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Custom High-Definition CGH (HD-CGH) Microarray

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

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Custom High-Definition CGH (HD-CGH) Microarray

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

  2. HD-CGH Custom MicroarrayHigh-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. Database Design Methodology • 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)

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

  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. CGH Custom Design Serviceshttp://brsfield.communications.agilent.com/CMDS/CMDS_Services_CGH.htm Database of ~ 4 million computationally validated CGH probes Design Requirements Probe design application (used by CMDS; soon available on eArray 4.0) Self-designed 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 • 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. Agilent-designed CGH Probes

  7. HD-CGH Example 1: ChrX Custom Design ChrX probes Catalog probes • 22580 probes spread across chrX • 20290 CGH catalog probes spread across genome • 50 replicate probes (10 X 5) • 1370 probes on CGH eQC grid

  8. HD-CGH Example 2: Chr17 Custom Design Chr17 probes Centromere probes Catalog probes • 38723 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. HD-CGH Example 2: Chr17 Custom Design Custom probes Catalog probes 1x density 1x density 3x density ERBB2 chr17:35110005 BRCA1 chr17:38451220 TOP2A chr17:35799136

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

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

  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. HD-CGH Example 4: Chr15 Design & Validation • 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 15 • 12 “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

  15. IDIC 15 - Isodicentric 15(47 chromosomes)

  16. HD-CGH Example 4: Chr15 Design & Validation 4 samples in dye-flip pairs DESIGN - ~ 38,000 probes on Chr15 (1 probe per 400-800bp). VALIDATION - Cell lines with known aberrations on Chr15. Karyotyping and FISH data available. 50kb Moving Average

  17. HD-CGH Example 4: Chr15 Design & Validation 4 samples in dye-flip pairs DESIGN - ~ 38,000 probes on Chr15 (1 probe per 400-800bp). VALIDATION - Cell lines with known aberrations on Chr15. Karyotyping and FISH data available. 50kb Moving Average

  18. HD-CGH Example 4: Chr15 Design & Validation 00_16 02_04 9930b 01_19 HI1539

  19. Chr15 HD-CGH custom array vs. Human CGH catalog array 01-19027/030 01-19280/333 9930b021/023 9930b270/279 Catalog CGH Array Chr15 HD-CGH Array

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