Reprint from

Biotechnology from bench to business Volume 30, Number 19 November 1, 2010

OMICS Drug Discovery Translational Medicine Bioprocessing Biobusiness

OMICS Tutorial

Improving LOH Detection on CGH Microarrays

Technology Strives to Bypass a Number
of Limitations of Current Products
Anniek De Witte and than 1,000-fold, microarrays can detect the role of varied chromosomal aberra-
Jeanene Swanson
much smaller genetic aberrations tions in susceptibility to diseases like

D
ue to its higher resolution,
oligo array CGH has recent-
ly made strong inroads in
the cytogenetic lab. Its not surprising.
With an increased sensitivity of more
A B
microduplications and microdele-
tionsthat may be linked to abnormal
phenotypes and diseases than can kary-
otype analysis.
As basic research studies revealing
C CGH microarrays is that they only
autism, mental retardation, and devel-
opmental delays continue to amass,
cytogenetic researchers are beginning to
understand the need to detect more and
smaller changes.
One problem, however, with array
detect copy-number changes, since they
measure total copies of alleles present
in a sample. Copy-neutral changes such
as loss of heterozygosity (LOH), uni-
parental disomy (UPD), and consan-
guinity, as well as balanced transloca-
tions, go undetected.
During cell division, recombination
between chromosomes might occur,
resulting in material exchange with
maintenance of the copy number. One
of the resulting possibilities is UPD,
where a person inherits two copies of

Anniek De Witte (anniek_de-witte@

Figure 1. Schematic for the Agilent CGH+SNP microarray workflow: Three possible cases for one SNP
example are shown: homozygous CC, heterozygous CA, and homozygous AA. For each case, homol- agilent.com) is CGH product manager
ogous regions of two chromosomes are shown, with the two possible alleles colored in red and/or and Jeanene Swanson is scientific
blue. (A) Neither of the SNP sites is cut by AluI or RsaI, which yields the highest signal level on the technical writer at Agilent Technolo-
microarray. (B) One of the SNP sites is cut by AluI or RsaI, which yields an intermediate signal level.
(C) Both of the SNP sites are cut by AluI or RsaI, which yields the lowest signal level. gies. Web: www.agilent.com.
a chromosome pair from one parent
and no copy from the other parent. If
the chromosomes involved are imprint-
ed such that the genes on these chro-
mosomes are monoallelically active
(i.e., only the maternal or paternal
allele of the pair is expressed), the
resulting phenotype may be abnormal.
UPD in tumor cells is often referred to
as acquired UPD or copy-neutral LOH
(cnLOH). Copy-neutral LOH is com-
mon in both hematologic and solid
tumors.
Being able to detect copy-neutral and
copy-number changes simultaneously
would go a long way toward improving
upon current efforts to create detailed
genomic profiles of disease susceptibili-
ty and disease states.
Agilent Technologies (www.agilent.
com/genomics) new SurePrint G3
CGH+SNP microarray was designed
to overcome the limitations of current
array CGH microarrays. It allows
cytogenetic researchers to detect both
copy-number and copy-neutral chro-
mosomal changes using one microar-
ray. The same labeling and hybridiza-
tion assay can be used, and an algo-
rithm has been incorporated into Agi-
lent Genomic Workbench 6.5 software
to report both copy-number and copy-
neutral changes.
The basis of Agilents new array is
the inclusion of both CGH and SNP
probes. Like with Agilents SurePrint
CGH microarrays, the CGH probes
measure the total number of alleles in a
chromosomal region. The SNP probes,
however, make it possible to measure
copy-neutral, allele-specific changes.
Using a restriction fragment enzymatic
digestion assay, SNPs located in the
enzymes recognition site can be geno-
typed. Regions of copy-neutral LOH or
UPD are then located by identifying
genomic regions with a scarcity of het-
erozygous SNP calls.
Protocol
The sample-preparation and
hybridization protocol is exactly the
same as that for CGH-only microar-
rays. We digested genomic DNA from
an unknown test sample and a control
sample with known genotype with AluI
and RsaI. Then we labeled the test sam-
ple with Cy5 dye and the control sam-
ple with Cy3 dye and hybridized both
samples to the same array. After wash-
ing, we scanned the slides at 3 micron
resolution on Agilents High Resolution
C Scanner and extracted and analyzed
the images using Agilent Genomic
Workbench 6.5 software.
Methodology
Our method is similar to restriction
fragment length polymorphism (RFLP).
In RFLP analysis a genomic DNA sam-
ple is digested by restriction enzymes
and the resulting fragments are separat-
ed by gel electrophoresis. Instead of
using gel electrophoresis we use a
microarray.
Restriction enzymes cut molecules of
DNA at specific recognition sites, so
cutting with a particular enzyme should
always produce the same size and num-
Figure 2. Agilent Genomic Workbench view of SNP data (number of uncut alleles, bottom panel), and
CGH data (log2 ratios, top panel) from a CGH+SNP array shows UPD of the entire chromosome 15. Set-
tings for CGH aberration calling: ADM-2, threshold 5, minimum of 3 probes 0.25 log2 ratio.
ber of fragments. However, when a site
is polymorphic, or existing as two alle-
les, the restriction enzyme will not rec-
ognize one allele and will not cut there,
leading to a length polymorphism.
The ~60,000 SNP probes on the
CGH+SNP microarray span variant
AluI or RsaI restriction enzyme recog-
nition sites and measure the copy num-
ber of the uncut allele at those loci (Fig-
ure 1). We measure the total copy num-
ber of the region encompassing the
SNP site by neighboring CGH probes.
The copy number of the cut allele can
then be inferred from the total copy
number and the copy number of the
uncut allele.
A SNP copy-number call is made
from the log ratio of the test sample
(Cy5 signal) versus a genotyped internal
reference (Cy3 signal). The copy num-
ber of each SNP in the reference sample
is known. In order to determine the
allele-specific copy number of the test
sample, the log ratios of the SNP probes
are adjusted by the copy number of the
reference sample.
The reference-adjusted log ratios
fall into three categories correspond-
ing to the copy numbers of the uncut
alleles in the sample, which corre-
spond to the three possible diploid
genotypes for the SNPs: AA, AB, or

