Step 1 Feature Extraction Featuer Extraction Feature Extraction Featuer Extraction Image Analysis Start>Programs>Agilent-Life Sciences>Feature Extract

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1 Agilent G2565AA Feature Extraction Step 1 Feature Extraction Step 2 Step 3 Step 4 ( ) Step 5 ( ) Step 6 Step 7 Step 8 Feature Extraction Step 9 Step 10 Feature Extraction Step 11 Feature Extraction

2 Step 1 Feature Extraction Featuer Extraction Feature Extraction Featuer Extraction Image Analysis Start>Programs>Agilent-Life Sciences>Feature Extraction Figure 1

3 Step 2 1. Open File>Open 2. Open.tif Open Image Analysis Figure 2 TIFF TIF Feature Extraction Image Analysis

4 Step 3 Feature Extraction 2 Cyanine3 Cyanine5 1. Window>New Window Select Image 2. Red Green Both 3. OK 4. (Red Green) Red Green False Color False Color with a white background Color>False Color w/ White Bgd

5 False Color Color>False Color Figure 3 False Color Color>Gray Scale Color>Reverse Gray Scale. Gray Scale Reverse Gray False Color Color>False Color Figure 4 1

6 Color>Use Log Color Scale Color>Use Log Color Scale Figure 5 TIFF Color > Set Data Range

7 Figure 6 Set Data Range Minimum Maximum Auto Scale Image Figure 7

8 1. Options>Set Image Defaults 2. One Image Color Palette 3. Two Image Color Palette 4. Put Red and Green channels in one window 5. Use Log Color Scale 6. OK 7. 8.

9 Step 4 1. View>Entire Histogram a. Options>Select Elliptical Area Figure 8 Options>Select Elliptical Area Figure 9 b.

10 Channel Selection dialog box 2. Red Green Figure 10

11 Step 5 Red Green 1. View>Select Row or Column Select Row/Col Figure 11 Select Row/Col 2. Row Number Column Row Number Column Number All Rows or Columns 5. OK

12 Figure 12

13 Step Figure 13 Esc. Feature Extraction Feature Extraction 2

14 Feature Extraction

15 Step 7 Feature Extraction View 1. File>Save Cropped View 2. Save Cropped Image Figure 14 Save Cropped View 3. New Array Identifier Left or single array _A01 _A02 Array Number Feature Extraction

16 1. File>Save Cropped View Create Barcode Figure 15 Create Barcode ID Save As ID OK 3. OK Feature Extraction

17 Step 8 Feature Extractor Feature Extractor Feature Extraction Configuration Feature Extraction Feature Extraction 1. A) Options>Set Design File Search Path B) Browse OK New Dir: C) Add 2. Feature Extraction>FeatureExtractor CD DesignID_P_yyyymmdd.xml yyyymmdd TIFF Feature Extraction Configuration TIFF Figure 16

18 TIFF Feature Extraction Configuration Feature Extraction Figure 17 Feature Extraction Configuration

19 Step 9 Feature Extraction Figure 18 Feature Extraction Configuration OK 3. Rosetta BioSoftware XML Visual results(.shp) Feature Extraction version 5.1 Visual results Visual results 5.1

20 Step 10 Feature Extraction Feature Extraction 1. Feature Extraction Configuration More More Changing module (algorithm) settings Figure 19 Feature Extraction Configuration Run Feature Extraction Progress Bar

21 Feature Extraction Figure 20 Feature Extraction Feature Extraction Cancel Feature Extraction Configuration

22 Step 11 Visual Results Feature Extraction Feature Extraction Extraction Results Find Tooltips Feature Extraction Feature Extraction Help>Feature Extraction Output Quick Reference Feature Extraction Figure 21 Feature Extraction

23 Outlier Simple Color Non-uniformity Outlier Population Outlier Non-uniformity Outlier Population Outlier Outlier Simple Color Non-uniformity Outlier Population Outlier Non-uniformity Outlier Population Outlier Outlier Outlier Outlier

