Download Document

Microarray Experimentation:
Design for a Meaningful Analysis
Toni Reverter
CSIRO Livestock Industries
Tissue Samples
Treat A
Treat B
Analysis
mRNA Extraction & Amplification
+
cDNA “A” Cy5
cDNA “B” Cy3
Image Capture
Laser 1
Hybridization
Laser 2
Optical Scanner
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Technical Concerns
Biochemist Level:
1. Preparation (Printing) of the Chip
2. RNA Extraction, Amplification and Hybridisation
3. Optical Scanner (Reading)
Quantitative Level:
1.
2.
3.
4.
Design
Image (data) Quality
Data Analysis
Data Storage
Replication:
1. Animal
2. Sample
3. Array
4. Spot
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Image/Data Quality Concerns: GP3xCLI
##############################################################
# GP3xCLI
#
# GenePix Processing Program by CSIRO Livestock Industries #
#
#
# Enquiries: [email protected]
#
# Copyright (c) 2003 CSIRO-LI
#
##############################################################
GPR Input:
Processed on:
F12.gpr
Tue Apr
8 13:40:01 EST 2003
=-=-=-=-=-=-= IMAGE QUALITY =-=-=-=-=-=-=-=
Total No. of Spots ------------------------> 19200
Spots
Spots
Red
Green
with Flag = -50 -------------------->
with Flag = -100 -------------------->
dye with Background >= Foreground --->
dye with Background >= Foreground --->
4720
12
892
915
Median to Mean Correlation Analysis:
DATA LEFT
RED
GREEN
Corr
Raw
Log2
Raw
Log2
______________________________________
> 0.00
19200 19200
19200 19200
> 0.20
19199 19200
19199 19200
> 0.40
19183 19200
19192 19200
> 0.60
19008 19200
19102 19200
> 0.80
17061 19199
18541 19198
> 0.85
14466 19193
17872 19196
> 0.90
10491 19137
15786 19181
=-=-=-=-=-=-= VALID SPOTS* =-=-=-=-=-=-=-=
Total No. of Valid Spots -----------------> 14433
Percentage of Valid Spots -----------------> 75.2
Total
Mean
Min.
Max.
No.
No.
No.
No.
of Genes ------------------------> 7220
Repetitions ----->
2 for 6600 Genes
Repetitions ----->
1 for
580 Genes
Repetitions -----> 24 for
8 Genes
Log(R/G) vs 0.5*Log(R*G)
________
____________
N
14433
14433
Mean
-0.017
10.327
Std
0.617
2.079
Min
-8.711
3.246
Max
4.030
15.994
Correlation
0.362
Log(R/G) across Intensity Values
Intensity
Spots
% <0
% >0
__________________________________
( 0 , 4)
4
100.0
0.0
( 4 , 8)
1499
74.1
25.9
( 8 , 12)
9847
40.4
59.6
(12 , 16)
3083
17.3
82.7
__________________________________
*NB: Valid Spot defined as spots with Background < Foreground for
both Red and Green channels and with a Quality Flag of 0.
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Data Storage: GEXEX
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Data Storage: GEXEX
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
4 Key Issues
Identify/Prioritise Questions
N of Available Samples
N of Available Arrays
Consider Dye Bias
More arrays
On key questions
Pooling?
Dye-Swap
Dye-Balancing
Self-Self
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
O
A
O
A
O
A
B
AB
B
AB
B
AB
Reference
Loop
All-Pairs
Variance of Estimated Effects
(Relative to the All-Pairs)
Main effect of A
Main effect of B
Interaction AB
Contrast A-B
Reference
1
1
3
2
Loop
4/3
1
8/3
1
All-Pairs
1
1
2
1
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Glonek & Solomon
Factorial and Time Course Designs for
cDNA Microarray Experiments
• Read pp 1-2
• Definition
A design with a total of n slides and design matrix X is said to be admissible
if there exists no other design with n slides and design matrix X* such that
ci*  ci
For all i with strict inequality for at least one i. Where ci* and ci are respectively
the diagonal elements of (X*’X*)-1 and (X’X)-1.
• Read pp 24
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Arrays:
(S-1)
to S·(S-1)
Samples:
S=3
2
6
S=4
3
12
S = 12
11
132
What is the No. of Possible Configurations?
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
SA-1
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Pie-Bald black
Non-Pie-Bald black
Normal
White
SA-1
=
53
= 125
Recessive
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
x5
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
0 hr
24 hr
SA-1 = 109 = 1 Billion!
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Opt 1: 10 Slides
Opt 2: 10 Slides
Opt 4: 9 Slides
Opt 5: 9 Slides
Opt 3: 11 Slides
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
R
24: 23 To 552
F
HS
M
TM
F
HS
G
G
R
R
G
R
R
G
pooling
14: 13 To 182
M
F
HS
TM
G
R
G
G
R
G
R
R
M
F
M
HS
HS
HS
R
G
R
G
G
R
G
G
R
R
G
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
RES
RES
SUS
0
3
24
M
F
HS
TM
8
-8
1
0
-1
-1.766
1.766
-3.866
3.866
8
-1
0
1
1.766
-1.766
3.866
-3.866
8
-4
-4
-1.335
1.335
0.666
-0.666
10
-6
-1.033
1.033
-0.468
0.468
10
2.368
-2.368
-0.198
0.198
6.247
-6.247
0.493
-0.493
6.247
-0.493
0.493
3.798
-3.798
SUS
0
3
24
M
F
HS
TM
Sum(ABS)
3.798
29.3
29.3
22.0
23.0
39.1
23.1
27.1
21.7
21.7
17.6
17.6
7.1
7.1
14.3
14.3
Reference Design
Sum(ABS)
26.8 26.8
17.3
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Pavlidis et al.(2003) The effect of replication on gene
Expression microarray experiments. Bioinformatics 19:1620
>= 5 Replicates
10-15 Replicates
Peng et al.(2003) Statistical implications of pooling RNA
Samples for microarray experiments. BMC Bioinformatics 4:26
Power: n9c9  95%, n3c3  50%, n9c3  90%
n25c5  n20c20
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Optimal use of the given amount of
Replication and Arrays
Pregnancy
Lactation
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Optimal use of the given amount of
Replication and Arrays
Lactation
Lactation
4 Arrays
8 Arrays
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Optimal use of the given amount of
Replication and Arrays
Lactation
Lactation
12 Arrays
6 Arrays
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Optimal use of the given amount of
Replication and Arrays
High Energy Diet
Low Energy Diet
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
v1
d5P
v2
d1L
d22P
v3
Virgin
d25P
d130L
d5L
d80L
Phase 1 Phase 2A
Pregnant
Wallaby
36 Comp. / 72 Arrays
d213L
d168L
d180L
Phase 2B
Lactating
d220L
d260L
Phase 3
i1
i2
i3
Involuting
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Experimental Design
Wallaby
36 Comp. / 72 Arrays
Sydney Symposium, 29-30/09/03
Microarray Experimentation: Design for a Meaningful Analysis
Statistical Analysis
My view ………educated?
Relaxed data acquisition criteria
S2N > 1.00
M2M > 0.85
Moving away from
Ratios
“heavy-duty” normalisation techniques
Mixed-Model Equations
Check residuals
Check REML estimates of VC
Process results Gene x Treatment
Mixtures of Distributions
Sydney Symposium, 29-30/09/03