Agilent G3 CGH+SNP Microarrays offer genome-

wide detection of deletions, duplications, and loss

of heterozygosity

Iman Kishawi, Ph.D

Application Scientist
November 11th , 2010

November 10, 2010

 Agilent breakthrough technology offers dual
CGH+SNP microarray
The first microarray that offers high resolution aberration calls focused
on your regions of interest in addition to LOH/UPD(loss of
heterozygosity/ uniparental disomy) detection

Run a single microarray to detect copy number changes and copy

neutral LOH/UPD

No need to choose between high resolution copy number data and

LOH/UPD or alternatively run two separate microarrays

Addition of SNP probes to the Agilent CGH SurePrint G3 CGH existing

probes enables the detection of blocks of LOH/UPD
CGH calls not affected by addition of SNP content
High quality of CGH calls remains

Not approved for use in diagnostic

procedures
WHAT ARE cnLOH AND UPD?
Not approved for use in diagnostic
procedures
UPD: uniparental disomy of a whole chromosome
Both members of a chromosome pair or segments of a chromosome
pair are inherited from one parent

UPD can result in an abnormal phenotype when the chromosomes

involved are imprinted, such that only the maternal or paternal allele of
the pair is active

Detected Detected

Not approved for use in diagnostic

procedures
UPD: uniparental disomy of a whole chromosome

Both members of a chromosome pair or segments of a chromosome

pair are inherited from one parent

UPD can result in an abnormal phenotype when the chromosomes

involved are imprinted, such that only the maternal or paternal allele
of the pair is active

parental samples needed to detect heterodisomy

Not approved for use in diagnostic

procedures
Examples of UPD associated with a Phenotype
maternal UPD15

paternal UPD15

paternal UPD6

maternal UPD7

paternal UPD11

maternal UPD14

Not approved for use in diagnostic

procedures
 LOH: loss of heterozygosity in cancer
LOH of arm
monosomic cell

LOH of
LOH of entire part of
chromosome normal cell
arm

Mitotic recombination

trisomic cell
interstitial regions of LOH

Not approved for use in diagnostic

procedures
 LOH: loss of heterozygosity in cancer

UPD in tumor cells is often referred to as

acquired UPD or copy neutral LOH (cnLOH)

Common in both hematologic and solid

tumors

Not approved for use in diagnostic

procedures
TECHNICAL INFORMATION
Not approved for use in diagnostic
procedures
No change in the workflow is required for CGH+SNP
Reference Test
DNA DNA

CGH data

SNP data

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&''( ( ( ' )' ' *+*,#

Not approved for use in diagnostic

procedures
Measuring SNPs using restriction enzymes

Probes are designed to include restriction digestion sites (AluI/RsaI)

that overlap with known SNP sites

If both alleles in target DNA are cut (0 uncut alleles) the signal will be
low. For a heterozygous SNP call (1 uncut allele) the signal will be
intermediate and a homozygous uncut alleles (2 uncut alleles) the
signal will be high

We measure the copy number of one allele at each SNP site relative to
a known reference. The resulting data will be 0 uncut (AA), 1 uncut
(AB), 2 uncut (BB)

Regions of LOH are located by finding genomic regions with a

statistically significant scarcity of heterozygous calls

Not approved for use in diagnostic

procedures
 Homozygous CC Heterozygous CA Homozygous AA

How it works CG CT CG CT AG CT
CG CT AG CT AG CT

Workflow
Restriction CG CT CG CT AG
CT
digestion CG CT AG AG
CT CT
(AluI & RsaI)

CG CT CG CT AG
Enzymatic CT
AG
labeling CG CT
CT AG
CT

Hybridization
Wash
CG CT

CG CT

CG CT

CT

CT
AG

AG
Scan

FE 10.10
Homozygous: Heterozygous: Homozygous:
TWO UNCUT COPIES ONE UNCUT COPY ZERO UNCUT COPIES
(0 cut copy) = (1 cut copy) = (2 cut copies) =
AGW 6.5 high signal intermediate signal low signal

