An enterprise of the University of
Utah and its Department of Pathology
Customized oligonucleotide FISH probes offer
increased sensitivity in characterizing
abnormalities detected by array CGH
L.S. Rector1, N.A. Yamada2, M.E. Aston1, M.C. Sederberg1 R.A. Ach2, P. Tsang2, E. Carr2, A. Scheffer-Wong2, N. Sampas2,
B. Peter2, S. Laderman2, L. Bruhn2, A.R. Brothman1,3
1ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Utah
2Agilent Laboratories, Agilent Technologies, Santa Clara, CA
3Departments of Pediatrics, Human Genetics and Pathology, University of Utah School of Medicine, Salt Lake City, Utah
Abstract
Array comparative genomic hybridization (aCGH) is now an accepted methodology for detection of genomic copy number changes. FISH is often used to confirm and further characterize abnormalities detected by aCGH. It is not unusual for
abnormalities detected by aCGH to be in a genomic region or of a genomic size which prohibits traditional FISH using large probes such as bacterial artificial chromosome (BAC) vectors. Our goal was to establish a robust method for visualizing FISH
probes on metaphase chromosomes to further characterize small abnormalities which would not be readily seen by conventional FISH techniques. Oligonucleotide FISH (oFISH) is an alternative to BAC FISH because it allows for higher resolution
visualization of smaller loci. In this study, libraries of long oligonucleotides (194mers) with high complexity (~>150 oligonucleotides per locus) were chemically synthesized. Probe generation involved PCR amplification, followed by the introduction of
fluorescent labels by chemical modification. By directing oFISH probes at the most informative elements of the region under investigation, we achieved high specificity and eliminated the need for Cot-1 or other suppressive hybridization reagents. Upon the
initial evaluation of 14 oFISH probes, 12 of these (86%) gave strong signals with good specificity and sensitivity. The two oFISH probes which did not hybridize well initially included regions at distal 4p and Xq which both contain a high frequency of repeat
sequences. When a modified in silico oligo selection method was used to enhance signal while maintaining specificity, easily detectable signal was achieved at the expected sites in the 4p and Xq regions even though as little as 10% (6.5 Kb) of the initial
region was included in the probe sequences. Ongoing evaluation of additional oFISH probes indicates that this method will be powerful for routine analysis of previously difficult genomic regions. The ease, reproducibility and high level accuracy of this
technique suggest that it may prove invaluable to the human genetics community.
Introduction
Results
Conclusion
In Silico Selected
FISH is one of the most common methods
used to confirm and further define array
findings. Regions for which commercial
probes are not available, regions that are
difficult to detect by using FISH, or regions
smaller than 50 kb make it challenging to
see many abnormalities identified by
aCGH.
Objectives of Study
• Develop a robust method for
generating FISH probes especially
those less than 50kb to characterize
abnormalities detected by aCGH
• Evaluate modified in silico selected
probe design
Simple Tiling Probes
Locus
Size (kb)
I
Sp
Se
Rep
Value
6p22.2
6.7
2
4
4
5
15
16p13.3
28.5
5
5
5
5
20
16p13.3
15.6
5
5
5
5
20
17p13.3
45.3
5
5
5
5
20
4p16.3
62.5
3
5
5
5
18
Xq27.1
31.0
1
------
------
------
1
Locus
size
(kb)
Tiled
(kb)
Xq27.1*
31
6.8
1
2q131
97
32
5
5
5
5
20
2q37.3*
77.1
8.1
4
1**
5
5
15
4p16.3*
62.5
6.5
2
5
5
5
17
4p16.3
23.1
8.6
4
5
5
5
19
Locus
Size (kb)
5p15.3
116.6
40
5
5
5
5
20
6p22.2
6.7
1
15
5p15.3
42.3
30
5
5
5
5
20
16p13.3
28.5
19
20
16p13.3
15.6
19
20
17p13.3
45.3
20
20
I
Sp
Se
Rep
Values
------
------
------
1
Comparison of Simple Tiling Values with In Silico Selected Values
Simple Tiling
In Silico
6p22.2*1
6.7
3.6
1
6p25.3***
110.3
55
5
1***
5
5
16
