1. The following slides have been adapted from http://www.tm4.org/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. THE INSTITUTE FOR GENOMIC RESEARCH TIGR TIGR Spotfinder: a tool for microarray image processing The Institute for Genomic Research Developer: Vasily Sharov

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

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

5. TIGROthers Slide Images.tif Gene Expression tables.mev.tav - outdated.gpr (GenePix).txt (tab-delimited, Excel) Gene Annotations and Array layout information.ann.gal Data File Formats

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

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 Demonstration Exploring 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 2 16 =65536 Each image size varies from 20 to 30 MB for scanning resolution 10  m/pixel

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

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

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. pin X pin Y Gridding Dimension Parameters

14. spot spacing Spot Spacing Parameter

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. Saturation, Pixel Value Limit Output: pixel value Input: fluorescence dye light signal 2 16 =65536

23. 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. Saturation Factor = (# good pixels in spot) (total number of spot pixels)

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

25. 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. shapesignal/noiseshapesignal/noise QC A QC B QC Score

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 med(BKG) 0 Pixel Values  *med(BKG) +  * SD(BKG) S/N factor = fraction of spot pixels exceeding: 2 16 SD(BKG)

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

29. Quality Control, RI Plot RI plot: log(I B /I A ) vs1/2log(I A *I B ) 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. 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 SpotFinder Flag Descriptions

33. UIDUnique identifier for this spot IAIntensity value in channel A IBIntensity value in channel B RRow (slide row) CColumn (slide column) MRMeta-row (block row) MCMeta-column (block column) SRSub-row SCSub-column Output data (.mev) per spot:

34. FlagATIGR Spotfinder flag value in channel A FlagBTIGR Spotfinder flag value in channel B SAActual spot area (in pixels) SFSaturation factor QCCumulative quality control score QCAQuality control score in channel A QCBQuality control score in channel B Output data (.mev) per spot:

35. BkgABackground value in channel A BkgBBackground value in channel B SDAStandard deviation for spot pixels in channel A SDBStandard deviation for spot pixels in channel B SDBkgAStandard deviation of the background in channel A SDBkgBStandard deviation of the background in channel B Output data (.mev) per spot:

36. MedAMedian intensity value in channel A MedBMedian intensity value in channel B MNAMean intensity value in channel A MNBMean intensity value in channel B X/YX resp. Y coordinates of the spot cell PValueAP-value in channel A PValueBP-value in channel B DBIDData Base ID (if UID is substituted) Output data (.mev) per spot: