Low Level Statistics and Quality Control Javier Cabrera.
-
date post
21-Dec-2015 -
Category
Documents
-
view
217 -
download
0
Transcript of Low Level Statistics and Quality Control Javier Cabrera.
![Page 1: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/1.jpg)
Low Level Statistics and Quality Control
Javier Cabrera
![Page 2: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/2.jpg)
Outline
1. Processing Steps.
2. Spotting the Raw Image
3. Panel quality
4. Array quality
5. Area problems
![Page 3: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/3.jpg)
Scanned Image
C1 C2
L
K
J
I
H
G
F
E
D
C
B
A
0.0
0.5
1.0A : 4 Genes
0.0
0.5
1.0B : 16 Genes
0.0
0.5
1.0C : 16 Genes
0.0
0.5
1.0D : 5 Genes
0.0
0.5
1.0E : 8 Genes
0.0
0.5
1.0F : 12 Genes
0.0
0.5
1.0G : 2 Genes
0.0
0.5
1.0H : 7 Genes
0.0
0.5
1.0I : 6 Genes
0.0
0.5
1.0J : 12 Genes
0.0
0.5
1.0K : 7 Genes
0.0
0.5
1.0L : 5 Genes
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Spotted Image
PreprocessingGene Exp. Mat.
StatisticalAnalysis
Data processing steps
![Page 4: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/4.jpg)
Preprocessing steps
- Microarrays are scanned
- The image is converted into spot intensities for analysis.
- Spot intensity: Reflects the amount of labeled probe that hybridized to it.
- Run a series of quality checks on the data.
- The spot intensity is adjusted for background effects.
![Page 5: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/5.jpg)
(A)
(B)
(C)
speckles
Light Background
Shape
Raw array
![Page 6: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/6.jpg)
Gridding: where are the spots?
Edge detection: Cho, Meer, Cabrera (1997)
Segmentation: which pixels correspond to the spot (signal) and which to background?
Measurement: what is the intensity at each spot?
Spot intensity = average pixel intensity within the spot
Background intensity = average pixel intensity immediately around the spot
Spotting the Raw image
![Page 7: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/7.jpg)
Segmentation
- Fixed circle segmentation: by fitting a circle with a constant diameter to all the spots.
- Adaptive circle segmentation: fitting circles with different diameters to different spots
- Seeded region growing algorithm (this works better)
(i) Seed specification.(ii) Region Growing.(iii)Stopping Rule.
![Page 8: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/8.jpg)
Segmentation
- Fixed circle segmentation: by fitting a circle with a constant diameter to all the spots.
- Adaptive circle segmentation: fitting circles with different diameters to different spots
- Seeded region growing algorithm (this works better)
(i) Seed specification.(ii) Region Growing.(iii)Stopping Rule.
![Page 9: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/9.jpg)
Spot quality: Assess the quality of the spots:Spot IntensityBackground IntensitySpot and background Std. Dev. Or CVSpot CircularitySignal to noise ratio
Use Scatter plot and scatter matrices to look for patterns, outliers.
Data example:ch1 Intensity Std Devch1 Background Std Devch1 Diameter ch1 Area ch1 Footprint ch1 Circularitych1 Spot Uniformitych1 Bkg. Uniformitych1 Signal Noise Ratio
1683.537231 88.588715 99.014877 5800 80.174225 0.883462 0.959335 0.99839 56.744021039.019409 45.566654 100.925308 5800 78.812447 0.895682 0.976974 0.999084 54.52414472.640381 58.376431 102.179092 5800 81.879074 0.93141 0.987099 0.99894 25.6764538.243103 70.611359 104.641586 5800 74.847275 0.912625 0.985878 0.998634 24.4041
1929.476563 47.217567 92.705818 5800 75.951424 0.909981 0.948647 0.999176 134.2162
Preprocessing the spot intensity values
![Page 10: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/10.jpg)
0 200 400 600 800 1000 1200
010
000
2000
030
000
4000
0
b1
x1
Channel 1 Signal Vs Background
0 2 4 6
56
78
910
log(b1)
log(
x1)
Channel 1 Signal Vs Background in log scale
0 1000 2000 3000 4000 5000
010
000
3000
050
000
b2
x2
Channel 2 Signal Vs Background
-4 -2 0 2 4 6 8
02
46
810
log(b2)
log(
x2)
Channel 1 Signal Vs Background in log scale
5 6 7 8 9 10
02
46
810
log(x1)
log(
x2)
Channel 2 Vs Channel 1 Log Signal
0 2 4 6
-4-2
02
46
8
log(b1)
log(
b2)
Channel Vs Channel 2 log Background
Exploring Channel 1,2 Intensities, Background
![Page 11: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/11.jpg)
Spot quality
-Spot Intensity-Background I-Circularity-Uniformity-Signal to Noise
The graphs show some outliers but there are not very dramatic.
![Page 12: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/12.jpg)
Panel and Array quality:
Assess the quality of the panels and the entire arrays
- Image plot - Boxplots of panels - Quality diagnostic plot from Bioconductor.
