflow Workflow status is displayed in the Summary Console tab during the run In addition the program highlights each step of the workflow as it occurs Both the data and results appear automatically in Genome Chromosome and Gene views and in the Data tab in Tab View See To display the results of a workflow on page 63 To display the results of a workflow After you configure a workflow and run it the results are displayed in the interactive tabs of Agilent Genomic Workbench Preprocessing Analysis Discovery Reports View Tool Workflow User Guide 63 2 64 Setting Up and Running Workflows e Switch to one of the interactive tabs to review the results that are generated by the completed workflows The workflow results appear as a node under the experiment created during the workflow run This experiment has the letter W on the folder during the run to let you know this experiment and its results were generated during a workflow run The W is not displayed after the workflow run is complete In addition you can use other programs to open the reports created by the workflow Use a spreadsheet program to display and further analyze xls report files Text Aberration and Text Penetration Summary
128. od Navigator in Normalization select the kind s of normalization you want the program to apply when it runs the workflow You can select either FE output or any combination of the others See the table below for a description of the method choices and instructions on how to set the specific parameter s for each Normalization method Description Instructions FE Output Blank subtraction By default the program uses the raw unprocessed feature intensity data from the output files of your feature extraction program If you use Agilent Feature Extraction you can use the processed intensity data from the output file instead No additional parameters are necessary Array normalization within Agilent Genomic Workbench and from FE output are not compatible If you select the FE Output option the other options are unavailable If you select any of the other normalizations the FE Output option is unavailable This kind of normalization corrects for non specific binding It first calculates the central tendency of the negative controls on the array for both the immunoprecipitated IP and whole cell extract WCE channels It then subtracts these central tendencies from the raw signal intensities of each feature on the array In the Analysis Method Navigator in Normalization select the check box next to Blank Subtraction The Median method is used to estimate central tendency of blank probes No other selection is possible
129. of how to use a workflow to automate feature extraction and data analysis You must have a Feature Extraction 10 7 or higher license and a CGH or ChIP license to run a feature extraction workflow To run a Feature Extraction workflow on CGH SNP microarrays you must have Feature Extraction 10 10 or higher You must have a CGH and or ChIP license to run an analysis workflow For information on how to run a SureSelect Target Enrichment workflow see the SureSelect Target Enrichment User Guide For details on how to activate a license see the Product Overview Guide or the User Guide for your analysis application ott Agilent Technologies 11 1 Getting Started Starting the Workflow Program This section describes how to open the Workflow program in Agilent Genomic Workbench and shows what the program window looks like To start the Workflow program 1 To open Workflow in the Open Application pane click the Workflow icon 2 See Figure 1 Genomic Viewer Application Type CGH 4 gt Open Application Product Overview Q Sample Manager Management console that enables easy association of meta data patient sample Help information with individual slides which is carried through the analysis process A DNA Analytics CGH Module Analysis application designed to examine data generated from CGH experiments License Help 2 Workflow Workflow utility that extracts image files with Agilent Feature Extraction and or Hel
130. og ratios to 1 This method multiplies the signal intensities of all data probes on the array by a correction factor This correction factor adjusts the central tendency of log ratios of the data probes on the array to 1 Note If Dye bias intra array Median normalization is selected Variance Stabilization normalization and Intra array Lowess normalization are not available Workflow User Guide 85 3 Setting up Workflow Analysis Methods Normalization method Description Instructions Intra Array Lowess Intra array normalization attempts to correct for artifacts caused by Intensity Dependent nonlinear rates of dye incorporation as well as inconsistencies in the relative fluorescence intensity between some red and green dyes The Lowess normalization algorithm normalizes the channels within each array using a nonlinear polynomial fit to the data and effectively normalizes by probes and by arrays Note If you are using feature extraction data that have been normalized by the Lowess approach you do not need to apply the intra array Lowess normalization here 1 Inthe Analysis Method Navigator in Normalization select Intra Array Lowess Intensity dependent Normalization 2 Inthe parameter panel select the data to be used to compute the regression curve Note If Intra array Lowess normalization is selected Dye bias intra array Median normalization and Variance Stabilization are not available Variance Stabilization
131. option see To use previously imported data as the workflow input on page 51 CGH microarray data from an existing Agilent Genomic Workbench experiment For instructions on how to set the parameters for this option see To use an experiment s arrays as the workflow input on page 52 Table 4 Workflow Input for ChIP Analysis Option Description Import Data Files Extracted microarray data that you have not yet imported into Agilent Genomic Workbench For instructions on how to set the parameters for this option see To select data files for the workflow to import on page 52 48 Workflow User Guide Setting Up and Running Workflows 2 Option Description Select Imported Data ChIP microarray data that you have previously imported into Agilent Genomic Workbench For instructions on how to set the parameters for this option see To use previously imported data as the workflow input on page 51 Select Experiment ChIP microarray data from an existing Agilent Genomic Workbench experiment For instructions on how to set the parameters for this option see To use an experiment s arrays as the workflow input on page 52 To select FE data files for the workflow to import Workflow User Guide When you do CGH analysis you can configure a workflow to import Feature Extraction data files and use them as the workflow input The design files for the extractions must be in Agilen
132. or probes and the stringency of the detection process The models consider probes in groups of three shown in Figure 10 In this figure two neighbor probes blue are located before and after a central probe red Distance between lt gt neigh ors bp Binding Signal P amp P xb Genomic Location bp Figure 10 Central probe flanked by two neighbor probes The program accepts the probe as bound if the p value of the composite error corrected ratio X of all three probes X is less than a set cut off value and if either of the following is true e The p values for the central probe and at least one of its neighbors are less than set cut off values e The p value of one or optionally another number of the neighbors of the central probe is less than a set cut off value For a detailed description of the statistical calculations involved in event detection see the ChIP Interactive Analysis User Guide Workflow User Guide 89 3 Setting up Workflow Analysis Methods To set the parameters for the Whitehead per array neighbourhood model 1 In the Analysis Method Navigator in Peak Detection and Evaluation click Whitehead Per Array Neighbourhood Model Parameters for the model appear in the parameter panel 2 Set any of the parameters in the parameter panel Default values appear for each parameter but you can change them Refer to Table 7 for descriptions of each parameter Tab
133. or the command ribbons of the Workflow tab see Workflow Command Ribbons on page 98 To create a new workflow 1 Start the Workflow program See To start the Workflow program on page 12 Click New The Create Workflow dialog box appears See Create Workflow on page 180 In Enter Workflow Name type a name If you intend to restrict access to this workflow select Apply Password Click OK If you selected the Apply Password check box the Set Password dialog box appears See Set Password on page 194 Type a password and click OK Set up the workflow See Setting up a Workflow for Feature Extraction on page 38 or Setting up an Analysis Workflow on page 45 Click Save To edit an existing workflow 1 Next to the Select Workflow list click the right arrow 2 Select a workflow name from the list 3 Edit the workflow See Setting up a Workflow for Feature Extraction on page 38 or Setting up an Analysis Workflow on page 45 Workflow User Guide Setting Up and Running Workflows 2 You can also create a new workflow from a selected one by saving it to another name See To save a workflow to a new name on page 37 To save a workflow e In the Workflow command ribbon click Save To save a workflow to a new name 1 Next to the Select Workflow list click the right arrow 2 Select a workflow name from the list 3 In the command ribbon click Save As
