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Page 1 Custom High-Definition CGH (HD-CGH) Microarray.

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Presentation on theme: "Page 1 Custom High-Definition CGH (HD-CGH) Microarray."— Presentation transcript:

1 Page 1 Custom High-Definition CGH (HD-CGH) Microarray

2 Page 2 HD-CGH Custom Microarray High-Definition Profiling, Customized Content and Format Designed for studying DNA copy number aberrations in specific regions of interest at unparalleled resolution.  High resolution tiling capability in regions of interest (~400bp)  Leverage ~ 4 million computationally validated CGH probes  Based on UCSC Genome Browser hg17 (NCBI Build 35)  Customizable content and resolution (single array or array-set)  Supporting format(s): 44K, 22K and 2x11K  Compatible with current Agilent microarray platform and CGH protocols We design it, so You Don’t have to

3 Page 3 Probes design in the database utilizes the same methodology as used for the catalog CGH arrays, but with a number of improvements: Improved candidate selection algorithm for choosing 45,000,000 candidates probes by screening ~1,400,000,000 (60-mer) seq’s. Improved performance of ProbeSpec (Homology search tool) - Homology search for Hs35 with 45,000,000 probes took 7 days. Improved model for combining metrics and ranking probes. A database of 3.7M prescreened “Semifinal” probes DB Includes 1 probe/exon for all known genes (~200,000 probes) Database Design Methodology

4 Page 4 Probe Design Application Retrieve probes by: Chromosomal coordinates Cytobands Accessions Filters: Best in interval By density Number of probes per annotation

5 Page 5 Probe Design Application Array Creation by: Select array format Corresponding eQC grid Select probe groups in array set (can be 1 or more arrays per array-set) Replicate probe groups Option to select CGH Catalog probes to fill the remaining array space for normalization

6 Page 6 CGH Custom Design Services Custom CGH Probe Design Service (G4431A) OR eArray 4.0 (or higher) Perfect for researcher who are interested in higher-resolution tiling capability in regions of interest, and would like to take advantage of Agilent’s predesigned, computationally-validated CGH probes Human only. Other model organisms follow. Database of ~ 4 million computationally validated CGH probes Custom CGH Microarray Layout Service (G4430A) OR eArray 3.5 (or higher) Tailored to researchers who have provided own CGH probes or designed CGH probes via G4431A, and want the probes printed on one of Agilent’s microarray formats. 44K now; 22K, 2x11K in fall’05 Probe design application (used by CMDS; soon available on eArray 4.0) Design Requirements Self-designed CGH Probes Agilent- designed CGH Probes

7 Page 7 HD-CGH Example 1: ChrX Custom Design probes spread across chrX CGH catalog probes spread across genome 50 replicate probes (10 X 5) 1370 probes on CGH eQC grid Catalog probes ChrX probes

8 Page 8 HD-CGH Example 2: Chr17 Custom Design Catalog probes Chr17 probes Centromere probes chr17probes: 1–78,000,000 bp at two different densities 3714 CGH catalog probes spread across genome 433 CGH catalog probes adjacent to centromere 50 replicate probes (10 X 5) 1370 probes on CGH eQC grid

9 Page 9 Catalog probes Custom probes BRCA1 chr17: ERBB2 chr17: TOP2A chr17: HD-CGH Example 2: Chr17 Custom Design 3x density 1x density

10 Page 10 HD-CGH Example 3: Chr2 Design & Validation Design: Generated ~23,500 probes covering a 22 MB region on chromosome 2 (1 probe/950 bp). Also include catalog probes across genome. Validation: Flow-sorted chromosome 2 DNA spiked into 46,XY samples at quantity equivalent to 2-copies/cell. 46,XY(+2 chromosome 2)/46,XX i.e. 2 copies of each chromosome and 4 copies of Chr2.

11 Page 11 46XY(+2 chr2)/46XX XY/XX +2chr2/chr2 HD-CGH Example 3: Chr2 Design & Validation

12 Page 12 HD-CGH Example 3: Chr2 Design & Validation Sample: A cell line containing known aberrations on Chr2q.

13 Page 13 Autism Overview Autism is a lifelong developmental disability - the result of a neurological disorder affecting the functioning of the brain. Usually diagnosed during the first 3 years of life. An accurate diagnosis requires a team of professionals, which may include a pediatrician, child psychologist, speech and occupational therapist, among others. The prevalence rate of 1 of every 250 makes autism one of the most common developmental disabilities affecting 500,000 individuals in the U.S. alone. Autism occurs 4 times more frequently in males than females, and frequently occurs in association with other disorders that effect the functioning of the brain, such as epilepsy or metabolic disorders. Interferes with speech, learning and social skills development. Echolalia, (persons with autism echoing words and phrases instead of forming his/her own) is a common characteristic of autism. Symptoms can include any combination of the following: Abnormal responses to sensations, such as touch or hearing; unusual ways of relating to people, objects, and events; disturbances in the rate of appearance of physical, social and language skills; delays in language development are common; resistance to change; lack of spontaneous or imaginative play; difficulties with skilled motor functions; social withdrawal. Manifests as a spectrum disorder. Individuals with autism may display characteristics of the disorder that range from mild to severe. U.S. Autism Population Estimate 08/2005 (ages 3-22 ) 199,022 To date, only 2-3% of autistic kids have genetically distinct anomalies - localized to chr15q12-q13. Hypothesis - Many more autistic chromosomal abnormalities exist, but requires higher resolution detection platform.

14 Page 14 Design: Chr15q11-15q13 with 9,986 probes (i.e. ~1 probe/1,442 bps) Remainder of chr15 with 28,316 probes (i.e. ~1 probe/2,434 bps) Total of 38,302 probes on chr15 and 4,268 probes across rest of genome Total 42,570 biological probes on all human chromosomes eQC control grid (1,370 probes) plus Epstein Barr Virus (350 probes) Validation: 12 autistic children who had identified IDIC “typical” children who did not present with IDIC 15 (controls) Peripheral Blood drawn and PB Lymphocytes were immortalized with EBV (Epstein Barr Virus) – cell lines established from these patients DNAs isolated from these established cell lines were used for the study HD-CGH Example 4: Chr15 Design & Validation

15 Page 15 IDIC 15 - Isodicentric 15 (47 chromosomes)

16 Page 16 DESIGN - ~ 38,000 probes on Chr15 (1 probe per bp). VALIDATION - Cell lines with known aberrations on Chr15. Karyotyping and FISH data available. 4 samples in dye-flip pairs 50kb Moving Average HD-CGH Example 4: Chr15 Design & Validation

17 Page 17 DESIGN - ~ 38,000 probes on Chr15 (1 probe per bp). VALIDATION - Cell lines with known aberrations on Chr15. Karyotyping and FISH data available. 50kb Moving Average 4 samples in dye-flip pairs HD-CGH Example 4: Chr15 Design & Validation

18 Page 18 00_ b 02_04 01_19 HI1539 HD-CGH Example 4: Chr15 Design & Validation

19 Page / / b 021/ b 270/279 Chr15 HD-CGH custom array vs. Human CGH catalog array Catalog CGH ArrayChr15 HD-CGH Array


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