cnv and a analysis strategies

Sample & Assay Technologies

Copy Number Variation & AlterationAnalysis Strategies

Krishnan Allampallam PhD, MBAGlobal Marketing Manager

PCR Array Technology

The qBiomarker Copy Number PCR Arrays are intended for molecular biology applications. This

product is not intended for the diagnosis, preventi on or treatment of a disease

Sample & Assay Technologies Agenda

� Copy Number Variation – brief introduction� Current methods to quantify Copy Number� Reference Genome in Copy Number Assays – current practice� ‘MRef’ - introduction and impact� qBiomarker Copy Number PCR Arrays

� Introduction� Product offering� Data Analysis

� Application� Testing gene dosage with Aneuploidy samples� Copy Number Variations in Liposarcoma� Resources

� Summary and Questions

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Sample & Assay Technologies What is copy number?

Copy Number changes are Genomic Structural Variations

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Copy Number changes are Genomic Structural Variations

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What is copy number?

Sample & Assay Technologies Facts about copy number variation

� Definition: DNA segment with 1 kb or larger variation in comparison to reference genome

� Copy Number Variation or Copy Number Polymorphism

� Frequent and occur semi-randomly throughout the genome

� Occurs in wide range of organismsHumans, Mice, Chimpanzees, Rhesus macaques,

Cows, Chickens, Arabidopsis thaliana, Fruit flies, C.elegans, Saccharomyces cerevisiae

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Sample & Assay Technologies Copy number variations

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Influences gene expression, phenotypic variation and adaptation by disrupting genes and altering dosage

■ Gene Expression■ Gastric cancer

■ Increase in CN # of 20q corresponds to frequent amplifications

■ Decrease in CN # in 9p corresponds to down regulation of 12 tumor suppressor genes1

■ Phenotye■ Bar gene in Drosophila melanogaster2

■ 1936 -First Association of CNV with phenotype■ X-linked duplication of Bar region■ As the # of bar increases, the size of the eyes become smaller

and bar sharped

■ Human salivary amylase gene (AMY1)Chimpanzee: Has two diploid copies

Humans: Ranges 6 – 15Might be an adaptation to a high- starch diet

Wild type B+B+

Heterozygous BarB+B

Homozygous BarBB

Double bar HeterozygousB+BD

1-Fan et al, PLoSone, e29824, April 2012; 2-Bridges CB, Science 83, 210-211, 1936;

Sample & Assay Technologies Copy number variations - impact

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Pharmacogenomics

� Drug Efficacy

� CYP2D6– Expressed in human liver– Critical in drug metabolism– Highly polymorphic - 75 alleles known– Affects wide range of drug classes –

antidepressants, beta blocking agents and endocrine therapy

– eg. Taxmoxifen

� Drug Toxicity

� CYP2D6 – affects Codeine

Pharmacogenomics – Patient Stratification

He Y et. al.,(2011) Trends Mol Med , 17 (5): 244-251.


� Copy Number Variation – brief introduction� Current methods to quantify Copy Number� Reference Genome in Copy Number Assays – current practice� ‘Mref’ - introduction and impact� qBiomarker Copy Number PCR Arrays




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Sample & Assay Technologies Current methods of copy number analysis

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Discovery

•Array CGH•SNP Chips•NGS

Validation

• FISH• qPCR

Pre-Screen

Different Methods for Different Experimental Questions

Diagnostic Test

Sample & Assay Technologies Array CGH: hybridization based mapping

Microarray : From image to copy number

CN=2

CN=2

CN=2

Affymetrix Mapping

250K Sty-I chip

~250K probe sets

~250K SNPs

CN=1

CN=0

CN>2

Tumor Normal

probe set (24 probes)

Deletion

Deletion

Amplification

Increased intensity DNA hybridization Greater DNA Copy number

Sample & Assay Technologies NGS : paired – end mapping

Next Generation Sequencing – difference between the two sequenced end points

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reference genome

sample

Deletion

sample

reference genome

Insertion

� Sequenced end points and the distance between the two point is known

� If distance between two ends in reference is greater than sample : deletion

� If distance between two ends in reference is less than than sample : insertion

Schrider and Hahn (2010) Proc. R. Soc. 277, 3213-3221

Sample & Assay Technologies qPCR

1. Determine the gene and reference genome2. Design the primers , amplify the gene by PCR, determine the Ct value3. As copy number increases Ct value decreases

1 x

2x

3 x

Sample 1

Sample 2

Sample 3

A B C D

A B C DB

A B C DB B

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Sample & Assay Technologies qPCR-based relative quantification of copy number

How does it work?