134. ormation about the probes in the current experimental result in tab separated value tsv format A probe report contains one row for each probe in the array or array set The program generates a separate file for each array See the ChIP Interactive Analysis User Guide for a description of the columns in the report You can display probe reports and perform further analysis on them with a spreadsheet program To set parameters for the Probe Report 1 Inthe Analysis Method Navigator in Reports select the box next to Probe Report 2 Inthe Probe Report Settings parameter panel click Browse A Select report folder dialog box appears Select a location for the report and if desired change the File name 4 Click Open The location of the Probe Report appears in Report Location ow Workflow User Guide 57 2 58 Setting Up and Running Workflows Table 6 ChIP module reports continued Report Description Instructions Gene Report ChIP QC Report This report contains information about the genes in the current experimental result in tab separated value tsv format It contains one row for each probe in the array or array set grouped by the genes to which the probes bind The program generates a separate file for each array It also includes loci represented by probes on the array that are not associated with genes The program creates the Gene Report in several formats See the ChIP Interactive Analysis User G
135. ort ChIP OC Report The gene report contains one row for each probe in an array grouped by the genes to which the probes bind It is a tab separated value tsv file that you open and analyze further with a spreadsheet program For multiple arrays the program creates a separate tsv file for each array The program creates gene reports in several formats See the ChIP Interactive Analysis User Guide for a description of the columns in each If you select this option parameters for the report appear in the parameter panel You can select a location for the report and customize its content See Gene Report Settings on page 145 The QC report summarizes the settings of the current analysis and the overall statistics of each array In addition to summary tables it also includes plots that summarize the data graphically The program creates the QC report in HTML format and you can display the report with your Internet browser For more information about the contents of the report see the ChIP Interactive Analysis User Guide If you select this option a parameter for the report appears in the parameter panel Select a location for the report See OC Report Settings on page 161 109 4 Workflow Reference Analysis Method Navigator This section describes the options for CGH analysis methods in the order in which they appear in the Analysis Method Navigator Analysis Method Navigator for CGH In Workflow mode
136. ow these instructions Comments Set up to combine replicates 1 In the Analysis Method Navigator under Combining Replicates select any of these options Intra Array Replicates Combines replicate probes within arrays Inter Array Replicates Combines replicate probes from multiple arrays that are marked as replicate arrays using one of the available attributes If you select the Inter Array option in the Parameters tab select an array attribute next to Group By For interarray replicates the program combines replicate arrays into groups by the value for the attribute you select in Group By Values must be the same If your array s contain probes that are replicated in the array you can combine them to increase the confidence of your analysis When the program combines replicates it selects probes with common probe names and calculates a weighted average of their values to create a single point If the probes are from arrays with the same polarity the algorithm used to combine replicates calculates a weighted average of the probes with the same name Otherwise it calculates a straight average Normalize the data To correct for artifacts by performing a regression fit to GC content ina specified region flanking the probes select GC Correction To normalize the data so that zero represents the most common ploidy select the Centralization check box GC Correction is recommended
137. ox appears where you type a password for the workflow See Set Password on page 194 180 Workflow User Guide Workflow Reference 4 Export Lookin C Agilent_sample_mgmt_demo za QCReport_Graphs 017130_D_F_20070627 xml E CGH Analysis Methods xml a Test2 xml File name JEGH2 xml Files of type XML EA Export Cancel Figure 62 Export dialog box Purpose Used to designate a location and file name to export analysis methods To open This dialog box opens when you click OK from the Export Analysis Method s dialog box Use the buttons at the top of the dialog box to change the display Lookin Click the arrow and select the folder where you wish to export the file Filename Type the name you wish to use for the exported file Files of type Shows the type of files displayed in the window Click the arrow to change the type of files displayed Export Click to export the currently selected analysis methods to the file Cancel Click to cancel the operation Workflow User Guide 181 4 Workflow Reference Export Analysis Method s Export Analysis Method s Select analysis method s to export Select All Deselect All Figure 63 Export Analysis Method s dialog box Purpose Used to select analysis methods for export To open On the command ribbon click Export Analysis Method s A list of analysis methods currently saved in the program appears Click the box next to an analy
138. p analyze data with CGH applications Figure 1 Open Application pane OR At the top of the Agilent Genomic Workbench window click the Workflow tab 12 Workflow User Guide Getting Started 1 Figure 2 Home Sample Manager Workflow Preprocessing Analysis Discovery Reports View Tool Help Workflow tab The Workflow window appears with the Workflow Navigator displayed on the left side of the window Agilent Genomic Workbench Lite Edition 65 CGH I l Home Sample Manager Create Edit Workflow workFlow Select Workflow Oanalysis Method EHI j Import FE Files Import UDF Files 8 Select Imported Data 3 Select Experiment O Image Files 9 Extraction F Feature Extraction fA CGH Aberration Report amp Probe Based Penetrance Sumr E CNR Report amp SNP Genotype Report Workflow Navigator Workflow Preprocessing Analysis Discovery Reports View Iool Help Switch Application Y 3 Command Ribbon Switch Application gt Workflow paeo Run gt Delete ox Save As we B Apply password Run Analysis Application Description Run a DNA Analysis Application Analysis Application DNA Analytics Application Type CGH Analysis Method CGH Parameter Panel mary Console CGH1 Status Application Type I Complete CGH Workflow Name I CGH Analysis Method il CGH1 Experiment Name Current Step CGH1 Summary Console
139. pair to be fused can be specified by setting the same value For the ArraySet Attribute Arrays from same design and already fused design cannot be fused Select Normalization None xa fHremove arrays from experiment after fuse Figure 32 Fuse Design Parameter Panel Purpose This parameter panel lets you select to have the workflow merge arrays from two different array designs into a larger virtual design This can make it easier to work with arrays that are part of an array set See the CGH Interactive Analysis User Guide for a description of the requirements that arrays must meet for you to fuse them To open The Fuse Design Parameter Panel appears when you select Fuse Design under Fuse in the Analysis Method Navigator In order to fuse designs the ArraySet microarray attribute for each array to be fused must have the same value The ArraySet attribute is entered in the microarray properties from one of the interactive tabs When you run the workflow the program fuses all of the arrays that have the same value for the ArraySet attribute Select Select to normalize the data The workflow always uses the Centralization Normalization algorithm to normalize the data in fused arrays For more information on this algorithm see the CGH Interactive Analysis User Guide Workflow User Guide 143 4 Workflow Reference Remove arrays If you select this option the workflow deletes the original individual from experiment arrays after i
140. parameter error model to compress the reported ratios of probes with weak signals after blank subtraction After the transform is applied the variance of the reported log ratios should be independent of the signal strength The additive component of the error model is estimated from the deviation of intenskies among negative control probes The Regression curve is fitted to atdsta robes FY Figure 53 Variance Stabilization Purpose This parameter panel is used to configure the regression curve for the variance stabilization method of normalization for ChIP workflow analysis To open This parameter panel is displayed when you select Variance Stabilization under Normalization in the ChIP Analysis Method Navigator Regression curve Select the data to use for the regression curve isfittedto All data probes Includes all of the data probes in the regression curve e All common probes Includes probes whose names start with LACO e Gene desert probes Includes data for probes whose names start with LACC GD 168 Workflow User Guide Workflow Reference 4 Whitehead Error Model Whitehead Error Model Description If selected per probe error estimates will be calculated as per Whitehead Error Model method Source of additivetintensity dependent error of each channel is Standard deviation of background pixels A The f value of one replicate of an array is the rate at which the multiplicative error increases w
