artifacts and effects in gene expression data carlo colantuoni april 12, 2006
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Artifacts and Effects in Gene Expression Data
Carlo Colantuoni
April 12, 2006
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Experimental Artifacts
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~200 microarrays ~100 samples
Nylon
NIA cDNA microarray Core Facility
P33
9600MGC
elements
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Uncorrected Intensities: MDS Colored by Batch
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Removing The Batch Effect
We Will Use These Dimensions for Additional Corrective Transformations
Much LikeRed:Green Analysis
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Uncorrected Intensities: MDS Colored by Batch
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Batch Subtracted Measures: MDS Colored by Batch
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MDS of All Array Experiments: Subject Replicates
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Hybridization Artifacts
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A “Simple” Pilot:
2 subjects in rep. = 4 arrays
Differing amounts of dye2-color (reference)
~48,000 probes
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4 arrays: Raw Log Intensities
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4 arrays: Raw Linear Intensities
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1 array: Ratio v. Intensity
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1 array: Ratio v. Intensity
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Biological Effects
… or are they?
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Big Effects:
Tissue Types and Growth Factor
Treatments
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Illumina 24K
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Smaller Effects:
Correlation of Gene Expression with
Biological Indices
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pH
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PMI
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age
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NylonP33
10K
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Illuminacustom
700
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More Subtle Effects:
Differential Gene Expression by Genotype
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COMT Val158Met SNP Affects Cognition and Risk for Schizophrenia
COMT enzyme activity
GeneticsCognition & Disease
Risk for Schizophrenia
Working Memory Performance
Patterns of Cortical Activation
Amphetamine & Tolcapone Response
VVVMMM
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p<0.00002
Over-Expression of HSP70 in VV Homozygotes
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VV-VM
Effect of COMT V158M on Gene Expression
NylonP33
10K
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MM-VM
Effect of COMT V158M on Gene Expression
NylonP33
10K
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VV-MM
Effect of COMT V158M on Gene Expression
NylonP33
10K
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VV-VM T-stat
MM
-VM
T-s
tat
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Looking Across Multiple Effects: Age and
Genotype
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N=15 genes across 80 subjects
p<7.34e-13
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Alternative Approaches
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COMT Activity as a Function of COMT Genotype
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-0.4 -0.2 0.0 0.2 0.4
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Distribution of Observed (black) and Permuted (blue) Correlations (r)
Correlation (r)
Den
sity
Correlation of COMT Activity with Expression
Permuted
Observed
Correlation (r)
N=64
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p<0.000089
r=0.45
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AcknowledgementsClinical Brain Disorders Branch, NIMH, NIH
Daniel Weinberger
Section on NeuropathologyJoel KleinmanThomas Hyde
Tissue ResourcesMary Herman
Amy Deep-SoboslayColleen Lynch
GenotypingRichard Straub
Bhaskar Kolachana
COMT ActivityJingshan ChenSamer Helem
RNA ResourcesJohanna CreswellClaudia AguirreRobert Fatula
Jeet BahraIsha Khan
Debora RothmondBarbara Lipska
Nick BeMariam Khan
National Institute on Drug Abuse, NIH, DHHSWilliam FreedElin Lehrmann
National Institute on Aging , NIH, DHHSKevin BeckerWilliam Wood
Diane Teichberg
Johns Hopkins School of Public HealthDepartment of Biostatistics
Scott ZegerZhianqan TanRafael Irizarry
Giovanni ParmigianiElizabeth Johnson
NHGRI Microarray FacilityAbdel Elkahloun
Iddil Berkov CBDB
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Beyond Individual Genes:Functional Gene Groups
• Borrow statistical power across entire
dataset
• Beyond threshold enrichment
• Systematic patterns throughout the dataset
-0.4 -0.2 0.0 0.2 0.4
01
23
Distribution of Observed (black) and Permuted (red+blue) Correlations (r)
Correlation (r)
Den
sity
Correlation of Age with Gene Expression
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Over-Expression of HSP70’s in VV Homozygotes
p<7.42e-08
T statistic
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3 Statistical Tests:
2
Kolmogorov-Smirnov
“Information”
Is THIS …
… Different from THIS?
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histogrambins
E
O
2
ED =
(O-E)2______
2 is the sum of D values where:
All Genes
Subset of Interest
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All Genes
Subset of Interest
Kolmogorov-Smirnov
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All Genes
Subset of Interest
Product of Individual Probabilities
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histogrambins
E
O
2
ED =
(O-E)2______
2 is the sum of D values where: E^0.5DPCA =
O-E______
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Dimension #1
Dim
ensi
on #
2
p value
0.0
>0.130
600
540
p<0.001
N = 20Pent.Phos.#30
p<0.032
N = 25Fruc.Mann.#51
p<0.097
N = 94Sphingo-Glycolip.#600
51
p<0.110
N = 96IP3#562
p<0.996
N = 44Pyrimid.Metabo.#240
p<0.999
N = 17Ribo-flavin#740
562
240
p<0.079
N = 3Lipo-Polysacch.#540
p<0.107
N = 4Lys.Biosyn.#300
740
300
Log10 Ratio Z-Score
Pro
port
ion
of G
enes
p<0.079
N = 24Aln.Asp.#252
p<0.133
N = 7C byfolate#670
252
670
N = 89 Gene Subsets
All Genes
The distribution of gene expression values for each gene group is passed to PCA as D^0.5 values and then plotted as a single point in low dimensional space.
Distance from center indicates deviation from distribution of all gene expression values in the microarray experiment
Proximity indicates similarity in the shape of distributions.
ED =
(O-E)2______
E^0.5DPCA =
O-E______
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Analysis of Gene Networks
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No Effect of Other COMT SNPs: P3224
Permuted
Observed
T statistic
1/1-1/2N=21 N=30
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0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.5
1.0
1.5
2.0
Distribution of p-values from Observed (Black) and Permuted Data
p-value
Den
sity
Distribution of p-values
Permuted
Observed
p-value
N=90
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-0.4 -0.2 0.0 0.2 0.4
01
23
Distribution of Observed (black) and Permuted (red+blue) Correlations (r)
Correlation (r)
Den
sity
Permuted
Correlation of Age with Gene Expression
Observed
Correlation (r)
N=90
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-0.45 -0.40 -0.35 -0.30
0.00
0.05
0.10
0.15
0.20
Distribution of Observed (black) and Permuted (red+blue) Correlations (r)
Correlation (r)
Den
sity
Permuted
Observed=
Correlation of Age with Gene Expression
FDR =False Pos.
Total Pos.
Permuted
Observed
Correlation (r)
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Correlation of GFAP Expression with Age
r=0.47
p<0.000002
Age (yr)
Ex
pre
ss
ion
: L
og
(Rat
io)
SD
Un
its
fro
m M
ea
n
(p<0.02)
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2 arrays(4 channels)