� Key assumptionCopy number of a reference gene (Ref) is consistent across

different samples

� Copy Number of Gene of Interest (GOI) is normalized to reference gene by ∆∆CT method

– CN of reference gene remains unchanged with CN of GOI(s)

– Assay for GOI(s)– Control sample(s) with known copy number of GOI(s)– Sample of interest with unknown copy number for GOI(s)

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Calculation of copy number gain or loss in sample of interest






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Sample & Assay Technologies Reference genome – current practice

■ Single copy reference genes� RNase P� TERT (Telomerase Reverse Transcriptase)

■ General recommendation � Quantifying the amount of DNA in your samples by

testing these genes against your samples before your GOI…..

■ Potential pitfalls � Copy Number Events� SNP–related effects on qPCR efficiency� Unstable Genomes


TERT - not a reliable reference genome

Variations don’t discriminate based on you experiment.

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Reference genome – current practice


RNase P also has documented Structural Variations

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Reference genome – current practice

RNaseP - not a reliable reference genome

Sample & Assay Technologies Inefficiencies associated with single gene reference assays

SNPs may effect qPCR efficiency and lead to altered CT values

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Genomic map of RNAse P (RPPH1) has at least 17 documented SNPs over 341 bases

Sample & Assay Technologies Reference genome – current practice

■ Single copy reference genes� RNase P� TERT (Telomerase Reverse Transcriptase)

■ General recommendation � Quantifying the amount of DNA in your samples by

testing these genes against your samples before your GOI…..

■ Potential pitfalls � Copy Number Events� SNP–related effects on qPCR efficiency� Unstable Genomes

Sample & Assay Technologies Reference gene copy number has dramatic effects

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Reference Assay

Copy Number of Reference

Gene

CT change (reference

assay)

GOI copy number (real)

GOI copy number

(calculated)

Copy Number Call

RNase P 2 0 2 2.00 No Change

2 + 1 -0.58 2 1.33 Loss

2 – 1 +1 2 4.00 Gain






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Sample & Assay Technologies Multicopy reference assay (MRef)

Benefit: Superior Assay For Input Normalization

The ideal reference assay should fulfill the following criteria:

■ Not be affected by a local change in the genome

‒ Copy Number or SNP

■ Copy number:

‒ >20 copies in a diploid genome

■ Location distribution:

‒ Located on different chromosomes

‒ ≤ 10% copies concentrated on a single chromosome

‒ For copies on the same chromosome, preferably on different arms

■ Sequence:

‒ Sequence stable in human genome

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Sample & Assay Technologies Genomic changes effect single gene references

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Reference Assay

Copy Number of Reference

Gene

CT change (reference

assay)

GOI copy number (real)

GOI copy number

(calculated)

Copy Number Call

RNase P

2 0 2 2.00 No Change

2 + 1 - 0.58 2 1.33 Loss

2 – 1 + 1 2 4.00 Gain

Multicopy Reference

Assay

40 0 2 2.00 No Change

40 + 1 -0.035 2 1.96 No Change

40 -1 + 0.035 2 2.04 No Change

Sample & Assay Technologies Multicopy reference assay yields increased accuracy

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Genomic DNA SamplesWildtype (Calibrator sample)SKBR3 cells

Gene Of Interest (GOI)GRB7 (qBiomarker Assay)

Reference GenomeRNaseP or Multicopy Reference Assay

CNA Experimental Setup


Single Copy Gene as a Reference (RNase P)

Multicopy Reference Assay

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ResultCalculated CNV ~ 2-fold different



Reference GenomeRNaseP or Multicopy Reference Assay

CNA Experimental Setup

Validation Experiment

Blend DNA samples at various ratios

Multicopy reference assay yields increased accuracy


Using a Single Copy Gene as a Reference (RNase P)