141. py Number for SNP Algorithm GC Correction and Centralization are recommended In the Analysis Method Navigator in SNP Algorithm select LOH The LOH Parameter Panel appears See LOH Parameter Panel on page 156 In Threshold type a value to use for the confidence level in the calculation For more information see the CGH Interactive Analysis User Guide 81 3 82 Setting up Workflow Analysis Methods To define an output experiment When you run a workflow the program creates an Agilent Genomic Workbench experiment that you can display in interactive mode 1 In the Analysis Method Navigator in Output click Experiment Two parameters appear in the parameter panel See Select Experiment Parameter Panel on page 163 By default the program gives the experiment the same name as the analysis method 2 You can type a new name for the experiment The name must not already exist in Agilent Genomic Workbench 3 In Description type an optional description for the experiment Workflow User Guide Setting up Workflow Analysis Methods 3 Setting up ChIP Analysis Methods This section describes how to set up analysis methods for ChIP analysis with a workflow To combine replicates ChIP You can configure a workflow to combine intra array or interarray replicate probes or both This increases the statistical power of your analysis For an explanation of the statistical model Agilent Genomic Workbench uses to com
142. r Guide 185 4 186 Workflow Reference Import Select All Deselect All 0K Cancel Select All Deselect All OK Cancel Figure 67 Import dialog box Purpose Used to select the analysis method s or workflow s to be imported from a selected workflow or analysis method file To open This dialog box appears when you select a file and click Import from the Import Workflow s or Import Analysis Method s dialog box A list of workflows or analysis methods in the selected import file is shown Click to select the workflows or analysis methods to be imported or use the buttons at the bottom of the dialog box Selects all displayed workflows or analysis methods Clears the selection from all workflows or analysis methods Once one or more workflows or analysis methods is selected click OK to import the workflows or analysis methods A status box that shows the status of the imported workflows or analysis methods is displayed Cancels the import operation and closes the dialog box Workflow User Guide Workflow Reference 4 isis Method s Import Analysis Method s Look in File name Files of type Import Workflow User Guide Import Analysis Method s Lookin Genomic Data BE Analysis Methods C Image Files L Reports File name Files of type xml Import Cancel Figure 68 Import Analysis Method s dialog box Purpose Used to select an analysis method file to be imported into the
143. reports CNVR report Probe and Gene reports Use Adobe Reader to display pdf format report files Cyto reports Use an Internet browser to display the QC report To export a workflow To save workflow in a file on your hard disk you must export it To export a workflow you must have one or more workflows saved in the program 1 On the command ribbon under Create Edit Analysis Method click Workflow 2 On the command ribbon click Export Workflow s A selection menu appears 3 To export workflow select Workflow s 4 In the Export Workflow s dialog box click to select the workflow s you wish to export See Export Workflow s on page 183 for more information The Export dialog box appears See Export on page 181 5 In the Export dialog box go to the location where you want to save the file and type the file name 6 Click Export Workflow User Guide To impor Workflow User Guide Setting Up and Running Workflows 2 ta workflow If you have exported one or more workflows and you want to import them into the program 1 On the command ribbon click Import Workflow s The Import Workflow s dialog box appears See Import Workflow s on page 188 2 Browse to the location where the file is located click to select it and then click Import The Import dialog box appears See Import on page 186 3 Click to select the workflow s to import 4 Click OK to import the workflow s 5 An
144. ring probes are probes to either side of the central probe The blue probes in Figure 55 are the neighbors of the central red probe e The default cut off value is 0 1 Decreasing the cut off value makes the selection more stringent The default value for n is 1 e The default cut off value for P X is 0 005 Decreasing the cut off value makes the selection more stringent Workflow User Guide 171 4 Workflow Reference Whitehead Per Array Neighbourhood Model Modified This model is exactly like the unmodified model except that in the modified model the number of neighbors includes the probe itself so the default value for n is 2 instead of 1 Purpose This dialog box lets you customize the parameters of the Whitehead per array neighbourhood model The ChIP application uses this model to make binding calls based on the p values of each probe and its neighbors To open This parameter panel is displayed when you click Whitehead Per Array Neighbourhood Model Modified under Peak Detection and Evaluation in the ChIP Analysis Method Navigator See To configure peak detection and evaluation ChIP on page 89 Z Score Parameter Panel zZ Score Parameter Panel Description Window Threshold Figure 56 Z Score Parameter Panel 172 Workflow User Guide Window Threshold Workflow User Guide Workflow Reference 4 Purpose This parameter panel lets you configure the Z Score aberration detectio
145. rt Description SNP Genotype Report Aberration amp LOH Report The SNP Genotype Report contains genotype and p values for SNP probes It generates reports in xls format for the entire genome or for each chromosome When you select this box the SNP Genotype Report Parameter Panel appears where you select the format and a location for the report See SNP Genotype Report Parameter Panel on page 167 The Aberration amp LOH Report contains aberration and log ratio information for significant intervals When you select this box the Aberration amp LOH Report Parameter Panel appears where you select the location for the report See Aberration amp LOH Report Parameter Panel on page 123 Table 16 Report selections for ChIP Option Description Probe Report The Probe Report contains information in tab separated value tsv about the probes in the workflow s arrays You can use a spreadsheet program to open this file A Probe Report contains one row for each probe in the array or array set See the ChIP Interactive Analysis User Guide for a description of the columns in the report If you select this option parameters for the report appear in the parameter panel Select a location for the report See Probe Report Settings on page 160 Workflow User Guide Workflow User Guide Workflow Reference Table 16 Report selections for ChIP 4 Option Description Gene Rep
146. rt FE Image Files Parameter Panel To correlate sample attributes when you add image files to the workflow if the Array ID is already in Sample Manager 1 In the Import FE Image Files Parameter Panel click the Sample ID lt Red Green Array ID gt field for a microarray 2 Click be and then select the correct Array ID from the list See the Sample Manager User Guide for more information 43 2 Setting Up and Running Workflows To set the output path for Feature Extraction results Once you have opened an image file and the images are displayed in the images list you must select a location where you want the FE output results to be saved 1 In the Navigator pane of the Workflow tab click the Extraction folder to open it 2 Click the Feature Extraction box to select it A check mark indicates Feature Extraction is selected 3 In the Set output path for FE File output pane type the path where you want the program to save results See Set Output Path for Feature Extraction Panel on page 165 4 Or click Browse and find the location to save results Click Save To run a Feature Extraction workflow you must have a license for Feature Extraction 10 7 or higher installed on your computer To run Feature Extraction for CGH SNP microarrays you must have Feature extraction 10 10 or higher installed on your computer To display or change the default FE parameters The Feature Extraction parameters that are used for automa