24 Figure 22 Outlier Extraction Results View>Extraction Results Figure 23 Extraction Results View Results Visual Results

25 View Outliers Only Outlier Hide Outer Local BG Ring Use Simple Colors Find Feature ignore 1. View>Find Feature Ctrl + F Find Feature 2. OK Figure 24 Find Feature Default Image Options Tooltips Tooltips Outlier Tooltip X Figure 25 Tooltip

26 Feature Extraction 1 Reverse Gray Scale Figure16 Feature Extraction tab-delimited Feature Extraction. Figure 26

Feature Extraction green red g (green) r (red) Outlier gmeansignal gbgused gbgsubsignal rmeansignal rbgused rbgsubsignal raw data - Outlier - Uniformity Outlier -

27 Feature Extraction green red g (green) r (red) Outlier gmeansignal gbgused gbgsubsignal rmeansignal rbgused rbgsubsignal raw data - Outlier - Uniformity Outlier - Population Outlier Outlier - Uniformity Outlier - Population Outlier raw data gdyenormsignal rdyenormsignal - - gprocessedsignal rprocessedsignal Surrogate 0 LogRatio

28 Outlier Outlier Uniformity Outlier Population Outlier Uniformity Outlier Population Outlier Agilent G2566AA Feature Extraction Software User Guide 47

29 Feature Extraction Feature (Green) Feature (Red) FeatureNum Row Col Start Sequence ProbeUID ControlType In Situ In Situ ID ProbeName GeneName SystematicName Description PositionX PositionY LogRatio GeneName SystematicName X Y Log(REDsignal/GREENsignal) (ProcessedSignal) SURROGATES -2 DyeNormRedSig 0 DyeNormGreenSig 0

30 Feature (Green) Feature (Red) 0 DyeNormRedSig 0 DyeNormGreenSig 0 LogRatioErro SURROGATES 0 DyeNormRedSig 0 DyeNormGreenSig 0 SURROGATES Lo RatioErro= LogRatio PvalueLogRatio LogRatio P 0 gprocessedsigna l rprocessedsignal LogRatio gprocessedsiger rprocessedsigerr ror or gnumpixolhi rnumpixolhi Outlier Outlier gnumpixollo rnumpixollo Outlier Outlier gnumpix rnumpix Inlier gmeansignal rmeansignal raw data gmediansignal rmediansignal raw data gpixsdev rpixsdev gbgnumpix rbgnumpix Inlier gbgmeansignal rbgmeansignal raw data

31 Feature (Green) Feature (Red) gbgmediansigna rbgmediansignal raw data l gbgpixsdev rbgpixsdev gnumsatpix rnumsatpix gissaturated rissaturated 1 0 PixCorrelation RedGreen Red Green BGPixCorrelation gisfeatnonunifo L risfeatnonunifo L 0 1 Uniformity Outlier Uniformity Outlier Uniformity Outlier gbgsubsigerror gbgsubsigerror BGSubSigCorrela tion RedGreen gisposandsignif RIsPosAndSignif 2-tail t P 0 1

32 Feature (Green) Feature (Red) gpvalfeateqbg rpvalfeateqbg Green Red Green(Red) P gnumbgused rnumbgused giswellabovebg riswellabovebg WellAbove g(r)isposandsignif g(r)bgsubsignal [ 2.6] IsUsedBGAdjust 0 1 gbgused rbgused g(r)bgsubsignal = g(r)meansignal g(r)bgused gbgsdused rbgsdused DelCtrlFeatNum DelCtrlProbeNam e gperfmatchsignal rperfmatchsignal =g(r)meansignal gdelctrlsignal rdelctrlsignal g(r)meansignal 0

33 Feature (Green) Feature (Red) gpermatcherror rpermatcherror =g(r)meanerror gdelctrlerror rdelctrlerror =g(r)meanerror 0 gpvalpermatche qdelctrl rpvalpermatcheq DelCtrl P gislowspecificity rislowspecificity g(r)perfmatchsignal t P<0.01 g(r)iswellabovebg gisgoodpm risgoodpm g(r)iswellabovebg t P<0.01 gpmdlratio rpmdlratio g(r)meansignal g(r)meansignal IsNormalization 0 1 gdyenormsignal rdyenormsignal gdyenormerror rdyenormerror DyeNormCorrelati on ErrorModel Red Green Feature Extraction

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