Page 1

Custom High-Definition CGH (HD-CGH) Microarray

Page 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

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

Page 4

Probe Design Application

Retrieve probes by:

•Chromosomal coordinates

•Cytobands

•Accessions

Filters:

•Best in interval

•By density

•Number of probes per annotation

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

Page 6

CGH Custom Design Serviceshttp://brsfield.communications.agilent.com/CMDS/CMDS_Services_CGH.htm

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

Page 7

HD-CGH Example 1: ChrX Custom Design

22580 probes spread across chrX

20290 CGH catalog probes spread across genome

50 replicate probes (10 X 5)

1370 probes on CGH eQC grid

BasePair0 50000000 100000000 150000000 200000000 250000000

chr1

chr10

chr11

chr12

chr13

chr14

chr15

chr16

chr17

chr18

chr19

chr2

chr20

chr21

chr22

chr3

chr4

chr5

chr6

chr7

chr8

chr9

chrX

chrY

Catalog probes ChrX probes

Page 8

HD-CGH Example 2: Chr17 Custom Design

Catalog probes Chr17 probes Centromere probes

BasePair0 200000000

chr1

chr10

chr11

chr12

chr13

chr14

chr15

chr16

chr17

chr18

chr19

chr2

chr20

chr21

chr22

chr3

chr4

chr5

chr6

chr7

chr8

chr9

chrX

chrY

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

Page 9

Catalog probes Custom probes

BRCA1 chr17:38451220ERBB2 chr17:35110005

TOP2A chr17:35799136

BasePair20000000 40000000 60000000

chr17

HD-CGH Example 2: Chr17 Custom Design

3x density1x density 1x density

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.

Page 11

46XY(+2 chr2)/46XX

XY/XX

+2chr2/chr2

HD-CGH Example 3: Chr2 Design & Validation

Page 12

HD-CGH Example 3: Chr2 Design & Validation

Sample: A cell line containing known aberrations on Chr2q.

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

Autism in the Literature

0

100

200

300

400

500

600

700

800

900

1997 1998 1999 2000 2001 2002 2003 2004 2005

Year

Ref

eren

ces

Autism

Autism + Genetics

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.

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 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

HD-CGH Example 4: Chr15 Design & Validation

Page 15

IDIC 15 - Isodicentric 15(47 chromosomes)

Page 16

DESIGN -

~ 38,000 probes on Chr15 (1 probe per 400-800bp).

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

Page 17

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

4 samples in dye-flip pairs

HD-CGH Example 4: Chr15 Design & Validation

Page 18

00_16 9930b 02_04

01_19HI1539

HD-CGH Example 4: Chr15 Design & Validation

Page 19

01-19280/333

01-19027/030

9930b021/023

9930b270/279

Chr15 HD-CGH custom array vs. Human CGH catalog array

Catalog CGH Array Chr15 HD-CGH Array