147. s e Probes should be spaced fewer than 300 bp apart depending on your shear distribution Table9 Parameters for predefined peak shape detection algorithm v2 1 Parameter Comments Thresholds p value threshold Maximum threshold for the nonparametric test for reporting peaks Value must be greater than 1 number of randomized runs Increase the value to find more peaks Score threshold Minimum threshold for extreme value distribution EVD based score Decrease value to find more peaks Peak Shape Parameters Estimated mean shear length Type a mean to be used in the gamma distribution calculation distribution of sample DNA for approximation of the distribution of sheared DNA fragments Workflow User Guide Table 9 Setting up Workflow Analysis Methods 3 Parameters for predefined peak shape detection algorithm v2 1 Parameter Comments Estimated standard deviation of the shear length distribution of sample DNA Other Algorithmic Parameters Precision of peak placement on the chromosome in base pairs Number of randomizations for determination of peak significance via nonparametric test and score Window size in bp for computing local baseline Desired spacing of interpolated data points between probe Automatically re run calculation after learning peak shape Use errors estimated by Error model Type a standard deviation to be used in the gamma distribution calculation of the di
148. s for setting up and running a CGH workflow continued Getting Started 1 To do this Follow these instructions Comments Create select an analysis method 1 On the Workflow command ribbon The name you type for the analysis under Create Edit Analysis Method method appears in the selection list click Analysis Method under Create Edit Analysis Method 2 Click New to create a new analysis e You can create more than one method analysis method for use in multiple A dialog box appears workflows 3 Type a Name for the analysis method then click Ok The Analysis Method window opens and the Experiment Parameter Panel contains a place to change the name of the experiment that is created By default the experiment name is the name of the analysis method 4 Type a name to change the name of the Experiment Experiment Parameter Parel Oesergtion When workflow nun is complete an experment vith peched name wil be created and can be hather anahyzed trom nteractive mode 7 Experiment Kome Expenment Desenpton To resed Enpero from wor iow Workflow User Guide 21 1 Getting Started Quick start instructions for analyzing CGH data in a workflow Table 1 Steps for setting up and running a CGH workflow continued To do this Follow these instructions Comments Set up to fuse designs In the Analysis Method Navigator under Fuse cl
149. s through the workflow Summary Console tab Summary Console FE_Hu22K FE_Hu22K1 f CGH_9_9 SNP Workflow1 if WFSNP3 WFSNP44 1 FE 2 FE 3 cah 4 cah 5 CGH No Application Type Status I Display Tab Failed Complete Workflow Name Experiment Name WorkFlowB FE_Hu22k CGH_9_9 SINPWorkFlowt WRSNPS Analysis Method Current Step es ta ba ba Workflow Workflow2_SNP SNP99 Workflow Complete Workflowt WorkFlowt Complete Complete 6 CGH eer WRSNP44 Workflow 5NP144 Complete Refresh Status Abort Workflows amp Clear Table No Application Type 174 Figure 57 Summary Console tab The Summary Console tab displays the workflows you run and gives basic identifying information and the workflow status It also lets you manage the workflows The Summary Console and workflow tabs include workflows for CGH ChIP and SureSelect Target Enrichment For information on SureSelect Target Enrichment workflows see the SureSelect Target Enrichment User Guide Read only The order in which the workflows were started from first to most recent Read only The basic application type CGH or ChIP for example for the workflow Workflow User Guide Workflow Name Analysis Method Experiment Name Status Current Step Display Tab Refresh Status Abort Workflows amp Clear Table KI Workflow User Guid
150. same probe Interarray replicates are features on different arrays that contain the same probe For expression arrays replicate probes are probes correlated with the same gene When you combine replicates you define how the program handles replicate probes The program can combine multiple biological and technical replicates within and among arrays Select any of these options Option Description Intra Array Replicates Inter Array Replicates Combines replicate probes within each array If you select this option the Intra Array Replicates Parameter Panel appears However no input parameters are required Combines replicate probes within designated groups of arrays When you select this option the Inter Array Replicates Parameter Panel appears Use this parameter panel to select the array attribute the program uses to group arrays when it combines interarray replicates See Inter Array Replicates Parameter Panel on page 153 Normalization Select any of these options Option Description GC Correction Centralization Corrects for artifacts by performing a regression fit to GC content in a specified region flanking the probes GC Correction is recommended for SNP Copy Number and LOH analyses Centralization recenters log ratio values It finds a constant value to subtract from or add to all values and ensures that the zero point reflects the most common ploidy state For a description of the c
151. se Design 111 Filter Before Analysis 111 Combining Replicates 112 Normalization 112 Aberration 113 SNP Algorithm 114 Filter After Analysis 115 Output 115 Analysis Method Navigator for ChIP 116 Combining Replicates 117 Normalization 117 Error Model 119 Peak Detection and Evaluation 120 Predefined peak shape detection algorithm 121 Output 121 8 Workflow User Guide Workflow User Guide Contents Parameter Panels 123 Aberration amp LOH Report Parameter Panel 123 Aberration Filter Parameter Panel 124 ADM 1 Parameter Panel 126 ADM 2 Parameter Panel 127 Array Level Filter Parameter Panel 129 Blank Subtraction Normalization 131 Centralization Parameter Panel 132 CGH Aberration Summary Report Parameter Panel 133 CNVR Report Parameter Panel 135 Cyto Report Parameter Panel 136 Design Level Filter Parameter Panel 137 Dye bias Intra Array Median Normalization 139 Experiment Parameter Panel 140 Feature Level Filter Parameter Panel 141 Fuse Design Parameter Panel 143 GC Correction Parameter Panel 144 Gene Report Settings 145 HMM Parameter Panel 146 Import Data Files Parameter Panel 147 Import FE Image Files Parameter Panel 149 Import UDF Files Parameter Panel 151 Inter Array Replicates Parameter Panel 153 Inter Array Median Normalization 154 Intra Array Lowess Intensity Dependent Normalization 155 LOH Parameter Panel 156 Predefined peak shape detection v2 1 157 Thresholds 157 Peak Shape Parameters 158 Other Algorithmic Parameters
152. sis Application DNA Analytics Application Type CGH Analysis Method KM_CGH3 ca New Edit Figure 47 Workflow Run Analysis Application panel Purpose This panel is used to display the analysis application and application type and to select create or edit the analysis method to use for the workflow To open This panel is displayed when Run Analysis is selected from Analysis in the Workflow Navigator for either CGH or ChIP analysis Analysis Method Click the arrows to select the Analysis Method to run in the workflow New Opens the Create Analysis Method dialog box where you type a name for the new analysis method The Analysis Method window then opens where you can create and save the new analysis method for the workflow Edit Opens the Analysis Method window where you can edit the selected analysis method 162 Workflow User Guide Workflow Reference 4 Select Experiment Parameter Panel Select Experiment Parameter Panel Description A new experiment will be created using the arrays in the experiment you select below Select Experiment Select Experiment ka Figure 48 Select Experiment Parameter Panel Purpose This parameter panel lets you configure the workflow to use the arrays from a CGH or ChIP experiment as input To open The Select Experiment Parameter Panel appears when you select Select Experiment under Input in the Workflow Navigator Select The names of the available experiments appear i
153. sis method to select it for export or use the buttons as described below Select All Selects all displayed analysis methods Deselect All Clears the selection from all analysis methods OK Once one or more analysis method is selected click OK to export those analysis methods to a file The Export dialog box is opened Cancel Cancels the export operation and closes the dialog box 182 Workflow User Guide Workflow Reference 4 Export Workflow s Select All Deselect All 0K Cancel Export workflow s Select workflows to export 5 pd_02Nov Select All Deselect All Cancel Figure 64 Export Workflow s dialog box Purpose This dialog box is used to select workflows for export To open On the command ribbon click Export Workflow s A list of workflows currently saved in the program appears Click the box next to a workflow to select it for export or use the buttons as described below Selects all displayed workflows Clears the selection from all workflows Once one or more workflow is selected click OK to export those workflows to a file The Export dialog box is opened Cancels the export operation and closes the dialog box Workflow User Guide 183 4 Workflow Reference Feature Extraction Preferences Feature Extraction Preferences Feature Extraction installed location folder FE Location l Browse Figure 65 Feature Extraction Preferences dialog box Purpose Used to designate the
