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Cytogenetics 101:
Clinical Research and Molecular
Genetic Technologies
Topics for Today’s Presentation
2
What aCGH? 2
1
3
What is NGS? 4
Classical vs Molecular Cytogenetics
What is FISH?
5 How can these work together?
CAG EMEAI | Agilent Restricted | Page 3 | | Page 3 S1
Classical Cytogenetic Analysis
Technology Advancements: Smaller Aberrations
4
3-5Mb 50-100kb 5-10Mb 0.5Mb >60bp 1bp
Li and Andersson:
J Pediatr. 2009 Sep;155(3):311-7.
5
1Mb Deletion
Technology Advancements: CGH vs Karyotype
Topics for Today’s Presentation
6
What aCGH? 2
1
3
What is NGS? 4
Classical vs Molecular Cytogenetics
What is FISH?
5 How can these work together?
Small fragments of DNA from known regions of each chromosome are arrayed onto glass microscope slides.
How does it work?
Three Agilent
platform
technologies
for molecular
analysis
SurePrint
CGH+SNP
microarrays
SureFISH
HaloPlex
based on
OLS (Oligo
Library
Synthesis)
Typical Microarray Workflow
Page 9
Sample Nucleic Acid
Purification
Hybridization Scanning Data Analysis
Sample QC Labeling
CANCER
• Blood
• Bone marrow
• Solid tumor:
• Frozen
• FFPE
Postnatal
• Blood
• Saliva
PRENATAL
• Amniotic Fluid
• Chorionic Villus Sampling
• POC
Sample types used in clinical CGH research
Page 10
CGH Data Interpretation
11
Trisomy 21
12
Miller el. al. Amer J. Human Genet 86, 749–764, May 14, 2010 749
Technology Advancements: CGH vs. Karyotype
aCGH Detects Net Copy Changes
Page 13
Topics for Today’s Presentation
14
What aCGH? 2
1
3
What is NGS? 4
Classical vs Molecular Cytogenetics
What is FISH?
5 How can these work together?
What is F.I.S.H?
FISH uses fluorescent molecules to paint genes or
chromosomes.
Involves the use of short single stranded sequences of DNA
which are labeled with fluorescent tags to bind to the
complimentary sequence of DNA
Region
Size (kb)
Sequence
Tiled (kb)
1 23.3 14.1
2 20.0 14.1
3 27.9 14.1
4 27.6 14.1
5 31.4 14.1
6 23.6 13.6 c-met locus divided into 6 regions
Repeat
Gaps
1
Red : SureFISH probe
Green: BAC CEP
Region 4
Regions 1-6 20/20 metaphase and 20/20 interphase cells showed this staining
Sure
FIS
H
2 3 4
5 6
Detection of Smaller Regions: confirming the
aCGH results
Pros of FISH
• Rapid results, ~5 days (STAT case-24hrs)
• High efficiency of hybridization and detection
• Lots of cells can be analyzed
• Cells do not need to be actively dividing
Cons of FISH
• Not a “Global” test
• Dependant on lateral testing
• Validation of new probes is time consuming and requires a lot
of tech time.
Topics for Today’s Presentation
20
What aCGH? 2
1
3
What is NGS? 4
Classical vs Molecular Cytogenetics
What is FISH?
5 How can these work together?
What is Next-Gen Sequencing: Brief History
• Frederick Sanger (Sanger Sequencing)
– “First Generation” (circa 1977)
• Radiolabeled Nucleotides (Sequencing Gels)
• Automated Capillary Electrophoresis
– “Second Generation”
• ABI 370 (500 Kb/day) ABI3730 (2.8 Mb/day)
• Helped drive the Human Genome Project
• Massively Parallel Sequencing
– “Next-Generation Sequencing”
• Does not use Sanger method
• High throughput with reduced cost
• Capable of Outputting >100 Gb/day!
• Desktop Sequencers & Beyond!
– “Next-Next-Generation Sequencing”
21
Target Enrichment: It’s just like fishing…
What is the concept?
1. Use oligos (i.e. baits) that are complementary to genomic regions of interest (i.e. ROI’s)
2. Baits will specifically “catch” (i.e. hybridize) to the ROI’s
3. Regions that are caught get sequenced, the rest is washed away
Why do it?
