agilent sureprint g3 cgh+snp microarray platform · pdf fileagilent sureprint g3 cgh+snp...

Agilent SurePrint G3 CGH+SNP

Microarray Platform

Your Complete Solution For Cytogenetic Research

For Research Use Only. Not for use in diagnostic procedures.

Complete workflow

• SurePrint G3 CGH+SNP microarrays

• Copy number changes and LOH/UPD on a single array

• Highest quality copy-number data

• Processing reagents and Automation

• Instruments and reagents optimized for Agilent microarrays, for greater

consistency of results

• Increased throughput with Bravo Automated Liquid Handling Platform

• Agilent CytoGenomics 1.5 analysis software

• Designed specifically for cytogeneticists


SUREPRINT G3 CGH+SNP


Breakthrough dual CGH+SNP microarray

• Single microarray that offers high resolution aberration calls in

addition to LOH/UPD (loss of heterozygosity/uniparental

disomy) detection

– No need to choose between high resolution copy number data and

LOH/UPD or alternatively run two separate microarrays

• Addition of SNP probes to the Agilent CGH SurePrint G3 CGH

arrays enables the detection of blocks of LOH/UPD

– CGH calls not affected by addition

of SNP content, high quality

copy-number data remains


UPD: uniparental disomy

• Both members of a chromosome pair or segments of a chromosome pair are

inherited from one parent

• UPD can result in an abnormal phenotype when the chromosomes involved are

imprinted, such that only the maternal or paternal allele of the pair is active

Remark: parental

samples needed

to detect

heterodisomy


LOH: loss of heterozygosity

• UPD in tumor cells is often referred to as acquired UPD or copy neutral LOH

(cnLOH)

• Common in both hematologic and solid tumors

Blood, Vol. 108, Issue 5, 1733-1743, September 1, 2006

normal cell

trisomic cell

LOH of entire

chromosome

LOH of arm

LOH of

part of

armmonosomic cell

interstitial regions of LOH


Measuring SNPs using restriction enzymes

• A subset of probes on a CGH array

measures SNPs in parallel to copy

number, on a single array

• Genotype SNPs using restriction

digestion (Alu/Rsa)

• Measure the copy number of one

allele at each SNP site relative to a

known reference

• Regions of LOH are located by

finding genomic regions with a

statistically significant scarcity of

heterozygous calls

• ~5-10 Mb LOH/UPD resolution

across the entire genome


DATA


Call rate at 95% confidence: >95%

Twelve samples hybridized against a single HapMap reference sample


Call accuracy: >99%

Three HapMap samples hybridized against a single HapMap


Detection of UPDe.g. NA20409: complete paternal isodisomy on chr. 15

SNP

data

CGH

data

No heterozygous SNPs

Copy-neutral LOH or UPD

Only TWO states of alleles appear:

1) 0 uncut copies

2) 2 uncut copies

Both copies were

inherited from the same

parent – UPD

BB

AB

AA


Confirmation of Copy Number Changese.g. NA09209: hemizygous deletion on chr. 17

SNP

data

CGH

dataDeletion at the

beginning of p-arm

Hemizygous deletion

TWO states of alleles appear (only one copy allele):

1) cut copy of the allele (0 copy)

2) uncut copy of the allele (1 copy)

B

A


Confirmation of Copy Number Changes e.g. NA04592: trisomy of chr. 21

Amplification

FOUR or more states

Each state corresponds to 0, 1, 2, 3 (or more) copies of the uncut allele

Amplification of chr. 21

SNP

data

CGH

data

BBB

ABB

AAB

AAA


Detection of Consanguinitye.g. NA06231: child from consanguineous parents

Extended segments of

homozygosity found

throughout the

genome

SNP

data

CGH

data

BB

AB

AA


Excellent correlation between Agilent and ILMN

93 LOH regions discovered in 3 consanguineous samples run on Agilent

CGH+SNP and Illumina 610 quad platforms

1

10

100

1 10 100Agile

nt C

GH

+S

NP

data

: L

OH

siz

e in M

b

Illumina Beadchip data: LOH size in Mb

LOH region size on Illumina and Agilent platforms

10.7 and 5.2 Mb regions

detected on Agilent but not

Illumina

5.3, 3.7, and 3.6 Mb

regions detected on

Illumina but not Agilent

r = 0.9952

Data generated at Baylor College of Medicine


AGILENT CYTOGENOMICS 1.5


Streamline data analysis for Cytogenetic labs

• Simple workflow to analyze samples processed on Agilent SurePrint G3

CGH and CGH+SNP microarrays.

