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