Cytogenetics 101: Clinical Research and Molecular Genetic Technologies

Cytogenetics 101: Clinical Research and Molecular Genetic Technologies

Topics for Today s Presentation 1 Classical vs Molecular Cytogenetics 2 What acgh? 3 What is FISH? 4 What is NGS? 5 How can these work together? 2

Classical Cytogenetic Analysis S1 CAG EMEAI Agilent Restricted Page 33

Technology Advancements: Smaller Aberrations 3-5Mb 50-100kb 5-10Mb 0.5Mb >60bp 1bp 4

Technology Advancements: CGH vs Karyotype 1Mb Deletion Li and Andersson: J Pediatr. 2009 Sep;155(3):311-7. 5

Topics for Today s Presentation 1 Classical vs Molecular Cytogenetics 2 What acgh? 3 What is FISH? 4 What is NGS? 5 How can these work together? 6

How does it work? Small fragments of DNA from known regions of each chromosome are arrayed onto glass microscope slides.

Three Agilent platform technologies for molecular analysis SurePrint CGH+SNP microarrays SureFISH HaloPlex based on OLS (Oligo Library Synthesis)

Typical Microarray Workflow Sample Nucleic Acid Purification Sample QC Labeling Hybridization Scanning Data Analysis Page 9

Sample types used in clinical CGH research CANCER Postnatal PRENATAL Blood Bone marrow Solid tumor: Frozen FFPE Blood Saliva Amniotic Fluid Chorionic Villus Sampling POC Page 10

CGH Data Interpretation Trisomy 21 11

Technology Advancements: CGH vs. Karyotype Miller el. al. Amer J. Human Genet 86, 749 764, May 14, 2010 749 12

acgh Detects Net Copy Changes Page 13

Topics for Today s Presentation 1 Classical vs Molecular Cytogenetics 2 What acgh? 3 What is FISH? 4 What is NGS? 5 How can these work together? 14

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

SureFISH Detection of Smaller Regions: confirming the acgh results Repeat Gaps Red : SureFISH probe Green: BAC CEP c-met locus divided into 6 regions 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 1 2 3 4 Region 4 5 6 Regions 1-6 20/20 metaphase and 20/20 interphase cells showed this staining

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 1 Classical vs Molecular Cytogenetics 2 What acgh? 3 What is FISH? 4 What is NGS? 5 How can these work together? 20

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 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 Lonigro et al. Neoplasia Vol13(11) 2011, pp.1019-1025. 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

Current Benefits of acgh over NGS for CNAs Well-established workflow and simple analysis Ability to detect exonlevel resolution, LOH, low-level mosaicism Decipher complex rearrangements Leverage data already generated by international consortia Cy5 Cy3 Red Green Exp Ref Amp Del -3-2 -1 0 +1 +2 +3 Lower cost and higher throughput for acgh compared to NGS acgh Source: Frederick National Laboratory for Cancer Research NGS 26

Topics for Today s Presentation 1 Classical vs Molecular Cytogenetics 2 What acgh? 3 What is FISH? 4 What is NGS? 5 How can these work together? 27

Applications: Targeted Cancer Panels Aim: To evaluate acgh as cost-effective alternative to multi-probe FISH for profiling and risk stratification in CLL Mapping 13q14 deletions in 41 cases 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 Journal Mol.Diagnostics, Vol. 11(5): January 2009 28

Applications: Targeted Cancer Panels 29

Applications: Low-level Mosaicism Aim: To identify the 10-15% missing alterations in EXT mutation-negative Multiple osteochondroma (MO) cases. Method: Custom 4x44k CGH array, tiling EXT1, EXT2 and 68 candidate genes Screen 17 patients with previously undetected mutations to identify other causative aberrations 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

Challenges: Complex Rearrangements Complex rearrangements can be detected by high resolution custom array that spans the MECP2 locus. The duplications and triplications are detected by arrays while inversion were detected by sequencing the break point. Duplication Triplication Inversion-Dup 31

Free Design Iteration

Free Design Iteration: CLL and GLI3 Mapping of 13q14 deletions in 41 CLL cases that had detectable deletions GLI3 Targeted CGH Array Journal Mol.Diagnostics, Vol. 11(1): January 2009 J Med Genet 2007 44 (1), e59 33

Free Design Iteration: Ch18 Array Heard P. et al. Am J Med Genet A. 2009 July; 149A(7): 1431 1437. 34

Free Design Iteration: Dystrophin gene del Gaudio D et al. Human Mutation 29(9),1100-1107,2008 35

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

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