154. ssay Follow these steps a In the Analysis Method Navigator select the check box next to Whitehead Error Model Parameters for the Whitehead Error Model appear in the parameter panel See Whitehead Error Model on page 169 b Set any of these parameters described below 87 3 Setting up Workflow Analysis Methods Parameter Comments Instructions Source of additive The choices for this parameter change the additive intensity dependent error of intensity dependent component of the estimate of the error each channel is in IP WCE Select one of these sources from the list Standard deviation of background pixels e Additive error as computed by Agilent Feature Extractor Observed spread of negative controls Custom defined f value The f value of one replicate of an array is the rate at which the multiplicative error increases with signal intensity Normally the ChIP module calculates f values automatically but you can define a custom value 1 Select Custom defined f value 2 Inthe box type the desired f value 88 Workflow User Guide Setting up Workflow Analysis Methods 3 To configure peak detection and evaluation ChIP The ChIP module uses two versions of the Whitehead Per Array Neighbourhood Model to make binding calls These models consider the p values of both the probe in question and its neighbors You can customize the parameters of the models that include the maximum distance between neighb
155. stribution of sheared DNA fragments This is the window within which the algorithm searches for potential positions for the peak center When you decrease this window the time it takes for the algorithm to run increases The program computes p value and peak score through a number of random samplings Increase the number of samples to increase the accuracy of the prediction however this also increases the time to do the calculation Use smaller number for smaller genomes Must be less than or equal to probe spacing on the array Selection increases accuracy but will double the runtime Select to use the estimated error for each probe to weight its contribution to the peak fit measurement Workflow User Guide To define output experiment ChIP When you run a workflow the program creates an Agilent Genomic Workbench experiment that you can display in interactive mode 1 In the Analysis Method Navigator in Output click Experiment Two parameters appear in the parameter panel See Select Experiment Parameter Panel on page 163 By default the program names the experiment with the name of analysis method 2 If desired type a new name for the experiment The name must not already exist in Agilent Genomic Workbench 3 In Description type an optional description to associate with the experiment 93 3 Setting up Workflow Analysis Methods To set the parameters for the predefined peak shape detection algorithm v
156. sword b a K e Op Extraction az J Preferences F Run Analysis Application Description Import FE Files Run a DNA Analysis Application O Import UDF Files 3 Select Imported Data O Select Experiment 9 Extraction B Feature Extraction Analysis Application DNA Analytics Application Type tun Analysis Method E Reports wv CGH Aberration Report B Probe Based Penetrance Sumr m 8 Cyto Report E cwe Report B SNP Genotype Report p Aberration amp LOH Report Summary Console cGH i Application Type Workflow Name Analysis Method Experiment Name Status Current Step CGH CGH1 CGH CGH1 Complete Refresh Status Abort Workflows amp Clear Table Figure 8 Workflow Window showing Run Analysis Workflow User Guide 45 2 46 Setting Up and Running Workflows To use Workflow for automatic analysis of data using CGH or ChIP you first create and save an analysis method that specifies all the parameters to use for the analysis For more information on how to create an analysis method see Setting up an Analysis Method on page 68 You then set up the analysis workflow as described below and then run the workflow To run an analysis workflow you must have a CGH and or ChIP application license installed To set up the Run Analysis To use a workflow to run a CGH or ChIP analysis you must select an analysis method 1 In the Workflow Run Navigator in the Anal
157. t Genomic Workbench database before you run the workflow For more information on how to import files see the CGH Interactive Analysis User Guide This input option is only available when CGH is selected as the application 1 In the Workflow Navigator under Input click the Import FE Files option The Import Data Files Parameter Panel appears See Import Data Files Parameter Panel on page 147 2 In the parameter panel click Add Arrays An Open dialog box appears 3 Select the file to import then click Open The array appears in the parameter panel You can add as many files as you want however all files must use the same genome build You can remove existing arrays from the program with the same names as the ones you import To enable this option select Overwrite arrays with duplicate names 49 2 50 Setting Up and Running Workflows To remove an array from the list in the parameter panel click its name then click Remove 4 In Dye Flip select either Normal or Flipped for each array Select Normal if e The test samples were labeled with cyanine 5 red e The control samples were labeled with cyanine 3 green e The imported ratio test control should be reported directly Select Flipped if e The test samples were labeled with cyanine 3 green e The control samples were labeled with cyanine 5 red e The imported ratio control test should be reported with the ratio inverted test control To se
158. t creates the fused array This minimizes the duplication of afterfuse data within the experiment GC Correction Parameter Panel GC Correction Parameter Panel Description GC Correction Description Window Size 2Kb Figure 33 GC Correction parameter panel Purpose To select the window size to use for GC content correction To open This panel appears when you click GC Correction under Normalization in the CGH Analysis Method Navigator Window Size Select a window size from the drop down menu of choices 144 Workflow User Guide Workflow Reference 4 Gene Report Settings Gene Report Settings Description This will generate a Gene report for peak detection results at the specified location IF experiment contains more than one array then reports would be created at the location in a Folder with provided name Show only gene names Show probe information Report Location Browse Workflow User Guide FA show only gene names amp Show probe information Report Location Pilacw Data Figure 34 Gene Report Settings Purpose This parameter panel lets you configure and select a location for the ChIP gene report To open These parameters appear when you click Gene Report under Reports in the ChIP Workflow Navigator If you select this check box the resulting gene report contains only accession numbers of genes or chromosomal locations for probe loci not associated with genes A mark in this check box
159. ta Results FEResults Browse F output location same as Image FE default parameters being used Figure 50 Workflow Output path for FE File output Purpose This panel is used to select the location for extracted FE files To open This panel is displayed when you select Feature Extraction under Extraction in the Workflow Navigator Output path for Type the path to be used for saving FE files To search for the location Feature Extracted click the Browse button See Open on page 189 files Workflow User Guide 165 4 Workflow Reference Browse Click this button to browse for the folder where you want to save the FE files Outputlocation Select this to set the output path for extracted files to the location of the same as Image image files FE default Click this to display the Feature Extraction Properties These are the parameters being parameters that are used when you run Feature Extraction using the used Workflow See Feature Extraction Properties on page 185 SNP Copy Number Parameter Panel SNP Copy Number Parameter Panel Description SNP Copy Number Parameter Panel Description SNP Conf Level 1 95 Figure 51 SNP Copy Number Parameter Panel Purpose This panel is used to type a confidence level value to use in the SNP Copy Number calculation To open In the Analysis Method Navigator for CGH under SNP Algorithm select SNP Copy Number SNP Conf Level Type a value for the confid
160. ted feature extraction during a workflow are set using the Feature Extraction program To display or change the basic default parameters currently in use for a workflow 1 In the Navigator pane of the Workflow tab click the Extraction folder to open it 2 Click the Feature Extraction box to select it A check mark indicates Feature Extraction is selected In the Output path for FE File output parameter panel click FE default parameters being used See Feature Extraction Properties on page 185 44 Workflow User Guide Setting Up and Running Workflows 2 Setting up an Analysis Workflow Setting up an Analysis Workflow This section describes how to set up a workflow for automatic analysis using CGH or ChIP If you have the Agilent Feature Extraction software and license installed you can use image files as the input and extract the files before you run the ChIP or CGH analysis in the workflow If you do not have the Agilent Feature Extraction license installed you can use existing FE files or UDF files imported data or an existing experiment For a detailed description of the main window of the Workflow tab see Main Window on page 96 Agilent Genomic Workbench Lite Edition 6 5 CGH Home Sample Manager Workflow Preprocessing Analysis Discovery Reports View ool Help Switch Application Y Create Edit Workflow rWorkflow Q workFlow Select Workflow a Import Export Feature analysis Method SEHI we E Apply pas