Using Multicopy Reference Assay

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Reference GenomeRNaseP orMulticopy reference assay

ResultCalculated CNV ~ 2-fold different

Validation Experiment


Multicopy reference assay yields increased accuracy

Sample & Assay Technologies Multicopy reference assay yields increased accuracy

Using a Single Copy Gene as a Reference (RNase P)

Using Multicopy Reference Assay

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Typical CNA Experimental Setup:


Measure GRB7 (qBiomarker Assay) and either RNase P or Multicopy Reference Assay

Result: Calculated Copy Number are almost 2-fold different

Validation Experiment:


Calculate Expected Copy Number

Measure GRB7 (qBiomarker Assay) and either RNase P or Multicopy Reference Assay

Compare Expected Copy Number to Observed Copy Number

Sample & Assay Technologies Why RNase P led to an overestimate of GRB7?

Above experiment uses MRef for Normalization of DNA Input and tests RNase P as the GOI

With cancer cells having increased genomic content (sometime 80 or more chromosomes), single copy genes “appear” like deletions because they are diluted.

Since RNase P, was the denominator in the last experiment the Copy Number value appears artificially high, while the multicopy reference assay better mirrrors the amount of DNA input.

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Sample & Assay Technologies Multicopy reference assay summary

Superior Assay For Input Normalization

�Multicopy Reference yields stable CT values in spite of local changes

�SNPs do not significantly effect CT values of Multicopy Reference Assay

�Large amplifications or deletions of DNA are more consistently normalized

�Stable Normalization Assay across human populations

�Relative ∆∆CT calculation yields calculated Copy Number

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� Copy Number Variation – brief introduction� Current methods to quantify Copy Number� Reference Genome in Copy Number Assays – current practice� ‘Mref’ (Multi Copy Reference)- introduction and impact� qBiomarker Copy Number PCR Arrays




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Sample & Assay Technologies qBiomarker copy number assays

Tiling the human genome at 200 base pair resolution

Assay design

2. Design a Primer assay within the tile

200 base pair Tile

Gene BGene A

1. “Virtually-Cut” the Genome into 200 bp tiles• Yielded ~11.6 million designable tiles

3. Lab-test the Primer assay for performance

4. Available as single assays or array content

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Sample & Assay Technologies qBiomarker copy number array layout

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■ qPCR Primer Assays are pre-plated

■ 1 sample per PCR plate/ring

■ 23 or 95-genes/loci tested per array

■ each # in a well represents a different gene

■ 1 qBiomarker Copy Number Assay / gene

■ Assay design■ Assays cover exons■ Assay position : close to center of a gene

■ 1 qBiomarker Multicopy reference assay (MRef)

■ Assay in technical quadruplicate for accuracy

Sample & Assay Technologies qBiomarker copy number arrays

Profile disease or pathway-focused copy number profiling

■ Platform-independent

■ Compatible with almost any qPCR instrument

■ Choose appropriate mastermix for instrument type

■ 23 or 95-genes/loci tested per array

■ 1 sample per PCR plate/ring

■ Gene Selection■ Arrays by disease■ Arrays by pathway■ Arrays by functional gene classes

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Sample & Assay Technologies Search for qBiomarker copy number assays

Users can search by

1.Gene SymbolTP53

2.Refseq/Transcript:NM_001126116

3.NCBI Gene ID:7157

4.DGV ID:Database like COSMIC

5.Chromosome Position:Chr. 17 7571801-7572001

6.Assay ID:VPH117-1234567A

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Diseases/Disorders Associated with CNA

Diseases/Disorders Associated with CNV

Pathway-Focused High Content Arrays

Breast Cancer Birth Defects Kinases & Phosphatases

Oncogenes & TSGs

Lung Cancer Intellectual Disability WNT signaling Pathway

Ovarian Cancer

Prostate Cancer

Gastric Cancer

Glioma

Pancreatic Cancer

Custom Copy Number Arrays are also available

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Sample & Assay Technologies Example qBiomarker copy number PCR array

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Description for the content of each array

Sample & Assay Technologies Example qBiomarker copy number PCR array

Technical Assay Details for each array are provided in Gene Table

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Sample & Assay Technologies Make it your own… with custom PCR arrays

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Profile disease or pathway-focused copy number profiling

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3

4

2

Isolate genomic DNA from fresh, frozen or FFPE samples using QIAamp or DNeasy kits recommended in the handbook.

