1. The 2007 Microarray Research Group Project
Comparison of Comparative Genomic Hybridization Technologies Across Microarray Platforms
The 2007 Microarray Research Group Project
Susan Hester
April 1, 2007

2. Two goals for this presentation:
Present a Comparative Genomic Hybridization
Study to the ABRF research community
2. Evaluate commercial CGH platforms for their
ability to detect known gains or losses

3. Background copy number differences between a reference genome and
Comparative Genomic Hybridization (CGH) measures DNA
copy number differences between a reference genome and
a test genome.
Early CGH experiments, the DNA targets were hybridized
to metaphase chromosome spreads in FISH assays.
This technology later evolved: DNA targets are hybridized
to microarrays containing cDNA fragments or bacterial
artificial chromosomes (BACs).
Commercial microarrays are characterized as whole-genome
CGH measures, obtaining copy number differences in DNA
across entire genomes.

4. What is CGH and what is it not?
CGH has nothing to do with gene expression.
CGH arrays are structural, not functional—they look for DNA that is
missing or duplicated, not DNA that is expressed.
In array CGH, thousands of probes are printed on a microscope slide.
These probes are bacterial artificial chromosomes, or BACs, which are
sequences of cloned human DNA Kb long that have been mapped
to specific locations on specific chromosomes.
To the array is added test DNA and reference or control DNA, each
labeled with a different dye. The essence of the experiment is that the two
DNA samples compete for hybridization to the BAC probes on the slide,
thus the "comparative" aspect of CGH.

5. The 2007 MARG Study:
The research question: How well do BAC and commercial CGH
arrays detect known copy number gains or losses in test and
reference DNA?
Analyzed Human leukemic HL60 DNA compared to reference with 5 platforms: RPCI BAC 19K arrays, Agilent 44K, Illumina HAP 550K, Affymetrix 500K, and Affymetrix U133 expression array.
DNA samples analyzed in quadruplicate.
3 different laboratory test sites; Roswell Park Cancer Institute, Memorial Sloan-Kettering Cancer Institute, and Columbus Children’s Hospital.
Quality assessment of each platform performed by each test site.

6. Known copy number gains and losses on cell line HL-60*
Amplification of the 8q24 locus, Trisomy 18
Deletions at loci 5q11.2-q31, 6q12, 9p21.3-p22,
10p12-p15, 14q22-q31, 17p12-p13.3, monosomy X
*Cancer Genet Cytogenet Nov;147(1):28-35; Peiffer et al.(2006)

7. The fundamental theory behind each platform*: nucleotide base-pairing
Before: hybridization:
DNA sample (varying locus concentrations reflecting inherent gains/losses)
Chromosome #1 probes
_ _ _ _ _ _ _ _ _ _ _ _
After hybridization:
DNA sample labeled and fragmented
_ _ _ _ __ _ _ _ __ _ _ _ _ __ _ _
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
gain gain gain
-_-- -
-_---
*in this example;
one color platform
loss loss

8. Chari et al, Cancer Informatics, 2006, 2, 48-58
Example of Array CGH Technology*
*in this example;
2-color platform
Chari et al, Cancer Informatics, 2006, 2, 48-58

9. Cont.

10. Platform Details Platforms # probes or probe sets (resolution)
annotation
platform resolution
protocol
replicates
primary data
BAC 19K1
~19,000 in duplicate
NCBI
Build 35 (hg17)
100kb
2-color
4 HL60/lung
Mean Log2
(test/ref)
Agilent2
~44,000
(~35 KB)
Sub 100kb
Affy U1333
54,675
1-color
4 HL60
4 lung
Log2(RMA)
Affy 500K2
~500,000
Build 35
(hg17)
0 lung
Reference database
Illumina2
~550,000
Log R ratio
The test sites were:
1Roswell Park Cancer Institute, Buffalo, NY,2Sloan-Kettering Institute Genomic Core, Rochester, NY
3Columbus Pediatric Hospital, Columbus Ohio

11. Study Questions: 1. What is the precision of each platform?
2. How accurately does each platform detect known copy number changes?
3. What is the resolution of array CGH?

12. Results: Assessment of precision- coefficient
of variation across replicates

13. Coefficient of variation statistics
BAC
Agilent
Illumina
Affy GE
Affy Nsp
Affy Sty
Mean
2.13
3.16
5.98
9.64
10.69
9.44
Median
1.96
2.66
5.23
8.71
9.91
8.47
Std Dev
1.09
2.43
3.52
4.78
5.24
5.60
Std Err
0.01
0.00
0.02
Size
18067
42897
555353
54676
524629
476709
Min
0.05
0.71
0.15
Max
16.87
77.89
98.05
59.57
48.97
50.59

