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The following slides have been adapted from tm4/ to be presented at the

The following slides have been adapted from http://www.tm4.org/ to be presented at the Follow-up course on Microarray Data Analysis (Nov 20-24 2006, PICB Shanghai) by Peter Serocka. TIGR. THE INSTITUTE FOR GENOMIC RESEARCH. TIGR Spotfinder: a tool for microarray image processing.

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The following slides have been adapted from tm4/ to be presented at the

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  1. The following slides have been adapted from http://www.tm4.org/ to be presented at the Follow-up course on Microarray Data Analysis (Nov 20-24 2006, PICB Shanghai) by Peter Serocka

  2. TIGR THE INSTITUTE FOR GENOMIC RESEARCH TIGR Spotfinder:a tool for microarray image processing Developer: Vasily Sharov The Institute for GenomicResearch

  3. Printer Scanner Image File Image Analysis Microarray Data Flow Raw Gene Expression Data Gene Annotation Normalization / Filtering Normalized Data with Gene Annotation Expression Analysis Interpretation of Analysis Results

  4. Printer Scanner Image File Image Analysis Microarray Data Flow .tif Raw Gene Expression Data .mev (.gpr) Gene Annotation Normalization / Filtering .ann (.gal) Normalized Data with Gene Annotation .mev (.gpr, .txt) Expression Analysis Interpretation of Analysis Results

  5. Data File Formats

  6. Cy3 Cy3-cDNA Cy5 Cy5-cDNA Process Overview Sample1 mRNA Cy3 intensity RT RT cDNA array Cy5 intensity Sample2 mRNA

  7. Basic Steps from Image to File 1.) Image File Loading 2.) Construct or Apply an Overlay Grid 3.) Computations • Find Spot Boundary and Area • Intensity Calculation • Background Calculation and Correction 4.) Quality Control 5.) Text File Output

  8. Basic DemonstrationExploring the Interface(Using An Existing Grid File)

  9. Microarray Image Parameters MA Scanner generates two 16 bit gray scale TIFF images: one image for each labeling probe (Cy3 and Cy5) 16 bit schema provides signal dynamic range from 0 to 216=65536 Each image size varies from 20 to 30 MB for scanning resolution 10 mm/pixel

  10. Typical layout of microarray image Image size 22 MB Image size 28 MB (images scanned at 10mm/pix resolution)

  11. Apply the Grid Determine Spot Boundary Calculate Spot Intensity Determine Background and Correct Intensity Processing Overview

  12. Applying an Overlay Grid • What does it accomplish? • The grid cells set a boundary for the spot finding algorithms. • The grid cells also define an area for background correction.

  13. Gridding Dimension Parameters pin X pin X pin Y pin Y

  14. Spot Spacing Parameter spotspacing

  15. Spot Finding Spot finding requires an estimated spot size. The spot can be drawn as an irregular contour, as an ellipse, or as unconnected pixels. Area inside contour is used for spot intensity calculation Area outside contour is used for local background calculation

  16. Processing Overview Apply the Grid Determine Spot Boundary Calculate Spot Intensity Determine Background and Correct Intensity

  17. Background Calculation Background intensity is calculated as the median pixel intensity from the area within the square and outside the spot. A separate local background is calculated for each spot using the non-spot pixels from it’s square. local background area

  18. Spot Definition and Calculations Spot Area, A = number of pixels within the defined spot boundary BKG = median pixel value within the cell (excluding the spot pixels) Integral = Sum of all spot pixels excluding saturated pixels Reported “Intensity”=Integral-BKG*A

  19. Spot Integration with Background Correction

  20. Quality Control Issues • Two measures of spot quality are reported by SpotFinder: • Saturation Factor • QC Score: reports shape and signal to noise ratio

  21. Saturation Examples Partially saturated spots can look like this: saturated area non-saturated area Completely saturated spots can look like this: fully saturated spot

  22. 216=65536 Saturation, Pixel Value Limit Output: pixel value Input: fluorescence dye light signal

  23. (# good pixels in spot) Saturation Factor = (total number of spot pixels) Saturation Factor -Partially saturated spots can be handled in SpotFinder by excluding the saturated pixels from spot area and intensity calculations. -Fully saturated spots can not be recovered in SpotFinder. In this case rescanning with lower excitation power or PMT gain could be considered. *Faint spots may possibly be lost.

  24. Saturation, RI Plot RI plot: log(IB/IA) vs 1/2log(IA*IB) clearly displays the saturation limits

  25. shape signal/noise shape signal/noise Quality Control, QC Score A QC Score is generated for each spot and is based on the spot shape and a measure of signal to noise ratio. QC Score QCA QCB

  26. Spot Shape Parameter Shape Factor = (Spot Area/Perimeter) Spots with large perimeters relative to spot area will have a low shape factor.

  27. Signal to Noise Ratio 216 S/N factor = fraction of spot pixels exceeding: *med(BKG) + * SD(BKG) Pixel Values med(BKG) SD(BKG) 0

  28. Quality Control Calculation QC Score = (QCA+QCB)/2 QCA=sqrt(QC shape*QC S/N) for channel A QCB=sqrt(QC shape*QC S/N) for channel B

  29. Quality Control, RI Plot RI plot: log(IB/IA) vs1/2log(IA*IB) plotted for means shows clearly low intensity distortion due to background overestimation. Data from earlier slide processed without QC filter

  30. Quality Control (data provided by E. Snesrud)

  31. Quality Control (data provided by E. Snesrud)

  32. SpotFinder Flag Descriptions A - Spot area is larger than 50 pixels B - Spot area is between 30 and pixels C - spot area is smaller than 30 pixels X - Spot rejected by QC based on spot shape and spot intensity relative to surrounding background U - Spot rejected (“flagged”) by user Y - Bad spot, background is higher than spot intensity Z - Spot was not detected by the program S - Warning: some spot pixels are saturated

  33. Output data (.mev) per spot: UID Unique identifier for this spot IA Intensity value in channel A IB Intensity value in channel B R Row (slide row) C Column (slide column) MR Meta-row (block row) MC Meta-column (block column) SR Sub-row SC Sub-column

  34. Output data (.mev) per spot: FlagA TIGR Spotfinder flag value in channel A FlagB TIGR Spotfinder flag value in channel B SA Actual spot area (in pixels) SF Saturation factor QC Cumulative quality control score QCA Quality control score in channel A QCB Quality control score in channel B

  35. Output data (.mev) per spot: BkgA Background value in channel A BkgB Background value in channel B SDA Standard deviation for spot pixels in channel A SDB Standard deviation for spot pixels in channel B SDBkgA Standard deviation of the background in channel A SDBkgB Standard deviation of the background in channel B

  36. Output data (.mev) per spot: MedA Median intensity value in channel A MedB Median intensity value in channel B MNA Mean intensity value in channel A MNB Mean intensity value in channel B X/Y X resp. Y coordinates of the spot cell PValueA P-value in channel A PValueB P-value in channel B DBID Data Base ID (if UID is substituted)

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