Not approved for use in diagnostic

procedures
 Sample
How it works (contd) No. uncut copies
0 uncut 1 uncut 2 uncut
Possible Sample vs Reference sample/reference

uncut combinations (in diploid

Reference
0 uncut 0/0 1/0 2/0

Known
regions) 1 uncut 0/1 1/1 2/1
2 uncut 0/2 1/2 2/2

1.2
1/1
1/1 ++
2/22/2 * SNPs for which the
no. of uncut copies for
Distribution of the SNP Probe 1 the reference is 0 are
excluded from the
raw log2 ratios analysis.
0.8
(Sample/Reference)
0.6

0.4 1/2
1/2
2/1
2/1
As we know the reference genotype
and the expected number of cuts at 0.2
0/1+ 0/2
+ 0/2
0/1 1/0
1/0++ 2/0
2/0 *
each SNP location (0, 1, 2)
0
-6 -4 -2 0 2 4 6
Log2Ratio

Not approved for use in diagnostic

procedures
 How it works (contd)
the SNP calls are corrected for the reference

Distribution of the SNP Probe Detection of a

reference-corrected log2 ratios LOH region

Non-human primate FISH

Statistically
significant
scarcity of
heterozygous
SNP calls

0 1 2
Number of uncut alleles

Not approved for use in diagnostic

procedures
 How many SNPs can be assayed with this method?
1. AluI & RsaI Cut Sites in Genome:
17,838,944

2. # SNPs in genome:
~17,804,034 (Sep 3rd 2009)

3. Overlap between 1 and 2: AluI & RsaI

800,000 SNPs HapMap Cut Sites
All SNPs
SNPs 17,838 k
4. # polymorphic HapMap SNPs: 17,804 k
4,165 k
~4,165,774

5. Overlap between 3 and 4:

~124,000 SNPs

6. MAF > 5% and empirical validation:

~65,000 SNPs

Corresponds to ~5-10 Mb LOH

resolution

Not approved for use in diagnostic

procedures
Position of SNPs and CGH probes
SurePrint G3 2x400K CGH+SNP microarray

chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
SNPs (~65,000)
17
18
CGH probes (~300,000)
19
20
21
22
X
Y

Not approved for use in diagnostic

procedures
ACCURACY OF DATA
Not approved for use in diagnostic
procedures
Call rate at 95% confidence: >95%

Call rate for 12 samples hybridized against a single HapMap reference sample

Not approved for use in diagnostic

procedures
Call accuracy: >99%

Call accuracy for three HapMap against HapMap hybridizations

Not approved for use in diagnostic

procedures
Excellent correlation between Agilent and ILMN
Agilent CGH+SNP data: LOH size in Mb LOH region size on Illumina and Agilent platforms
r2 = 0.995
100 10.7 and 5.2 Mb regions
detected on Agilent but
not Illumina

10

5.3, 3.7, and 3.6 Mb

regions detected on
Illumina but not Agilent
1
1 10 100

Illumina Beadchip data: LOH size in Mb

93 Absence of Heterozygosity regions discovered in 3

consanguineous samples run on Agilent CGH+SNP and
Illumina 610 quad platforms Data generated at Baylor College of Medicine
Not approved for use in diagnostic
procedures
EXAMPLES
Not approved for use in diagnostic
procedures
Some examples with expected aberrations

Not approved for use in diagnostic

procedures
Relationship of genotype to SNP status
- number of uncut alleles

In this example, B is uncut allele

Not approved for use in diagnostic

procedures
UPD on Chromosome 15, paternal isodisomy

BB
AB
AA

Not approved for use in diagnostic

procedures
Detection of consanguinity on chromosome 11

BB
AB
AA

Not approved for use in diagnostic

procedures
 Hemizygous deletion on chromosome 17

B
A

Not approved for use in diagnostic

procedures
Trisomy of chromosome 21

BBB
ABB
AAB
AAA
WHAT IS AGILENT GENOMIC
WORKBENCH 6.5?