161. ters in the analysis See To select to filter the results after analysis CGH on page 80 Output When you run a workflow the program creates a new experiment that you can display in the interactive tabs By default the program uses the name of the workflow as the name of the new experiment The program always creates an output experiment when it successfully runs a workflow To customize the name or description of the experiment click Experiment in the Analysis Method Navigator The Experiment Parameter Panel appears See Experiment Parameter Panel on page 140 115 4 116 Workflow Reference Analysis Method Navigator for ChIP The Analysis Method Navigator is used to set or edit the ChIP analysis method parameters As you select each option you set the parameters for it as they appear in the Parameters Panel You can select different options and change parameter settings later chip2 ks Save As Analysis Method D Data E O Combining Replicates am Intra Array Replicates EH Normalization F FE Output fA Blank Subtraction am Inter Array Median A Dye bias intra array Median am Intra Array Lowess Intensity Dependent t B Variance Stabilization E Error Model i FE Error Model t Whitehead Error Model 29 Peak Detection and Evaluation be T9 Whitehead Per Array Neighbourhood Model 6 Whitehead Per Array Neighbourhood Model Modified Pre defined Peak Shape detection v2
162. their original values click Reset 4 Click Update to save the filter 5 Click Close Workflow User Guide Setting up Workflow Analysis Methods 3 To configure the analysis method to apply a feature level filter 1 In the Analysis Method Navigator in Filter Before Analysis select Feature Level Filter The Feature Level Filter Parameter Panel appears See Feature Level Filter Parameter Panel on page 141 2 In Name select the Default Feature Filter or any filter previously defined in interactive mode or click New to create a new filter See To create or modify a feature level filter on page 75 To configure the analysis method to apply an array level filter 1 In the Analysis Method Navigator in Filter Before Analysis select Array Filter The Array Filter Parameter Panel appears See Array Level Filter Parameter Panel on page 129 2 In Name select any filter previously defined in interactive mode or click New to create a new filter See To create or modify an array level filter on page 76 To create or modify a feature level filter 1 In the Analysis Method Navigator in Filter Before Analysis select Feature Level Filter The Feature Level Filter Parameter Panel appears See Feature Level Filter Parameter Panel on page 141 2 Do one of the following e To create a new filter click New In the dialog box that appears type a name for your new filter then click OK e To edit an existi
163. ther a region is statistically different from a log ratio value of 0 In Nesting Level select the level of nesting In Nesting Level the Nesting Filter text box is enabled Type the maximum number of iterations for the ADM algorithms Click Fuzzy Zero to apply the Fuzzy Zero algorithm See the CGH Interactive Analysis User Guide for more information 79 3 80 Setting up Workflow Analysis Methods To configure the analysis method to apply the CBS algorithm e In the Analysis Method Navigator in Aberration select CBS The CBS algorithm is selected for the analysis method See the CGH Interactive Analysis User Guide for more information To configure the analysis method to apply the HMM algorithm 1 In the Worklfow Navigator in Aberration select HMM The HMM Parameter Panel appears See HMM Parameter Panel on page 146 Select the number of states and type an FDRQ value to be used THE HMM Hidden Markov Model algorithm is selected for the analysis method See the CGH Interactive Analysis User Guide for more information To select to filter the results after analysis CGH Agilent Genomic Workbench can apply a post analysis filter to aberrant regions This filter will ignore small spurious or low quality aberrations and is important for commonly aberrant region and CNVR detection For example you may want to consider only aberrations that contain three or more probes or have a minimum log ratio or hav
164. thm works see the CGH Interactive User Guide 71 3 78 Setting up Workflow Analysis Methods To select to Centralize the data CGH You normalize microarray data to correct it for known factors that cause the reported log ratios to differ from the true log ratios 1 In the Analysis Method Navigator in Normalization select Centralization The Centralization Parameter Panel appears See Centralization Parameter Panel on page 132 2 In Centralization Threshold type a value as a threshold for probes to be included in the centralization algorithm 3 In Centralization Bin Size type a value for the number of contiguous probes to be averaged together across the genome Many statistical algorithms for aberration detection use log ratio values that are centered around zero if no aberration occurs that reflects no change between the reference and sample channels In samples with a high aberration percentage this can lead to erroneous results because the measured center of the data deviates from a log value of zero The centralization algorithm finds a constant value to add to or subtract from all log ratio measurements to recenter the log ratio values This causes the zero point to reflect the most common ploidy state For a discussion of the statistical algorithms the program uses to normalize data see the CGH Interactive Analysis User Guide To select aberration algorithms CGH A variety of detection algorith
165. tion to which the workflow saves the CGH Aberration Location Report To select a location click Browse An Open dialog box appears Select a location and type a name for the report then click Open Overwrite if file If you select this option the workflow deletes an existing file if it has the exists same name and location as a generated CGH Aberration Report Report Flat Select this to have aberration intervals reported without any nested Intervals structure Generate report Select this to generate a report after each microarray is analyzed This lets perarray you look at the results for individual microarrays before the workflow is completed 134 Workflow User Guide Workflow Reference 4 CNVR Report Parameter Panel CNYR Report Parameter Panel Description Report will be created at the specified location CNVR Node Name Select File Location Overwrite if file exists Workflow User Guide pCNVR Node Name Select File Location Browse overwrite if file exists Figure 26 CNVR Report Parameter Panel Purpose You use the CNVR Report Parameter Panel to select a location for the report You also provide a name for the CNVR node that appears in the results folder of the output experiment generated by the workflow A CNVR report contains a list of the copy number variant regions CNVRs detected by an analysis saved as an xls file The report also contains a list of the parameters of the analysis You can op
166. tive Analysis User Guide 128 Workflow User Guide Workflow Reference 4 Array Level Filter Parameter Panel Array Level Filter Parameter Panel Description You can select an already existing filter From the drop down below or can create anew filter Edit Array Level Filters Name Array Filter ry Attribute Operator value Logical Oper New Condition Amt Cy3 used ug mc 0 wan e include matching values Ob Exclude matching values New Update Reset Delete Rename Figure 22 Array Level Filter Parameter Panel Purpose This parameter panel lets you create or edit array level filters An array filter excludes arrays from the workflow based on selected criteria To open The Array Filter Parameter Panel appears when you select Array Level Filter in Filter Before Analysis in the Analysis Method Navigator Name Select the name of the array filter you want to edit To create a new filter and add its name to the list click New Filter conditions Displays the conditions defined for the selected array level filter When table you create or edit the filter for each condition row select options from the lists In Value select an option from the list if available Otherwise type a value then press Enter To add another row to the table click New Condition Each condition has these elements e Attribute The array attribute evaluated by the filter Workflow User Guide 129 4 Workflow Reference
167. to abort all workflows Click Yes A warning that workflow information will be lost is displayed for each workflow Click Yes to confirm each All workflows are aborted and the Summary Console Progress tabs pane is cleared of all workflow information To run an existing workflow on a new set of data You can use an existing workflow to analyze many different sets of data For each set of data you change the input and output parameters of the workflow Workflow User Guide Setting Up and Running Workflows 2 Choice of input will depend on whether the CGH or ChIP application is selected 1 On the command ribbon in Select Analysis Method select an analysis method 2 In the Workflow Navigator in the Input folder select the source of data for the workflow See To select workflow input on page 47 3 Set the specific parameters for the selected source of data For more information see these topics e To select data files for the workflow to import on page 52 e To select FE data files for the workflow to import on page 49 e To select UDF data files for the workflow to import on page 50 e To use previously imported data as the workflow input on page 51 e To use an experiment s arrays as the workflow input on page 52 4 In the Workflow Navigator in the Output folder click Experiment In Tab View the Experiment Parameter Panel appears By default the name of the workflow appears in Experime