Upload CT values to Data Analysis Webportal

Standard 40 cycle PCR on most real-time Thermocyclers.

1 sample goes on 1 plateAssays in technical quadruplicate

Add qBiomarker SYBR mastermix to genomic DNA• 400 – 1000 ng fresh/frozen DNA• 800 – 1200 ng FFPE DNA

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Sample & Assay Technologies Included data analysis






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Sample & Assay Technologies Validated copy number assays yield accurate results

Testing Gene Dosage with Aneuploidy Samples

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Aneuploidy samples that have different numbers of X chromosomes

Samples acquired from Coriell Cell Repositories

Use qBiomarker Copy Number Assays for Androgren Receptor (AR) and Methyl CpG binding Protein 2 (MECP2) that are single copy genes on the X chromosome

Use qBiomarker Multicopy Reference Assay to normalize for DNA Input

Use ∆∆CT to calculate relative copy number changes compared to the XX sample

Cell line Copy number

Coriell NA 13619 (XY) 1

Coriell NA 0192 (XX) 2

Coriell NA 03623 (XXX) 3

Coriell NA11226 (XXXX) 4






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Sample & Assay Technologies Liposarcoma collaboration

Thirty (30) liposarcoma samples were tested by aCGH for copy number events

Results from one of those samples (T50) is shown• Analysis with Partek software• Deletions on Chromosome 11• Amplifications on Chromosome 12

Initial screen yielded a list of 23 genes with copy number changes.

All samples were re-tested using Custom Copy Number PCR Array

Initial Screen for Copy Number Changes

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Data courtesy of Kara Pascarelli and Dominique Broccoli, Memorial University Medical Center, Savannah, GA, USA; and Lesley Ann Hawthorne, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA)


0

5

10

15

20

25

30

35

Cop

y N

umbe

r

Chr 9 9 9 9 12 12 12 12 12 12 12 11 11 11 11 1 1 1 1 1 1 1 19

**

**

*

* *

*

*

* *

***

qBiomarker

aCGH

Copy number assay in liposarcoma

Copy Number PCR Array Data for Sample T50

Data courtesy of Kara Pascarelli and Dominique Broccoli, Memorial University Medical Center, Savannah, GA, USA; and Lesley Ann Hawthorne, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA)






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Sample & Assay Technologies Protocol & product overview

DNA Isolation- QIAamp DNA Mini Kit (51304 or 51306)- DNeasy Blood & Tissue Kit (69504 or 69506)- QIAamp DNA FFPE Tissue Kit (56404)

Whole Genome Amplfication (optional)- Repli-G

Individual Assays - qBiomarker Copy Number PCR Assays- qBiomarker Multicopy Reference PCR Assay- qBiomarker SYBR Green Mastermix (based on instrument)

Copy Number Profiling- qBiomarker Copy Number PCR Arrays- Custom qBiomarker Copy Number PCR Arrays- qBiomarker SYBR Green Mastermix (based on instrument)

qBiomarker Data Analysis

What do you need to measure copy number by qPCR?

Sample & Assay Technologies Search for qBiomarker copy number assays

Use SABiosciences.com

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Sample & Assay Technologies Summary

Copy Number Alterations/Variations are important biological changes with ramifications for human health

Experimental Solutions for Copy Number DeterminationDiscovery experiments- Arrays, beadchips, etc.Validation / Pre-Screen / Hypothesis-driven experiments- qPCR

Better normalization assays yield better data- Multicopy reference assay vs. single gene

Bench-validated assays at highest resolution

Copy Number Profiling- Pathway-focused- Custom designs

From Discovery to Validation

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Promo Code: FDK-WN20WA3

Questions?Contact Technical Support: 9 AM – 6 PM M – F ETContact: 1-800-742-4368 OR [email protected]


Contact Technical Support9 AM – 6 PM Eastern M – F

Telephone: 888-503-3187

Email: [email protected]

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor

Thank you!

Krishnan [email protected]

Questions

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