Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric...
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Transcript of Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric...
![Page 1: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/1.jpg)
Detection and Restoration of Hybridization Problems in Affymetrix
GeneChip Data by Parametric Scanning
Tomokazu Konishi
Akita Pref. Univ.
![Page 2: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/2.jpg)
a monolith?
!
![Page 3: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/3.jpg)
praise and censurefor microarray technology
毀毀毀毀
Admired:the comprehensiveness
Criticized:the low reproducibility
Origin of the failure:the intelligent framework for data processing
![Page 4: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/4.jpg)
values without units
obtained from hybridization images
Index CHI11 69715.252 55335.523 89216.684 145717.85 128202.26 1143737 75725.448 39021.069 115491.510 97384.4711 10182312 194268.713 114838.714 118911.315 45748.0516 113630.717 53177.5518 65225.819 117009.420 32688.52
excitation light fluorescence
![Page 5: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/5.jpg)
Requirements for understanding unitless values
philosophy or
metaphysics
framework
ex. International Systems of Units (SI)
![Page 6: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/6.jpg)
desirable framework for microarray
• Measurements–Standard–Scale
• Interpretations–direct link to cell functions
![Page 7: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/7.jpg)
Measurements
• Standard• Scale
are available from the data distribution
Parametric Normalization (2003-4)
data service is available through Skylight Biotech Inc.
![Page 8: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/8.jpg)
Human fibroblast
(Iyer et al. 1999 Science)
ord
ere
d re
spo
nse
va
lue
t h e o re t ica l- 3 -2 -1 0 1 2 3
z -s c o re
-3
-2
-1
0
1
2
3
raw data
SuperNORM
lognormal distribution can be foundby subtracting proper background
distortedby saturation
noise-affected
normalized
![Page 9: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/9.jpg)
statistical framework
• Standard• Scale
are available from the data distribution
Parametric Normalization (2002-4)
data service is available through Skylight Biotech Inc.
![Page 10: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/10.jpg)
desirable framework for microarray
• Measurements–Standard–Scale
• Interpretations–direct link to cell functions
statistical framework
![Page 11: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/11.jpg)
cell
link to cell functions
![Page 12: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/12.jpg)
• Most of the factors are well characterized
• Bottom-Up approach
– nucleotide sequence recognition factors
– controlling the rate limiting steps
– concrete physics
The cell is not a black-box
cell
![Page 13: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/13.jpg)
[mRNA]s are on the balancesrates of synthesis and degradation
Pseudo Equilibrium
Cytosol
Interactions among factorschange the rate
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energies describe the interactions
]Eregulator[em0p KkCE
]regulator[cr00
p KkCΔGRate of synthesis
Rate of degradation
energies = k [factors]
![Page 15: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/15.jpg)
energies determine the [mRNA]
kc = ApP0[polymerase]/Ad
T
EGEk
Rexp][mRNA d
0pp
cg
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link to cell functions
Thermodynamic Model of
Transcriptome Formation (2005)
(the theory and some supporting evidences)
![Page 17: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/17.jpg)
desirable framework for microarray
• Measurements– Standard
– Scale
• Interpretations– direct link to cell functions
physical framework
![Page 18: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/18.jpg)
The framework for microarray data
science
Common to
physics and
biochemistry
![Page 19: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/19.jpg)
feedback to the wet methods
Parametric Scanning
![Page 20: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/20.jpg)
The GeneChip system
• detects nucleotide hybridization,– Measure mRNA levels– Find SNPs
• has 1,000,000 probes (=cells) synthesized in situ
column ~ 1,000 cells
row ~ 1,000 cells
![Page 21: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/21.jpg)
pseudo image: comparison with a standard
![Page 22: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/22.jpg)
scratchy noise
pseudo image: comparison with a standard
![Page 23: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/23.jpg)
pseudo image: comparison with a standard
malfunction region
![Page 24: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/24.jpg)
pseudo image: comparison with a standard
air bubble?
troubles should be removedin prior to data analyses
![Page 25: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/25.jpg)
ideal standard
ideal standard
group of chips
brief normalization&
trimmed mean
![Page 26: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/26.jpg)
finding problems
ideal standard each chip
(z-score)(z-score)
Δz
Normalize with robust parameters
![Page 27: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/27.jpg)
distribution of Δz
in >85% of data,
Δz : N(0, 12)
![Page 28: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/28.jpg)
scanning the image by using moving windows
![Page 29: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/29.jpg)
scanning
• Any of large Δz can be large signal
• Clusters of large Δz should be extremely rare
medians in the moving window are challenged by the test
![Page 30: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/30.jpg)
distribution of the medians of windows
N(0, 12)
(central limiting theorem)
![Page 31: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/31.jpg)
test level is given as the expectation of cancellation
expect = 2
Number of windows:
n=500,000
Double sided test
test=qnorm(1/n)= -4.61
![Page 32: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/32.jpg)
cancellation
expectation = 2
![Page 33: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/33.jpg)
improvement in reproducibilityPMdata, repeated measurements
![Page 34: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/34.jpg)
Some innocent data are cancelled accompanying
only a limited portion!
![Page 35: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/35.jpg)
Now we can handle the .cel data
0
20-2
-2
22 4 7 3 9 3 _ a t
exp
1
exp 2
exp
1
0
-2
22 4 6 0 0 4 _ a t
20-2exp 2
verification by the cells
finding splicing variants
![Page 36: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/36.jpg)
Cancellation by dChip packagePMdata, repeated measurements
After cancellation Cancelled data
![Page 37: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/37.jpg)
improved reproducibility
MAS 5.0 SuperNORM
Exp. 1 (logratio)
Exp
. 2 (
logr
atio
)
Exp. 1 (logratio)
Exp
. 2 (
logr
atio
)
![Page 38: Detection and Restoration of Hybridization Problems in Affymetrix GeneChip Data by Parametric Scanning Tomokazu Konishi Akita Pref. Univ.](https://reader036.fdocuments.in/reader036/viewer/2022062408/56649ea05503460f94ba30ce/html5/thumbnails/38.jpg)
commercially available from …
Skylight-biotech Inc.
http://www.super-norm.com