168. tput folder of the Workflow Navigator If you select this option you must also select Feature Extraction This option lets you select the image files to extract during the Feature Extraction workflow See Import FE Image Files Parameter Panel on page 149 Table 14 Input for ChIP Option Description Import Data Files Select Imported Data Configures the workflow to import ChIP microarray data files When you select this option the Import Data Files Parameter Panel appears in the parameter panel Use this panel to select data files for import See Import Data Files Parameter Panel on page 147 To use this option you must have already imported the representative design files into the program In Workflow mode the ChIP module supports the import of Agilent microarray data files only To use Axon files in a workflow first import them from the Genomic Viewer See the ChIP Interactive Analysis User Guide for more information Then in Workflow use the Select Imported Data option in the Workflow Navigator to select them Configures the workflow to use ChIP microarray data that you previously imported into Agilent Genomic Workbench When you select this option the Select Imported Data Parameter Panel appears in the parameter panel Use this tab to select previously imported arrays for the workflow See Select Imported Data Parameter Panel on page 164 Workflow User Guide Workflow User Guide Workflow
169. tructions CGH Aberration To set parameters for the report Report 1 Inthe Workflow Navigator in Reports select CGH Aberration Report Three settings appear in the parameter panel See Variance Stabilization on page 168 2 Under Report Type select to generate a Probe Based report an Interval Based report or both a Probe amp Interval Based report 3 Under Output Format select if the report file will contain output from the Complete Genome or if individual files will be generated Per Chromosome 4 Under Select File Location select Report Flat Intervals to have aberration intervals reported without any nested structure 5 Under Select File Location select Generate report per array to generate reports as each microarray sample is analyzed This lets you look at results for samples even though the workflow has not completed the entire analysis 6 Under Select File Location click Browse The Select report folder dialog box appears 7 Select a location for the report and if necessary change the File name 8 Click Open The location of the CGH Aberration Report appears in the parameter panel in Select File Location 9 Under Select File Location select Overwrite if file exists to overwrite a previous report saved as the same filename and location Probe Based To set parameters for the report Penetrance 1 Inthe Workflow Navigator in Reports select Probe Based Penetrance Summary Report Summary Report Two settings appear in
170. ts allele specific copy numbers for SNP probes lt LOH Detects regions that show loss of heterozygosity In order to select SNP Copy Number you must also select an aberration algorithm other than z score or HMM In order to select LOH you must first select SNP Copy Number Select filters after the analysis Under Filter After Analysis select the Aberration Filter check box Select the DefaultAberrationFilter or another filter from the list or create a new one Suggested filter for CNV analysis 2 probes 0 25 log ratio To create a new filter see instructions in the CGH Interactive Analysis User Guide 24 Workflow User Guide Getting Started 1 Quick start instructions for analyzing CGH data in a workflow Table 1 Steps for setting up and running a CGH workflow continued To do this Follow these instructions Comments 1 To change the name of the workflow By default Workflow creates an experiment under Output click experiment and gives it the name of Experiment the analysis method unless you Select experiment 2 Type the name you want to use for the change the experiment name in the f experiment analysis method or output 3 Type a description for the experiment 1 Click Save To save the analysis method with a new name click Save As and then Save the analysis type the name of the new analysis method method Workflow User Guide 25 1 Getting Started Table 1 Steps for
171. uide for a description of these formats and the columns in each You can display gene reports and perform further analysis on them with a spreadsheet program To set parameters for the Gene Report 1 Inthe Analysis Method Navigator select the box next to Gene Report Three settings appear under Gene Report Settings 2 Select one of these check boxes Show only gene names The resulting gene report contains only accession numbers of genes or chromosomal locations for probe loci not associated with genes This check box overrides the next one Show probe information The resulting Gene Report contains additional information about the probes in the array 3 Under Gene Report Settings click Browse A Select report folder dialog box appears 4 Select a location for the report and if desired change the File name 5 Click Open The location of the Gene Report appears in Report Location This report summarizes the settings of the current analysis and the overall statistics of each array In addition to summary tables it includes plots that summarize the data graphically The program creates the QC Report in HTML format and generates a separate folder for each array For more details about the contents of the report see the ChIP Interactive Analysis User Guide To set parameters for the QC Report 1 Inthe Analysis Method Navigator select the box next to ChIP QC Report One setting appears under OC Report Settings 2 Inthe OC Report Sett
172. un a workflow refer to Quick start instructions for analyzing CGH data in a workflow on page 18 Even though many of the individual settings for ChIP analysis methods are different than those for CGH analysis methods you set them up the same way and set up and run the workflow the same way On the next page is a typical ChIP workflow analysis To learn more about the individual settings for ChIP analysis methods see Setting up ChIP Analysis Methods on page 83 Workflow User Guide 31 1 Getting Started To change settings for ChIP Workflow Analysis Import data design files Y Create experiments Create report templates Create or select a Create analysis workflow method Home Reports Tabs Set up and run workflow Select input data Monitor progress Select Feature of workflow run s Extraction or not Select analysis Configure method Combine replicates optional Select normalization Select error model Peak detection amp evaluation Select reports Select experiment for output Click Run Display results under i newly created Navigator Genomic Viewer Save analysis method Figure 6 Typical ChIP Workflow analysis pathway 32 Workflow User Guide Getting Help Getting Started To get help within Agilent Genomic Workbench Help guides are opened in Adobe Reader software Agilent Genomic Workbench has several help resources 1 Help Resource Description Instructions
173. ure 10 are the neighbors of the central red probe The default value is 0 1 To make detection more stringent decrease the value The default value for n is 2 The default cut off value for P X is 0 005 To make detection more stringent decrease the value Workflow User Guide 91 3 92 Setting up Workflow Analysis Methods To set the parameters for the predefined peak shape detection algorithm v2 1 This peak detection algorithm slides a peak shape through the data and searches for good fits The peak shape is computed from the estimated mean and standard deviation of DNA lengths of the shear distribution and the significance of a potential fit is judged by comparing it to fits on randomized data with a nonparametric rank significance test For each peak that satisfies the nonparametric test a score is computed by testing the quality of the fit under the assumption of an extreme value distribution of the qualities of the fits to randomized data The significance derived from this test is converted to a score by computing log10 significance for the peak fit This algorithm assumes that e You are trying to detect relatively rare events that occur over small genomic intervals for example transcription factor binding e Genomic regions to be analyzed must be covered by stretches of several consecutive probes It will not work well with Agilent 44k Proximal Promoter 2 set design