November 1, 2010 genengnews.com Genetic Engineering & Biotechnology News

BB. Regions of copy-neutral LOH or
UPD are then located by identifying
genomic regions with a statistically
significant scarcity of heterozygous
SNP calls.
Results/Conclusions
With high-quality DNA samples, the
SNP call rate is greater than 95% with
greater than 99% accuracy, and the
presence of SNP probes does not affect
the performance of CGH probes. The
number and quality of copy number
aberrations detected on the SurePrint
G3 CGH+SNP microarrays is compara-
Genetic Engineering & Biotechnology News
ble to detection using SurePrint G3
CGH-only microarrays with the added
benefit of simultaneous identification of
copy-neutral aberrations.
For a normal diploid region of the
genome, one expects the 0, 1, and 2
SNP copy numbers of the uncut allele
to be randomly distributed. In a
diploid genome carrying a copy-neu-
tral LOH or UPD aberration, the SNP
probes will only report alleles that are
homozygously cut and uncut (0 and 2
uncut alleles) and, therefore, only two
states are found. Figure 2 shows an
example of UPD observed in an indi-
genengnews.com
vidual known to have genomic aber-
rations associated with Angelman
syndrome.
In conclusion, the SurePrint G3
CGH+SNP microarray affords the pre-
cision of SNP detection to measure
copy-neutral genomic changes with
~510 Mb resolution as well as the
reproducibility of Agilents CGH plat-
formall on a single microarray. As a
result, researchers no longer need to
choose between high-resolution, high-
quality CGH data and the detection of
LOH/UPD or alternatively run two sep-
arate experiments.
November 1, 2010