14. Platform Visualizations:
Chromosome 8 gain
Chromosome 17 deletion
Chromosome 5 deletion

15. RPCI BAC 19K array
chr8 -2 -1 1 2 3
chr17 3
+8q24
-17p12 2 1 -1 -2

16. Agilent 44K
Chr. 5q deletion

17. Visualization of each platform: Illumina HAP 550K
Chr. 5q deletion
Replicate 1
Replicate 2
Replicate 3
Replicate 4
-5q11

18. Affymetrix U133 Gene Expression Array
Chromosome 17

19. Partek: Affy500K Chr 17p deletion Replicate 1 Replicate 2 Replicate 3

20. Results: Assessment of Accuracy: concordance with known gains/losses
Gain/Loss
-5q11
-6q12
+8q24
-9p21
-10p12
-14q22
-17p12
Tri 18
monoX
BAC Y N
Agilent
Affy U133 GE
Affy 500K
Illumina
red=amplification
black=loss

21. Results: Assessment of platform resolution
Circular binary segmentation analysis (CBS)*
This approach is used to translate noisy intensity
measurements into regions of equal copy number.
Detects the point at which signal changes.
Log ratios of normalized signal intensities are used.
log2 (signal HL60/ref DNA)
ex: normal state is 2copies/2copies=1
log2 (1)=0
*Olshen, A.B. et al. (2004)-Circular Binary Segmentation for the Analysis
of array-based DNA Copy Number Data

22. Adopted;

23. Circular Binary Segmentation
CBS
Calculation
Point of transition
in signal
Simulated
Intensity
Ratio
Segment

24. Assessment of platform resolution on a known
deletion or gain:
RPCI BAC 19K
Agilent 44K
Illumina HAP550
Affymetrix U133 Gene Expression
Affymetrix 500K

25. If 1 copy present in HL-60 and 2 in ref;
then intensity ratio= ½=0.5; log2(0.5)= -1
Log2 between
0 & -1= 1cn loss
0 & 0.5= 1cn gain
0.5 & 1= 2cn gain

26. CBS Summary Details for known gains/losses:
Deletion Chr.5 and Gain Chr.8
Platform
chromosome
loc.start
loc.end
num.probes*
seg.mean
Est. Copy number
RPCI BAC 5
53,817,423
139,426,041
491 -1
Agilent
53,787,209
139,475,226
1003
Illumina
53,729,928
113,350,609
10438
Affy GE
53,536,915
139,532,618
1212
Affy 500K**
53,925,665
73,549,145
13886 8
98,584,025
130,781,748 14
1.2942
>2
126,357,705
130,632,571 26
0.3993
>1
126,302,381
130,771,462
428
1.1630
126,335,997
128,498,243 22
1.612
126,000,000
131,000,000
1192
1.7650
*used only probes>10
**Data generated using Partek Software to determine gains/losses; Green= deletion
Red=gain

27. Did Agilent underestimate the change?
Chr.8 -entire view
zoomed-in view of amplification
at 120 to 130Mb
follows

28. of our Agilent data compared to other platforms;
CBS
2.011
CBS
1.1630
CBS
1.612
CBS
0.3993
Results:
lowest CBS ratio value and small dynamic range
of our Agilent data compared to other platforms;
huge number of segments in Illumina data

29. CBS analysis finds 3 novel gains or losses**
BAC& Affy500K agree -Chromosome 2
Illumina, Agilent, Affy GE, BAC and Affy500K
all agree on Chromosome 16
Affy500K and Affy GE agreement on Chromosome 19
** Agreement means that 2 of the 5 platforms found the same gain or
losses in the same location (start to end location)

30. Table of Novel Gains and Losses
 Platform
Chr.2
Chr.16
Chr.19
BAC Y N
Agilent
Affy U133
Affy 500K
Illumina
Red=gain
Black=loss
 Platform
Chr.2
Chr.16
Chr.19
BAC Y N
Agilent
Affy U133
Affy 500K
Illumina

31. Conclusions
Good precision across all platforms: CVs ranged from 2.1 to 10.7% across the platforms
Illumina and Affymetrix 500K identified 9 of 9 (100%) of the known losses and gains in HL60 DNA
Agilent, Affymetrix U133 gene expression, and RPCI BAC arrays identified 8 of 9 (89%) of known losses and gains in HL60 DNA
CBS used to assess resolution of each platform; all platforms detected known gains/losses
3 novel changes were also detected by at least 2 or more platforms

32. Acknowledgments The Microarray Research Group (MARG):
Susan Hester, Laura Reid, Agnes Viale, Norma Nowak, Herbert Auer, Kevin Knudtson, Bill Ward, Jay Tiesman, Caprice Rosato, Aldo Massimi, Greg Khitrov, and Nancy Denslow
Laboratory test sites:
Roswell Park Cancer Institute, Memorial Sloan-Kettering Cancer Institute,
and Columbus Children’s Hospital
Data analysis efforts:
US Environmental Protection Agency
Roswell Park Cancer Institute
Columbus Children’s Hospital
Expression Analysis
Funding support for this project:
The Executive Board of the Association of Biomolecular Resource Facilities

33. MARG poster number is RG4-S