4p16.3
62.5
17
18
8q23.3
38.2
29
3
5
5
5
18
Xq27.1
31.0
1
1
9p24.3
75.5
31
5
5
5
5
20
9q34.31
79.5
34
5
5
5
5
20
10q23
123.8
27
5
5
5
5
20
Locus
Array Findings
oFISH Findings
16p13.3*
28.5
16
4
5
5
5
19
2q13
Deletion
Deletion
16p13.3*1
15.6
10
4
5
5
5
19
2q37.3
Deletion
Deletion
4p16.3
Duplication
Duplication
17p13.3*
45.3
25
5
5
5
5
20
4p16.3
Deletion
Deletion
19q12
33.1
17
4
5
5
5
19
5p15.3
Duplication
Duplication
5p13.3
Duplication
Duplication
6p25.3
Duplication
Duplication
9p24.3
Duplication
Duplication
9q34.3
Duplication
Duplication
10q26.3
Duplication
Duplication
------
------
------
I = Intensity; Sp = Specificity; Se = Sensitivity; Rep = Reproducibility
*In silico probes available **Cross hybridized to 10q *** This locus represents a known
polymorphic site and was shown to hybridize to two separate regions
1Representative Examples
• Characterize and validate these
probes with a rigorous objective
criteria
1
aCGH Samples
97 kb NPHP1 (2q13) deletion seen by aCGH
Abnormality detected by aCGH
(uArray, ISCA Consortium design Agilent)
Probe design based on genomic coordinates
Probe generation by chemical synthesis,
involving PCR and fluorescent labeling
Co-denaturation/hybridization of probes
and slides using standard FISH methods
FISH and inverted DAPI banding of metaphase cell confirms NPHP1
deletion (arrow indicates deleted chromosome)
79.5 kb 9q34 duplication seen by aCGH
Analysis of probes
Each probe was tested for within- and between-run
reproducibility, in addition to a specificity and sensitivity
study resulting in 6 separate slides evaluated per probe
Analysis Criteria:
Category
Specificity
Sensitivity
Reproducibility
• In silico selected probes appear to
improve signals over simple tiling
probes (5/6).
By using the oFISH method, we have
been able to visualize small probes or
those which were either unavailable or
unacceptable using traditional FISH
methods. The in silico selected design
displays an overall better performance
over
unselected
sequences
while
maintaining
specificity.
This
methodology for probe design should be
considered for future applications. As
research in the field of molecular
genomics advances, we are likely to see a
greater number of small regions which
will require characterization in whole
cells; the oFISH method is well suited to
play a significant role in that endeavor.
Rationale:
Score
1
Signal
Intensity
• Difficult genomic regions not
previously
detectable
by
traditional FISH methods are
visible using oFISH, such as the
NPHP1 region at 2q13. (see poster
PB#1092)
Discussion
Representative Examples
Methods
• oFISH is highly reproducible and
produces robust signals in 83%
(15/17) of the initial probes studied
and confirmed aCGH findings in
100% of samples tested.
Interpretation
Non-existent
2
Poor
3
Average
4
Fair
5
Good
1
<50%
2
60-69%
3
70-79%
4
80-89%
5
>90%
1
<50%
2
60-69%
3
70-79%
4
80-89%
5
>90%
1
20%
2
40%
3
60%
4
80%
5
100%
In cases where no signal was detected,
there were no values for any category
Metaphase cell identified location of probe
at 9q34 (arrow indicates likely duplication)
Interphase cell
confirmed duplication
Specificity = # of signals seen at correct location
Total # of signals
Sensitivity = # of signals seen at correct location
# of expected signals
# of slides with same
Reproducibility = intensity, sensitivity, and
specificity
Total # of slides scored
Value = sum of individual scores
6p22 oFISH probe and inverted DAPI banding (6.7kb)
16p13.3 oFISH probe and inverted DAPI banding (15.6 kb )
References
• Bailey, Kidd, Eichler Human copy number
polymorphic genes. Cytogenet. Genome Res.
123:234-243, 2008
• Baldwin et al. Genet Med 10:415-429, 2008
• International Standard Cytogenomic Array
Consortium (ISCA,
https://isca.genetics.emory.edu/iscaBrowser/)
• Wolff, DJ et al. Guidance for Fluorescence in
Situ Hybridization Testing in Hematologic
Disorders. Journal of Molecular Diagnostics
2007, 9:134-43.