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Mark Hartnett Software Support Engineer December 7, 2001

Mark Hartnett Software Support Engineer December 7, 2001. Introduction to Analysis and Feature Extraction Software. Launching the Image Analysis Software. Double-click the Image Analysis icon on the desktop OR Click Start > Programs > Agilent Life-Sciences > Image Analysis.

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Mark Hartnett Software Support Engineer December 7, 2001

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  1. Mark HartnettSoftware Support EngineerDecember 7, 2001 Introduction to Analysis and Feature Extraction Software

  2. Launching the Image Analysis Software • Double-click the Image Analysis icon on the desktop OR • Click Start > Programs > Agilent Life-Sciences > Image Analysis Agilent Restricted

  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 Agilent Restricted

  4. Accessing the Image Analysis Software Version • Click Help > About from the Image Analysis menu bar • The About Analysis dialog displays the software version Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 compress scale - raise min & lower max expand scale - lower min & raise max auto scale Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  18. Histogram Plot - Sample Elliptical histogram for this feature Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  22. How to Check the Memory Status • Click Help > Memory Status on the Image Analysis menu bar • The Memory Status dialog is displayed Agilent Restricted

  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 Agilent Restricted

  24. Line Graph - Sample Line graph for a row of pixels through these features Agilent Restricted

  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 Agilent Restricted

  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 in Zoom out 100 percent Agilent Restricted

  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 Agilent Restricted

  28. Viewing Scan Image File Information • Click View > FileInfo • select General tab • The scanner reads the barcode and generates the Array Identifier Agilent Restricted

  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 Agilent Restricted

  30. Viewing Scan Image File Information • Click View > FileInfo • select the Versions tab • Scanner software versions are stored with the scan image Agilent Restricted

  31. Viewing Scan Image File Information • Click View > FileInfo • select the Image tab • Scan regions and PMT Voltages are displayed Agilent Restricted

  32. Viewing Scan Image File Information • Click View > FileInfo • select the Image 2 tab • Scan resolution is shown in the MicronsPerPixel field Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 For version 1: # of digits in the design identifier field Version of the barcode format Design identifier Serial number 16NNNNNNNNNNNN Agilent Restricted

  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 barcode Agilent Restricted

  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 D indicates Design file, P indicates Printing file Two-digit Month Four-digit Year Design Identifier Two-digit Day NNNNNN_D_YYYYMMDD.xml Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  46. How to Manually Specify a Design File Warning 1 Design File Loading Logic Warning 2 Agilent Restricted

  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 Agilent Restricted

  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 Agilent Restricted

  49. Viewing Feature Extraction Results • Click View > Extraction Results • Three on / off toggles • View Results • Hide Good • Simple Colors Agilent Restricted

  50. Loading Feature Extraction Results • Feature Extraction visual results can be saved • makes re-extraction unnecessary • click Feature Extraction > Load Results Agilent Restricted

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