Center for Human Genetics
Oligonucleotide based array CGH test performance for detection of
chromosomal imbalances in single cells
Melotte C, Dimitriadou E, Boogaerts A, Vermeesch J
Center for Human Genetics, University Hospitals Leuven, Leuven, Belgium
Introduction
Microarray technology is rapidly replacing traditional FISH-based Pre-implantation Genetic Diagnosis (PGD) for carriers of reciprocal
translocations. To overcome family specific workup and errors related to technical issues affecting the accuracy of result interpretations, most
laboratories use BAC based array technology.
In this study, we present the development and validation of an oligonucleotide array CGH (aCGH) platform for the detection of chromosomal
imbalances in single cells.
Materials & methods
The CytoSure™ Embryo Screen Array (8x60k) from Oxford Gene Technology is an aCGH platform which utilizes 60-mer oligonucleotide probes.
The probe design of this array is based on sequencing information of single cell DNA amplified with the PicoPlex® WGA kit (Rubicon Genomics).
This special probe design ensures that the array content mirrors the amplified DNA resulting in an optimal hybridization and minimal possibility of
obtaining misleading results. The array is accompanied by a complimentary CytoSure interpret software which includes a dedicated PGS analysis
tool for simple detection of aneuploidies and large unbalanced structural aberrations in single cells.
PicoPlex whole genome DNA was amplified from unbalanced translocation products from 3 different PGD indications with multiple single
blastomeres per indication. In addition multiple single EBV cells from 3 different cell lines with known imbalances were amplified and tested on the
CytoSure™ Embryo Screen Array.
Cell type
blastomere
blastomere
blastomere
PGD indication
46,XY,t(14;19)(p12;q13.1)
45,XX,rob(13;14)(q10;q10),t(6;16)(q21;q13)
46,XY,t(5;9;13)(p13.3;q22.33;q22.3)
Cell type
EBV cell
EBV cell
EBV cell
Table 1
Conventional karyotype
46,XX, dup(7p21.3)
46,XY der(20)t(18;20)(p1121;p13)
46,XX,der(18),t(9;18)(p13;p11)
Table 2
Table 1 & 2: Origin of single cells used for testing the CytoSure™ Embryo Screen Array.
Sample preparation
& amplification
Purification &
measurement
• PicoPLEX® WGA Kit
(Rubicon Genomics)
DNA labeling
2 hours
• High Pure PCR
product purification
(Roche)
• CytoSure Genomic
DNA Labelling
O/N Hybridisation
• Mai Tai® (Oxford
Gene Technology)
Posthybridisation
wash & scanning
Feature extraction
& data analysis
• Little Dipper® (Oxford
Gene Technology)
• Microarray scanner
G2565CA (Agilent)
• CytoSure Interpret
Software (Oxford
Gene Technology)
Figure 1: Proces of the used oligonucleotide array technology for single cell analysis.
Results
Array CGH results of 50 different single cells were analysed and compared with genetic profiles obtained via BAC aCGH. This analysis
showed that all aberrations previously characterized by BAC aCGH were also detected by the oligo aCGH platform. They consist out of 10
different aneuploidies, 7 different unbalanced translocations and 18 different segmental imbalances.
Considering the aneuploidy detection, there was 1 inconsistency, in which the oligo aCGH platform detected a trisomy of chromosome 2
which was not present in the BAC aCGH profile.
Chromosome
1
2
7
8
14
5
9
10
11
13
18
Imbalance
Trisomy
Trisomy
Trisomy
Trisomy
Trisomy
Monosomy
Monosomy
Monosomy
Monosomy
Monosomy
Monosomy
Bac aCGH










Oligo aCGH











Table 3: Different aneuploidies detected.
Chromosome
6
13
16
14
19
19
19
Cytogenetic band
q21q27
q11q34
q13q24.3
q11.2q32.33
p13.3p11
q11q13.43
q13.11q13.43
Size (Mb)
55
96
26
88
23
31
24
Imbalance
loss
loss
gain
gain
loss
loss
gain
Bac aCGH







Oligo aCGH







Table 4: Different unbalanced translocations detected.
Visualisation of result in CytoSure interpret software
Chromosome Cytogenetic band Size (Mb) Imbalance
20
p13
1
loss
11
q24.3q25
4
loss
18
p11.32p11.23
7
loss
18
p11.32p11.23
8
gain
7
p23.3p21.1
10
gain
16
q22.1q23.2
11
gain
8
q24.22q24.3
15
loss
9
p24.3p22.1
19
gain
9
p24.3p21.3
23
loss
10
q24.1q26.3
38
loss
7
q31.31q36.3
39
loss
8
q22.1q24.3
51
loss
3
q13.33q29
77
gain
18
p11.32q23
78
loss
7
q21.11q36.3
79
loss
7
q11.21q36.3
89
gain
7
q11.21q36.3
89
loss
7
p22.3q33
137
gain
Bac aCGH


















Oligo aCGH


















Table 5: Different segmental imbalances detected.
Conclusion
Based on the data obtained from this validation study we
conclude that the CytoSure™ Embryo Screen Array
platform from Oxford Gene Technology allows reliable
detection of aneuplodies and unbalanced translocations in
DNA amplified from single cells.
Aneuploidy summary plot
Figure 2: Array result of female sample with trisomy 1(yellow) , partial duplication and deletion on chromosome 8
(green), partial duplication 19q (red).
Therefore this technology offers a generic alternative to
FISH for the detection of chromosomal imbalances in Preimplantation Genetic Diagnosis.