1. Sequence only your regions of interest!
2. Focus on a smaller subset
3. Sequence more samples per run (i.e. Multiplex)
4. Save time and money
5. Faster time to data
6. Identify variants in samples with increased
reliability and accuracy
Technology Advancements:
CGH vs. Next Gen Sequencing
Lonigro et al. Neoplasia Vol13(11) 2011, pp.1019-1025.
• Publications have explored WGS
for CNAs as well as Exome Capture
• Detection of CNAs using targeted
exome capture sequencing in
cancer
• 17 prostate tumor-normal pairs
(SSEL 38Mb vs. Agilent 244k CGH)
• Data sets highly concordant
23
SAT Analogy Question
Karyotyping : FISH
oKaryotyping : aCGH
oaCGH : NGS
oFISH : NGS
oKaryotyping : r-banding
SAT Analogy Question
Karyotyping : FISH
oKaryotyping : aCGH
oaCGH : NGS
oFISH : NGS
oKaryotyping : r-banding
Source: Frederick National Laboratory for Cancer Research
• Well-established
workflow and simple
analysis
• Ability to detect exon-
level resolution, LOH,
low-level mosaicism
• Decipher complex
rearrangements
• Leverage data already
generated by
international consortia
• Lower cost and higher
throughput for aCGH
compared to NGS
Current Benefits of aCGH over NGS for CNAs
-3 -2 -1 0 +1 +2 +3
Cy5
Cy3
Red
Green
Amp
Del
Exp
Ref
NGS aCGH
26
Topics for Today’s Presentation
27
What aCGH? 2
1
3
What is NGS? 4
Classical vs Molecular Cytogenetics
What is FISH?
5 How can these work together?
Applications: Targeted Cancer Panels
Journal Mol.Diagnostics, Vol. 11(5): January 2009
Mapping 13q14 deletions in 41 cases
28
Method: • 62 CLL samples (pilot) followed by 38
blinded samples (>25% CLL) to compare
w/ FISH and/or, G-banding karyotype
• Agilent custom 4x44k array genome-wide
coverage w/ high density in 15 CLL target
regions
Aim: To evaluate aCGH as cost-effective
alternative to multi-probe FISH for
profiling and risk stratification in CLL
29
Applications: Targeted Cancer Panels
Method: • Custom 4x44k CGH array, tiling EXT1,
EXT2 and 68 candidate genes
• Screen 17 patients with previously
undetected mutations to identify other
causative aberrations
Aim: To identify the 10-15% missing alterations
in EXT mutation-negative Multiple osteo-
chondroma (MO) cases.
Results: • Somatic mosaicism in EXT genes in 3
previously negative cases.
• Aberrations detected at 10-15%
mosaicism.
Hum. Mutat., 32: E2036–E2049. doi: 10.1002/humu.21423
Applications: Low-level Mosaicism
31
Complex rearrangements can be detected
by high resolution custom array that spans
the MECP2 locus.
Challenges: Complex Rearrangements
Inversion-Dup Duplication Triplication
The duplications and triplications are
detected by arrays while inversion were
detected by sequencing the break point.
Free Design Iteration
Journal Mol.Diagnostics, Vol. 11(1): January 2009
Mapping of 13q14 deletions in 41 CLL
cases that had detectable deletions
33
J Med Genet 2007 44 (1), e59
GLI3 Targeted CGH Array
Free Design Iteration: CLL and GLI3
Free Design Iteration: Ch18 Array
Heard P. et al. Am J Med Genet A. 2009 July; 149A(7): 1431–1437.
34
del Gaudio D et al. Human Mutation 29(9),1100-1107,2008
35
Free Design Iteration: Dystrophin gene
Three Agilent
platform
technologies
for molecular
analysis
SurePrint
CGH+SNP
microarrays
SureFISH
HaloPlex
based on
OLS (Oligo
Library
Synthesis)
• Agilent HaloPlex is “For Research Use Only. Not for use in
Diagnostic Procedures."
• Agilent CGH+SNP Microarrays are “For Research Use Only. Not
for Use in Diagnostic Procedures. User is Responsible for US FDA
Approval or Clearance Prior to Diagnostic Use.
• Agilent SureFISH probes are Analyte Specific Reagents. Analytical
and performance characteristics are not established.
37
Thank you
38