Not approved for use in diagnostic

procedures
Agilent Genomic Workbench
What is it?
The Agilent Genomic Workbench is a desktop application that makes
available the functionality of multiple applications through a single user
interface, and streamlines the data analysis process.

Not approved for use in diagnostic

procedures
Software Companion Products
1. Agilent Genomic Workbench 6.5 (includes FE 10.10)
New SNP and LOH analysis algorithms
SNP data is different from CGH data: raw SNP log ratios from FE file
cannot be plotted, upfront analysis needed
Offers CGH+SNP microarray design (eArrayXD )
2. eArray
Researcher can easily design custom CGH+SNP microarrays
Choose from 28 million Human CGH probes; and 65,000 SNPs
Design files are directly downloadable for catalog & custom arrays
SurePrint G3 High Density arrays only (not HD)

Not approved for use in diagnostic

procedures
Agilent Genomic Workbench
How is the Software packaged?

Not approved for use in diagnostic

procedures
Agilent Genomic Workbench
eArrayXD

Not approved for use in diagnostic

procedures
Both eArray and Agilent Genomic Workbench:
eArrayXD offer SNP and CGH probe search

Search by probe ID

Not approved for use in diagnostic

procedures
Agilent Genomic Workbench
DNA Analytics

Not approved for use in diagnostic

procedures
New SNP Panel in Chromosome & Gene View

Not approved for use in diagnostic

procedures
CGH + SNP Analysis: SNP QC Metrics

Not approved for use in diagnostic

procedures
EXPERIMENTAL DESIGN

Not approved for use in diagnostic

procedures
 Need for a single genotyped reference sample
Advantages Disadvantages

HapMap sample: Genotypes available from database Because of differences in DNA extraction
NA18507 Yoruban Male method used for the experimental and the
Copy number and LOH/UPD data
reference sample, extra care needs to be taken
NA18517 Yoruban Female measured from the same hybridization
to accurately quantitate the DNA. Highly
NA12891 European Male
recommended to use Qubit fluorometer.
NA12878 European Female
NA18579 Chinese Female Need to rely on supply from Coriell

Individual reference CGH data quality will be very good Genotypes of reference sample need to be
sample determined beforehand by:
Copy number and LOH/UPD data
determined from the same hybridization hybridizing it against the 5 supported
HapMap samples on the Agilent
CGH+SNP microarrays (this experiment
only needs to be performed once, also
use Qubit fluorometer, see above)
or sequencing
Pooled reference CGH data quality will be very good (CNVs Genotypes of pooled reference sample can not be
sample are diluted out) determined
NOT SUPPORTED Easy supply of DNA Copy number and LOH/UPD calls are determined
from different hybridizations:
experimental sample vs. pooled reference
HapMap sample vs. pooled reference
LOH/UPD data is obtained by combining
experimental sample and HapMap sample in silico

Not approved for use in diagnostic

procedures
How to get started
1. Decide which reference to use
Option A: Supported HapMap sample: NA18507 (Yoruban Male), NA18517 (Yoruban Female),
NA12891 (European Male), NA12878 (European Female), or NA18579 (Chinese Female).
Option B: Genotype your own reference (single individual) by hybridizing against the 5 supported
HapMap samples on the Agilent CGH+SNP microarrays. This experiment only needs to be
performed once.
Option C: Genotype your own reference (single individual) by sequencing
2. For Option 1A and 1B: order samples from Coriell: 1, 2 (=1 male + 1 female), or all 5

3. For Option 1B: genotype the unknown single individual reference(s) by hybridizing it
against the 5 HapMaps on the CGH+SNP microarrays. Remark: use Qubit fluorometer
to verify concentrations of all DNA samples.

