Mark Hartnett Software Support Engineer December 7, 2001 Introduction to Analysis and Feature Extraction Software

Title of Presentation 1 March, 2001 Agilent Restricted Page 2 Launching the Image Analysis Software Double-click the Image Analysis icon on the desktop OR Click Start > Programs > Agilent Life-Sciences > Image Analysis

Title of Presentation 1 March, 2001 Agilent Restricted Page 3 Accessing the Image Analysis User Manual Double-click the Agilent-Life Sciences icon on the desktop OR Click Help > Help from the Image Analysis menu bar

Title of Presentation 1 March, 2001 Agilent Restricted Page 4 Accessing the Image Analysis Software Version Click Help > About from the Image Analysis menu bar The About Analysis dialog displays the software version

Title of Presentation 1 March, 2001 Agilent Restricted Page 5 How to Open a Scanned Image Click the Open icon on the Image Analysis toolbar OR Click File > Open and select the image file in the Open dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 6 How to Save a Copy of a Scanned Image Click the Save As icon on the Image Analysis toolbar OR Click File > Save As and name the image file in the Save As dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 7 How to Crop a Scanned Image Left-click and hold the mouse and then draw a box around the array Release the mouse button The cropped image appears in a new window

Title of Presentation 1 March, 2001 Agilent Restricted Page 8 How to Crop a Scanned Image (Continued) Click the Save Cropped View Icon in the new image window OR Click File > Save Cropped View on the Image Analysis menu bar Complete the information in the Save Cropped View dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 9 How to Close an Image Click the Close icon on the scan image window OR Click File > Close on the Image Analysis menu bar

Title of Presentation 1 March, 2001 Agilent Restricted Page 10 Changing the Color Range of the Image Display Click the Set color range icon in the toolbar of the scan image OR Click Color > Set Data Range from the Image Analysis menu bar In the Set Data Range dialog, enter the minimum and maximum values or choose Auto Scale Image

Title of Presentation 1 March, 2001 Agilent Restricted Page 11 Setting the Data Range for the Color Scale Changing the data range only affects the visualization of the array Underlying pixel intensities - raw data - do not change Compressing the data range brightens the array Expanding the data range dims the array expand scale - lower min & raise max compress scale - raise min & lower max auto scale

Title of Presentation 1 March, 2001 Agilent Restricted Page 12 Changing between Log and Linear Color Scale Click the Toggle Log Scale Icon to switch between Log and Linear color scale OR Click Color > Use Log Color Scale to toggle between Log and Linear color scale

Title of Presentation 1 March, 2001 Agilent Restricted Page 13 Viewing Images with Log or Linear Color Scales Click Color > Use Log Color Scale Toggle between log and linear color scale Log Scale Linear Scale

Title of Presentation 1 March, 2001 Agilent Restricted Page 14 Changing the Color Palette Click on Color in the Image Analysis menu bar False Color and False Color with White BG are available for two-color images Gray Scale and Reverse Gray Scale are available for single- color images

Title of Presentation 1 March, 2001 Agilent Restricted Page 15 Histogram Graphs of Pixel Intensities Histograms can be plotted for the entire view or a selected region for entire view - click View > Entire Histogram for a selected region - right-click and draw the region Selected region can be rectangular or elliptical use the toggle button to switch between shapes For two color images - one or both colors can be plotted

Title of Presentation 1 March, 2001 Agilent Restricted Page 16 How to Select an Elliptical Area for a Histogram Click the Toggle Histogram Shape icon on the image toolbar OR Click Options > Select Elliptical Area on the Image Analysis menu bar

Title of Presentation 1 March, 2001 Agilent Restricted Page 17 How to Select Graph Defaults for Histograms Click Options > Set Graph Defaults on the Image Analysis menu bar Specify either the Bin Size or the Maximum Number of Bins in the Set Bin Defaults dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 18 Histogram Plot - Sample Elliptical histogram for this feature

Title of Presentation 1 March, 2001 Agilent Restricted Page 19 Histogram Plot - Bin Size Bin size is set automatically by the Analysis software Users can change the bin size by two methods specifying the number of pixel intensity counts per bin specifying the maximum number of bins click Options > Set Graph Defaults -OR- Histogram > Change Bins

Title of Presentation 1 March, 2001 Agilent Restricted Page 20 How to Select Default Image Options Click Options > Set Image Defaults on the Image Analysis Toolbar Specify the desired default parameters in the Default Image Options dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 21 How to Set the Design File Search Path Click Options > Set Design File Search Path Click Browse in the Set XML Path dialog Locate the directory containing the design file in the Browse for Folder dialog Click Add in the Set XML Path dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 22 How to Check the Memory Status Click Help > Memory Status on the Image Analysis menu bar The Memory Status dialog is displayed