Preprocessing the spot intensity values
2 2 2
2 22
5log log ( 5) log ( 3)
3
log ( 5) log ( 3)log 5 3
2
CyM Cy Cy
Cy
Cy CyA Cy Cy
![Page 13: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/13.jpg)
Target InfoProbe Info = mrrayLayout : Structure,
construction marrayInfo: Probe Anotation Intensity Measures : maRf, maGf, maRb, maGb,
maW
Preprocessing the spot intensity values
2 2 2
2 22
5log log ( 5) log ( 3)
3
log ( 5) log ( 3)log 5 3
2
CyM Cy Cy
Cy
Cy CyA Cy Cy
![Page 14: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/14.jpg)
6Hs.166.gpr: image of A1 2 3 4
12
11
10
9
8
7
6
5
4
3
2
1
0
1.8
3.6
5.3
7.1
8.9
11
12
14
16
![Page 15: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/15.jpg)
Pla
te 1
Pla
te 2
Pla
te 3
Pla
te 4
Pla
te 5
Pla
te 6
Pla
te 7
Pla
te 8
Pla
te 9
Pla
te 1
0P
late
11
Pla
te 1
2P
late
13
Pla
te 1
4P
late
15
Pla
te 1
6P
late
17
Pla
te 1
8P
late
19
Pla
te 2
0P
late
21
Pla
te 2
2P
late
23
Pla
te 2
4P
late
25
Pla
te 2
6P
late
27
Pla
te 2
8P
late
29
Pla
te 3
0P
late
31
Pla
te 3
2P
late
33
Pla
te 3
4P
late
35
Pla
te 3
6P
late
37
Pla
te 3
8P
late
39
Pla
te 4
0P
late
41
Pla
te 4
2P
late
43
Pla
te 4
4P
late
45
Pla
te 4
6P
late
47
Pla
te 4
8P
late
49
Pla
te 5
0P
late
51
Pla
te 5
2P
late
53
Pla
te 5
4P
late
55
Pla
te 5
6P
late
57
Pla
te 5
8P
late
59
Pla
te 6
0P
late
61
0
5
10
15
6Hs.166.gpr
Plate
A
![Page 16: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/16.jpg)
![Page 17: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/17.jpg)
Systematic Effects
Changes in measurement scale due to: • Time: Day effect or other order• Operator• Batch
![Page 18: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/18.jpg)
X1 X5 X9 X14 X19 X24 X29 X34 X39
45
67
8
Example of day Effect
Day Two
Day One
![Page 19: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/19.jpg)
68
10
12
14
16
81
01
21
41
6
Day effect
Day effect removed via linear model + normalized
Removal of Systematic Effects
![Page 20: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/20.jpg)
Array quality: Assess the quality of the array - area problems
Area Effects
![Page 21: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/21.jpg)
0 5 10 15 20 25 30
05
01
00
15
0
0 5 10 15 20 25 30
05
01
00
15
0
Array QualityImage plot of a good array
Signal Background
![Page 22: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/22.jpg)
0 5 10 15 20 25 30
05
01
00
15
0
0 5 10 15 20 25 30
05
01
00
15
0
Image plot of a defective array
Signal Background
![Page 23: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/23.jpg)
Area Problems(i) Large spots covering a good part of the area of
the background image. These spots show higher or lower intensities than the rest of the image.
(ii) Vertical or horizontal strips on the background image that show higher or lower intensities.
(iii) Diagonal strips again showing higher or lower background intensities.
(iv) A ramp in the background intensities going across the array.
(v) Bleeding in the spotted image showing sequences of consecutive.
![Page 24: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/24.jpg)
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
![Page 25: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/25.jpg)
Algorithm:Step 1. Split the image into high intensity and low intensity spots:
Yrc=1 for high intensity Irc > cYrc=0 for low intensity Irc c
c = (I0.05 + I0.95)/2
Step 2. Fit a quadratic discriminant function to the binary response Yrc using the spot coordinates (r,c) on the microarray as predictors.
Step 3. Let q=proportion of correct classifications.
Step 4. Generate M=300 images by randomly permute the rows and columns of {Irc} and for each image calculate the corresponding q1,…,q300.
Step 5 Use prop{qi >q } as a quality measure of the array or p-value.
![Page 26: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/26.jpg)
Intensity
Fre
qu
en
cy
2 4 6 8
05
00
10
00
15
00 Low High
Histogram of intensities
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
![Page 27: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/27.jpg)
Row profile.
Col profile.
High and Low
p-val= 0
1 22 4 6 9
Microarray with an area problem
![Page 28: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/28.jpg)
Row profile.
Col profile.
High and Low
p-val= 0
1 22 4 5 8
Microarray with a ramp
![Page 29: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/29.jpg)
Row profile.
Col profile.
High and Low
p-val= 0.328
1 1.3 1.8 2.3 3 3.9
Good Microarray
![Page 30: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/30.jpg)
Image Quality Graph
Row profile.
Col profile.
High and Low
p-val= 0.116
1 1.3 1.6 1.9 2.4 2.9
![Page 31: Low Level Statistics and Quality Control Javier Cabrera.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a334e9/html5/thumbnails/31.jpg)
Microarray Graph
D
C
B
A0.0
0.5
1.0A : 47 Genes
0.0
0.5
1.0B : 8 Genes
0.0
0.5
1.0C : 24 Genes
0.0
0.5
1.0D : 27 Genes
00 1010 30 60