4. Decide which microarrays to use

Catalog microarrays: 4x180K or 2x400K
Custom microarrays: design arrays in eArray
5. Scan on Agilent C-scanner and Analyze data in AGW 6.5 (includes FE 10.10)

Not approved for use in diagnostic

procedures
Example of typical 1st CGH+SNP experiment
Array Cy5 sample Cy3 sample Purpose
1 NA18507 Customer reference (single Genotyping of reference
individual)
2 NA18517 Customer reference (single Genotyping of reference
individual)
3 NA12891 Customer reference (single Genotyping of reference
individual)
4 NA12878 Customer reference (single Genotyping of reference
individual)
5 NA18579 Customer reference (single Genotyping of reference
individual)
6 NA18507 NA12891 Call rate and call accuracy
7 Unknown #1 Customer reference (single Real data
individual)
8 Unknown #2 Customer reference (single Real data
individual)

Not approved for use in diagnostic

procedures
PRODUCT INFORMATION
Not approved for use in diagnostic
procedures
Products
Part # slides
Type Product Description
Number per kit
SurePrint G3

G4842A SurePrint G3 CGH+SNP Array Kit 2x400K 5

Catalog

G4890A SurePrint G3 CGH+SNP Array Kit 4x180K 3

G4882A SurePrint G3 Custom CGH+SNP 1x1M 1

SurePrint G3
Custom

G4883A SurePrint G3 Custom CGH+SNP 2x400K 1

G4884A SurePrint G3 Custom CGH+SNP 4x180K 1

G4885A SurePrint G3 Custom CGH+SNP 8x60K 1

Not approved for use in diagnostic

procedures
Catalog 2x400K vs. 4x180K
Catalog 2x400K Catalog 4x180K
CGH+SNP CGH+SNP
Number of CGH probes ~300,000 ~120,000
Median CGH probe ~7 Kb ~25 Kb
spacing
ISCA content none ISCA 8x60K
Exon biased yes no
Number of SNPs ~65,000 ~60,000
Number of SNP probes ~120,000 ~60,000
Number of SNP probes 2 1
per SNP
Copy-neutral LOH ~5-10 Mb ~5-10 Mb
resolution

Not approved for use in diagnostic

procedures
Updated Sample Prep Protocols
Enzymatic Labeling for Blood, Cells or Tissues
Main protocol for CGH+SNP microarrays
AluI/RsaI restriction digestion no longer optional but required, C-scanner required
WGA not supported
Manual P/N: G4410-90010 Version 6.3

Bravo Automated Liquid Handling Platform with Enzymatic Labeling

Methods
Enzymatic labeling on Bravo will be supported for CGH+SNP microarrays
Manual P/N: G4410-90040 Version 1.1

ULS Labeling for Blood, Cells, Tissues or FFPE

ULS labeling will not be supported for CGH+SNP microarrays (note was added to the
manual)
Manual P/N: G4410-90020 Version 3.2

Not approved for use in diagnostic

procedures
 Overview of Bravo Protocol
Steps in blue use the Bravo
Automated Liquid Handling
Platform.

Not approved for use in diagnostic

procedures
Supported sample types
Human only

Non-WGA (whole genome amplification)

Non-FFPE

Non-cancer
Challenges associated with cancer samples:
Normal clone contamination
Subclones, i.e. tumor heterogeneity
Copy number: 0, 1, 2, 3, 4, 5
Samples not necessarily mostly diploid
Some cancer samples only available as FFPE tissue
Algorithm development for cancer ongoing

Not approved for use in diagnostic

procedures
http://genomics.agilent.com/CollectionOverview.aspx?PageType=Applicatio
n&SubPageType=ApplicationOverview&PageID=2394

Not approved for use in diagnostic

procedures
James Lupski Seminar
http://www.cnpg.com/Video/flatFiles/1517/index.aspx

Not approved for use in diagnostic

procedures
Questions

Not approved for use in diagnostic

procedures