Title of Presentation 1 March, 2001 Agilent Restricted Page 23 Line Graphs Line graphs show a horizontal or vertical slice through the image To open the Select Row/Col dialog box click View > Select Row or Column for horizontal slice - click for vertical slice - click Choose Row -OR- Column -OR- All Rows or Columns

Title of Presentation 1 March, 2001 Agilent Restricted Page 24 Line Graph - Sample Line graph for a row of pixels through these features

Title of Presentation 1 March, 2001 Agilent Restricted Page 25 Exporting Images to other Applications Scan images, histogram plots, and line graphs can be exported to other applications Export methods high-resolution - click File >Save As and specify bitmap file type low-resolution - click the copy button to send the image to the clipboard

Title of Presentation 1 March, 2001 Agilent Restricted Page 26 Zooming In and Out Click View > Zoom from the menu select a magnification Mouse shortcuts To zoom in - Ctrl + left double click on the image To zoom out - Ctrl + right double click on the image Toolbar buttons Zoom inZoom out100 percent

Title of Presentation 1 March, 2001 Agilent Restricted Page 27 Scan Image File Information Information regarding the scan image is stored within its TIF file chip barcode scan date and time scanner software versions scan region PMT voltage

Title of Presentation 1 March, 2001 Agilent Restricted Page 28 Viewing Scan Image File Information Click View > FileInfo select General tab The scanner reads the barcode and generates the Array Identifier

Title of Presentation 1 March, 2001 Agilent Restricted Page 29 Viewing Scan Image File Information Click View > FileInfo select the General tab The scanner reads the barcode and generates the Array Identifier user can modify if necessary

Title of Presentation 1 March, 2001 Agilent Restricted Page 30 Viewing Scan Image File Information Click View > FileInfo select the Versions tab Scanner software versions are stored with the scan image

Title of Presentation 1 March, 2001 Agilent Restricted Page 31 Viewing Scan Image File Information Click View > FileInfo select the Image tab Scan regions and PMT Voltages are displayed

Title of Presentation 1 March, 2001 Agilent Restricted Page 32 Viewing Scan Image File Information Click View > FileInfo select the Image 2 tab Scan resolution is shown in the MicronsPerPixel field

Title of Presentation 1 March, 2001 Agilent Restricted Page 33 Feature Extraction Input Files Input Files - Required Scan image of Agilent microarray in 16-bit TIFF format 16-bit TIFF images are generated by the Agilent scanner. Scan images converted to other formats can not be feature extracted. Scan images of non-Agilent microarrays can be viewed - but not feature extracted - with the Agilent Image Analysis and Feature Extraction software. Array Design file - also called Pattern file - in GEML XML format Describes the layout and the probes on the microarray XML - Extensible Markup Language GEML - Gene Expression Markup Language

Title of Presentation 1 March, 2001 Agilent Restricted Page 34 Feature Extraction Input Files (Continued) Input Files - Optional Printing File - applies to cDNA microarrays. This file contains information on the cDNA clones spotted on the microarray. Bad clones on the microarray are ignored and excluded from analysis. If no Printing File is found, the below dialog is displayed In-situ microarray customers should disregard this message cDNA microarray customers should place the appropriate Printing File in the Design File Search path

Title of Presentation 1 March, 2001 Agilent Restricted Page 35 Feature Extraction Output Files XML - Contains the gene expression profile for the microarray in GEML format. Expression data in the GEML format can be loaded directly into the Rosetta Resolver Gene Expression Analysis software. Tab-text - Contains the gene expression profile for the microarray in tab-delimited text format. This format is intended for customers who do not use Resolver for gene expression analysis. The data will load into other gene expression analsysis software with a minimum of manipulation. Visual Results - Contains the “shapes” annotation generated by the Feature Extraction software. This file allows for subsequent viewing of the “shapes” annotation without having to re-extract the scan image. From the Feature Extraction UI

Title of Presentation 1 March, 2001 Agilent Restricted Page 36 How to Crop a Scanned Image Left-click and hold the mouse and then draw a box around the array Release the mouse button The cropped image appears in a new window

Title of Presentation 1 March, 2001 Agilent Restricted Page 37 How to Crop a Scanned Image (Continued) Click the Save Cropped View Icon in the new image window OR Click File > Save Cropped View on the Image Analysis menu bar Complete the information in the Save Cropped View dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 38 How to Manually Input the Slide Barcode The Agilent scanner reads the barcode for each slide and stores that information inside the TIFF scan image In rare cases, the Agilent scanner does not read the slide barcode correctly The user must then manually enter the slide barcode when saving a cropped microarray scan image