174. workflow continued To do this Follow these instructions Comments 3 Select any of the following Reports See the CGH Interactive Analysis CGH Aberration Report Gives User Guide for more detailed overall deletion and amplification instructions and for the column Select reports tabular results along with p values formats of each report log 10 Select Probe Based or You must type a name and location Interval Based report type for all the reports Probe based Penetrance Summary If no report template exists for the Report Gives percent penetrance Cyto Report you must set up the for each probe across all the report template under Reports See selected arrays for amplification or the CGH Interactive Analysis User deletion Guide Cyto Report Gives deletion and amplification tabular and graphical results with all the parameter settings in pdf format Select a report template CNVR Report Reports the CNV regions found during analysis Type a CNVR Node Name This node will appear under the experiment in the Experiment pane You can accept this name for the report or not SNP Genotype Report Reports genotype and p values for SNP probes in the microarray Aberration amp LOH Report Reports SNP data on a per interval basis San You can run multiple workflows 3 Click the Run button Eii maximum simultaneously only on a The Provide Workflow Identifier dialog 64 bit computer You can start more box opens than one wor
175. xperiment 4 Change the name of the experiment if the named experiment exists in 5 6 7 the Experiment pane Save the workflow Click Run Repeat steps 2 5 until you are complete See To monitor workflow runs on page 61 To run a series with the same workflow 1 2 Run the first workflow Follow the instructions in To run a workflow on page 59 Click Run again Workflow User Guide 4 5 Setting Up and Running Workflows 2 An Input dialog box appears with the name of the experiment in the workflow plus an increment of 1 This will be the name of the experiment created for the second run of the same workflow You can change the name if you want Click OK The Provide Workflow Identifier dialog box appears See Provide Workflow Identifier on page 190 Enter the name for the second workflow progress tab and click OK Repeat steps 2 through 4 until you are complete See To monitor workflow runs on page 61 The Summary Console tab at the bottom of the window in the Summary Console Progress tabs pane displays the status of all the in progress or completed workflow s See Summary Console tab on page 174 To monitor workflow runs You can monitor workflow runs in the Summary Console Progress tabs pane by viewing the Summary Console tab and or the progress tabs Click Summary Console to monitor the status of all the workflows See Summary Console tab on page 174 Click t
176. xperiment select the experiment from the list To select data files for the workflow to import For ChIP analysis you can configure a workflow to import data files and use them as the workflow input Because a single workflow can process many data files the array design file s must be available in the Agilent Genomic Workbench database Array design files can only be imported from one of the interactive tabs so it may be necessary to temporarily switch tabs to load a design file for use in an analysis See the ChIP Interactive Analysis User Guide for more information on how to import design files This input option is only available when ChIP is selected as the application Workflow mode supports these microarray data files e Agilent Feature Extraction txt array files e Axon gpr array files 1 In the Workflow Navigator under Input click the Import Data Files option The Import Data Files Parameter Panel is displayed See Import Data Files Parameter Panel on page 147 2 In the parameter panel click Add Arrays An Open dialog box appears 3 Select the file to import then click Open Workflow User Guide Setting Up and Running Workflows 2 The name of the file appears in Name in the parameter panel You can add as many files as you want You can remove existing data files from the program with the same names as the ones you import To enable this option select Overwrite arrays with duplicate names To remov
177. xperiment Description Experiment Name 140 Experiment Description Figure 30 Experiment Parameter Panel Purpose This parameter panel lets you customize the name of the output experiment for the workflow and edit the description that the program saves with the experiment To open The Experiment Parameter Panel appears when you click Experiment in Output in the Analysis Method Navigator or after you create a new workflow Edit the name if desired By default the program creates a new experiment when it runs a workflow and gives the new experiment the same name as the analysis method After you run the workflow the program makes the experiment available under this name in the Navigator Experiment pane in the interactive tabs Type or edit an optional description for the experiment Workflow User Guide Workflow Reference 4 Feature Level Filter Parameter Panel Feature Level Filter Parameter Panel Description You can select an already existing filter From the drop down below or can create a new filter Edit Feature Level Filters Name Feature Attribute Operator Value Logical Oper New Condition LogRatio rIsSaturated Delete Condition include matching values OeExclude matching values New Update Reset Figure 31 Feature Level Filter Parameter Panel Purpose This parameter panel lets you select a feature level filter display a description of its filtering criteria or
178. y password Lets you type your password for a password protected workflow Workflow Run Starts the workflow Import Lets you select to import workflow s or analysis method s Export Lets you select to export workflow s or analysis method s Feature Extraction Preferences Lets you set the path for the installation folder of your Feature Extraction Software 99 4 Workflow Reference Command ribbon for Analysis Method Command ribbon for Analysis Method Create Edit Analysis Method zae IF Mp QwWorkFlow Select Analysis Method New Delete Edit mem Feature mec oo a Biter essen Fe Figure 13 Command ribbon for Analysis Method The commands in this ribbon are described in the following table 100 Workflow User Guide Workflow Reference 4 Table 12 Commands for Analysis Method command ribbon Command Purpose Workflow Analysis Method Select to change the command ribbon for Workflow or Analysis Method Select Analysis Method Shows the existing analysis methods in the program To use an existing analysis method select one from this list New Opens the Create Analysis Method dialog box To create a new analysis method type a name for the analysis method in Enter Analysis Method Name then click OK The Analysis Method window opens where you select parameters for the analysis method Delete Available only if an analysis method is selected Opens a Confirm dialog box that asks if you want to delete the
179. you use the Analysis Method Navigator to configure an analysis method The Analysis Method Navigator is displayed in the Analysis Method window that appears when you create or edit an analysis method As you select each option you set the parameters for it as they appear in specific parameter panels You can select different options and change parameter settings later Analysis Method Sy Data hn iy Fuse amp Fuse Design Filter Before Analysis i amp Design Level Filter f fj Feature Level Filter amp Array Level Fiter Combining Replicates bas 5 Intra Array Replicates am Inter Array Replicates D Normalization a TA GC Correction Seve wv Centralization Aberration Z Score H ADM 1 H aDM 2 ces O HMM SNP Algorithm SNP Copy Number m h Fiter After Analysis 5 Aberration Fiter Output ie A Experiment Figure 15 CGH Analysis Method Navigator with selections for SNP analysis 110 Workflow User Guide Workflow User Guide Workflow Reference 4 To select an analysis method option click the option button or select the check box next to its name To display the parameters for an analysis method option without changing its selection status click the name of the option Fuse Design If you have two or more arrays that use different design files you can combine them into one larger virtual array This can make it easier to work with multiple array that are part of an array set
180. ysis folder click the box to select Run Analysis The Run Analysis Application panel appears See Run Analysis Application Panel on page 162 for more information The Analysis Application is the software application that is used to analyze the data in your workflow The Application Type is CGH or ChIP depending on what application is selected To change this see To change the Application Type on page 47 Click Analysis Method and select the analysis method to use for the workflow analysis of the data If there is no existing analysis method you must create one See Step 4 below optional To edit the selected analysis method click Edit See To edit an existing analysis method on page 70 for more information optional To create a new analysis method click New See To create a new analysis method on page 69 for more information Workflow User Guide Setting Up and Running Workflows 2 To change the Application Type The application type for a workflow can be CGH or ChIP The CH3 application does not apply to Workflow The current application is displayed in the Run Analysis Application pane To change the application type 1 On the tab menu click Switch Application A pop up list appears that displays the applications 2 Click to select the application You can run a workflow for Feature Extraction without selecting any analysis application You can also run a workflow that includes both Feature Extra

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