• Fast 5-minute data analysis for a normal sample.

• Convenient input and output support connection to LIMS.

Sample info

Laboratory LIMSCyto report

Triage

& sign off

Run data

analysis


Powerful algorithms for calling CNC, LOH and UPD

Feature

Extraction

ADM-1

ADM-2

SNP CN & LOH

Log2 ratio of

intensities to call CNCs

No. of uncut alleles

to call LOH/UPD


Sample triage

Not approved for use in diagnostic

procedures

Preloaded tracks:

DGV-CNV, OMIM

Custom tracks

Auto updated

tracks based on

aberration

classification in

CytoGenomics

Database

Add notes to

aberrations

View aberrations

in UCSC

genome browser

Classify

aberrations

Showing number

of samples with

similar aberrations

in database

Suppress

aberrations

Right click on Classification allows query for

overlapping aberrations in database: results will

be shown as new track

Link out from gene

to external

sources: DGV,

OMIM, Entrez


Additional features in Agilent CytoGenomics 1.5

• Interval classification and database queries for similar aberrations

• Workflow Automation Mode for automatic processing of TIFF images and

report generation

• Upload data to Cartagenia bench

• Supports multiple versions of genome build and ability to lock into a specific

genome build

• Links to OMIM and OMIM morbid entries from aberrant intervals

• Multiple levels of user privilege settings

Download CytoGenomics free trial @ https://earray.chem.agilent.com/earray/


PUBLICATIONS


Evaluation of high-resolution array platforms

Agilent arrays:

• show the highest dynamic

range

• slope of the measurements

closest to the theoretical

values

Plot of measured log2 ratios compared to theoretical

values

Kresse et al. BMC Res Notes. 2010;3:223


• More probe density does not mean more array resolution

• Even with half the number of probes the Agilent 1M array has:

– better exon/gene coverage

– lower DLRSD

– less signal variation

– higher copy-number detection rate

• Higher DLR values in SNP6.0

reduces the resolution of

abnormality detection

– SNP6.0 unable to call a focal gain

on PAX5 identified by 8 and 31

probes using Agilent 244K and

Agilent 1M arrays, respectively

Agilent 244K, 1M, Affymetrix SNP6.0 comparison

Braggio et al. AACR 101st Annual Meeting, April 2010


DNA copy number array technologies assessed

• Agilent was the best performing platform and for the SNP-CGH platforms,

Affymetrix tended to outperform Illumina.

• Nimblegen failed to detect numerous aberrations that were clear in the other

platforms even when probes were tiled in the region of interest.

• Nimblegen exhibits 2-4 fold greater variance amongst replicate probes and

variances an order of magnitude greater for replicate array comparisons.

Clarity of signal Agilent > Affymetrix > Illumina > Nimblegen

Curtis et al. BMC Genomics. 2009;10:588


Automation of the CGH sample preparation with

the Bravo Automated Liquid Handling Platform

• Agilent Automation Solutions (formerly Velocity 11) Bravo platform was

successfully used in the WTCCC 19,000 sample landmark CNV study

• 19,000 samples in 19 weeks


PRODUCT INFORMATION


SurePrint G3 CGH+SNP Products

TypePart

NumberProduct Description

CGH

probe

focus

# slides

per kit

SurePrint G3

Catalog

G4842A SurePrint G3 CGH+SNP Array Kit 2x400K Genes and

exons5

G4890A SurePrint G3 CGH+SNP Array Kit 4x180K ISCA 3

SurePrint G3

Custom

G4882A SurePrint G3 Custom CGH+SNP 1x1M Custom 1

G4883A SurePrint G3 Custom CGH+SNP 2x400K Custom 1




Agilent Catalog ISCA CGH+SNP Microarray

CGH probesSNP probes

60K SNP probes ~5-10 mb LOH

4x180K SurePrint G3 CGH+SNP kit P/N G4890A


Design your Custom ISCA CGH+SNP Microarray

Visit: https://www.agilent.com/genomics/earray

• Use the ISCA targeted

regions probe groups

• Add your own content in

eArray


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