Title of Presentation 1 March, 2001 Agilent Restricted Page 39 Agilent Slide Barcode Nomenclature The Agilent microarray barcode standard consists of 14 digits The six-digit design identifier is the same for all microarrays printed from the same pattern The six-digit serial number serves as a unique identifier for all microarrays printed from the same pattern The barcode uses Code 128C encoding 16NNNNNNNNNNNN Version of the barcode format Design identifier Serial number For version 1: # of digits in the design identifier field

Title of Presentation 1 March, 2001 Agilent Restricted Page 40 Agilent Microarray Identifier Nomenclature The Agilent microarray identifier is based on the slide barcode Slides contain either one 22K-feature microarray or two 8.4K-feature microarrays 22K-feature microarray - one microarray per slide Suffix _A01 appended to the slide barcode 8.4K-feature microarray - two microarrays per slide Suffix _A01 appended to slide barcode - Left array Suffix _A02 appended to slide barcode - Right array Example: 16NNNNNNNNNNNN_A02 barcod e

Title of Presentation 1 March, 2001 Agilent Restricted Page 41 Agilent Design and Printing File Nomenclature Agilent design and printing file naming nomenclature is based on the design ID and date Printing files are only generated for cDNA microarrays and contain information about bad clones on the array Do not rename the design or printing files! Deviating from the nomenclature prevents automatic association of microarray scan images with their design and printing files NNNNNN_D_YYYYMMDD. xml Design Identifier D indicates Design file, P indicates Printing file Two-digit Month Four-digit Year Two-digit Day

Title of Presentation 1 March, 2001 Agilent Restricted Page 42 How to Set the Design File Search Path Feature Extraction checks the design file search path to find the microarray scan image’s design file Click Options > Set Design File Search Path Click Browse in the Set XML Path dialog Locate the directory containing the design file in the Browse for Folder dialog Click Add in the Set XML Path dialog

Title of Presentation 1 March, 2001 Agilent Restricted Page 43 How to Feature Extract a Scan Image Make sure the design file is in the design file search path Crop and save the microarray scan image (and manually enter the slide barcode if necessary) Click Feature Extraction > Feature Extractor Specify the directory path for the output files Click the Load All Defaults button and the Run button

Title of Presentation 1 March, 2001 Agilent Restricted Page 44 How the Feature Extraction Software Searches for Design Files The design ID is part of the microarray identifier, which is embedded in the microarray scan image The design ID is embedded in the filename of the design file The design ID is also embedded in the filename of printing files - applicable to cDNA microarray design files For all directories in the design file search path: the Feature Extraction software searches for design and printing files with embedded design IDs that match the design ID in the microarray scan image

Title of Presentation 1 March, 2001 Agilent Restricted Page 45 How to Manually Specify a Design File If the microarray design file is not found in the design file search path, the user is prompted to manually specify a design file Slide printing files can not be loaded using this method and therefore no clone information will be available The user must ensure that the manually-specified design file represents the microarray in the scan image If the manually-specified design file does not represent the microarray in the scan image, an erroneous gene expression profile will result

Title of Presentation 1 March, 2001 Agilent Restricted Page 46 How to Manually Specify a Design File Warning 1 Warning 2Design File Loading Logic

Title of Presentation 1 March, 2001 Agilent Restricted Page 47 Locating Corner Features in the Feature Extraction Software The Feature Extraction software automatically locates the corner features of the microarray and uses these as references to locate all other features In rare cases, the Feature Extraction software fails to locate the corner features of the microarray The user must then manually provide the locations of three corners of the microarray, to allow the software feature extract the microarray

Title of Presentation 1 March, 2001 Agilent Restricted Page 48 How to Manually Locate the Corner Features in a Microarray Scan Image Zoom in on the top-left corner feature on the microarray Place the mouse pointer over the center of the corner feature Left-click the mouse Click the enter button for Upper Left in the Find Spots tab of the Feature Extraction user interface The row and column coordinates are pasted into the user interface Repeat the process for the upper-right and lower-left corner features top-left corner feature

Title of Presentation 1 March, 2001 Agilent Restricted Page 49 Viewing Feature Extraction Results Click View > Extraction Results Three on / off toggles View Results Hide Good Simple Colors

Title of Presentation 1 March, 2001 Agilent Restricted Page 50 Loading Feature Extraction Results Feature Extraction visual results can be saved makes re-extraction unnecessary click Feature Extraction > Load Results