CytoGenomics 3 - Agilent...Agenda CYTOGENOMIC TRAINING Onstead Auditorium BSRB S3.8012 8:30-9:00...

CytoGenomics 3.0

For Research Use Only. Not for Use in Diagnostic

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Agenda

CYTOGENOMIC TRAINING Onstead Auditorium BSRB S3.8012

8:30-9:00 Sign in

9:00-9:30 Introduction and Installation Help

9:45-10:30 Feature Extraction

10:40-11:30 Microarray QC Metrics

11:30-12:30 Lunch Provided

12:30-1:30 Analysis Workflow

1:40-4:00 Interpretation in Triage View

4:00-4:30 Wrap-up


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Agenda


8:30-9:00 Sign in







4:00-4:30 Wrap-up


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What is array-CGH?

Array-CGH

• Microarray based Comparative Genomic Hybridization

• Used to detect gains and losses of genomic sequences

180K feature array = ~12,000 bands

High resolution G-bands = ~800 bands


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Agilent SurePrint Microarray

Platform

Oligonucleotide Probes

– DNA sequences from Synthesized oligonucleotides

• Usually 60-mers of nucleotides, varies by array

• Very high resolution (<20kb varies by design)


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Dual CGH+SNP microarray •Single microarray that offers high resolution aberration calls in

addition to LOH/UPD (loss of heterozygosity/uniparental

disomy) detection

• 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


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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 •

Agilent Dual CGH+SNP

Microarray Platform


Procedure

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How to translate an image into data

Sample Nucleic Acid

Purification Labeling

Hybridization Scanning

Sample QC

Extraction & Data Analysis


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Analysis using CytoGenomics Software


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Navigating in CytoGEnomics 3.0


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Live Demo

Agenda


8:30-9:00 Sign in







4:00-4:30 Wrap-up


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Translate pixels of an Image into

numerical data points



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Typical Workflow

• Grid Placement

optimize fit

define features and background pixels

• Flag Outliers

• Compute Background/Error and Correct Dye Bias

• Compute Ratios and SNP Allele calls

• Calculate overall Array QC Metrics

• Generate Output for Further Analysis


definitions.


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• Grid Placement

optimize fit of grid file (gal file)


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• Grid Placement

define features and background pixels



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• Flag Outliers

Non-uniformity

Outliers

Population

Outliers



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•Compute Background/Error and Correct Dye Bias

A dye bias is the difference in the red and green signals caused by

different efficiencies in labeling, emission or detection.

1. Select features used to normalize

(flagged features, inner quartile,

rank consistency)

2. Select method of normalization

(Rank Consistency method,

Linear and or Lowess



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Log base 2 (Log2) Ratio of Sample to Reference is used due to the

assumption that there are 2 copies (alleles) in a normal genome.

Sample 2 = Log2 Ratio 0

Reference 2

Sample 3 = Log2 Ratio 0.6

Reference 2

Sample 1 = Log2 Ratio -1

Reference 2


• Generate Output for Further Analysis


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Live Demo

Agenda


8:30-9:00 Sign in







4:00-4:30 Wrap-up


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High level info available on number of metrics that need

evaluation

Summary of QC in the sample review tab


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Metrics to inform on

quality of the sample, but

also on proper CN2 peak

assignment

SNP Associated QC metrics

CGH QC metrics

SNP QC metrics


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Live Demo

Agenda


8:30-9:00 Sign in







4:00-4:30 Wrap-up


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LUNCH

Agenda


8:30-9:00 Sign in







4:00-4:30 Wrap-up


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CGH Data Analysis


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Aberration (copy number change) detection methods:

CGH Data analysis

Proven Algorithm CNV detection.

ADM-2 algorithm: The Aberration Detection Method 2 (ADM-2) algorithm identifies

all aberration intervals in a given sample with consistently high or low log ratios

based on a statistical score. The score is proportional to the absolute log2 ratio and

to the length of the interval that significantly deviates from log2 ratio of zero. The

ADM-2 algorithm searches for intervals in which a statistical score based on the

average quality weighted log ratio of the sample and reference channels exceeds a

user specified threshold. ADM-2 reports contiguous genomic regions of arbitrary size

as aberrant regions.

ADM algorithm 4 probe filter is the ISCA/ICCG consortium recommended analysis

method.


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SNP Data analysis

Detection of LOH. CytoGenomics detects statistically significant absence

of 1 and 2 uncut alleles.


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Detection of a LOH

region

0 1 2

Number of uncut

alleles

Restriction Digestion & SNP Arrays

SNP + CGH Arrays


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Live Demo

Agenda


8:30-9:00 Sign in







4:00-4:30 Wrap-up


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Analysis Software


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Log2 ratio of

intensities to call CNCs

No. of uncut alleles

to call LOH/UPD

Chromosome view Gene view

Aberrations

Customer defined

tracks

Preloaded tracks

Tracks

Genome view


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Live Demo

Agenda


8:30-9:00 Sign in







4:00-4:30 Wrap-up


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Customer webinar

CGH+SNP Array Can Identify Clonal Evolution in Acute

Myelogenous Leukemia

Excellent webinar by Dr. Barbara Zehentner from Hematologics

on the use of Agilent CytoGenomics software to identify

multiple clones in hematological cancers

Very good explanation of use of manual peak assignment

Recorded on September 12, 2012

Link:

https://agilenteseminar.webex.com/agilenteseminar/lsr.php?AT

=pb&SP=EC&rID=5739957&rKey=7a21427e0f7dcac3

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Procedure

https://agilenteseminar.webex.com/agilenteseminar/lsr.php?AT=pb&SP=EC&rID=5739957&rKey=7a21427e0f7dcac3

https://agilenteseminar.webex.com/agilenteseminar/lsr.php?AT=pb&SP=EC&rID=5739957&rKey=7a21427e0f7dcac3

Questions


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In Conclusion…

Thank you for attending!

To learn more about how Cytogenomics Software can

help you, please contact us!

Jean Jasinski Doug Blake

[email protected] [email protected]

Jennifer Jones Iman Kishawi


Katie Weaver Genomic Support



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CytoGenomics 3 - Agilent...Agenda CYTOGENOMIC TRAINING Onstead Auditorium BSRB S3.8012 8:30-9:00...

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