Cnv and a analysis strategies

Sample & Assay Technologies

Copy Number Variation & Alteration
Analysis Strategies

Krishnan Allampallam PhD, MBA
Global 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, prevention
or treatment of a disease


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

2


What is copy number?
Copy Number changes are Genomic Structural Variations

3


What is copy number?
Copy Number changes are Genomic Structural Variations

4


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 organisms
Humans, Mice, Chimpanzees, Rhesus macaques,
Cows, Chickens, Arabidopsis thaliana, Fruit flies,
C.elegans, Saccharomyces cerevisiae

5


Copy number variations
Influences gene expression, phenotypic variation and adaptation
by disrupting genes and altering dosage
■ Gene Expression
■

Wild type B+B+

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
Heterozygous Bar
B+B

■
■
■
■

Homozygous Bar
BB

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 – 15
Might be an adaptation to a high- starch diet

Double bar Heterozygous
B +B D
1-Fan et al, PLoSone, e29824, April 2012; 2-Bridges CB, Science 83, 210-211, 1936;
6


Copy number variations - impact
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.
7


Agenda

‘Mref’ - introduction and impact
Introduction
Product offering
Data Analysis
Application
Resources

8


Current methods of copy number analysis
Different Methods for Different Experimental Questions

Pre-Screen

Discovery

Validation

•Array CGH
•SNP Chips
•NGS

•
•

Diagnostic Test

FISH
qPCR

9


Array CGH: hybridization based mapping
Microarray : From image to copy number

Increased intensity

DNA hybridization

Normal

Affymetrix Mapping

Greater DNA Copy number

Tumor

250K Sty-I chip
~250K probe sets
~250K SNPs
probe set (24 probes)

CN=2
Deletion

CN=2

CN=1

Deletion
CN=0

CN=2

Amplification

CN>2


NGS : paired – end mapping
Next Generation Sequencing – difference between the two
sequenced end points

Deletion
sample

reference genome

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

Insertion

sample

reference genome

Schrider and Hahn (2010) Proc. R. Soc. 277, 3213-3221
11

qPCR


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

Sample 1
A

B

C

D
1x

Sample 2
A

B

B

C

D
2x

Sample 3
A

B

B

B

C

D
3x

12


qPCR-based relative quantification of copy number
How does it work?
Key assumption
Copy 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)

Calculation of copy number gain or loss in sample of interest

13


Agenda

Introduction
Product offering
Data Analysis
Application
Resources

14


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


Variations don’t discriminate based on you experiment.

TERT - not a reliable reference genome

16


RNase P also has documented Structural Variations

RNaseP - not a reliable reference genome
17


Inefficiencies associated with single gene reference assays
SNPs may effect qPCR efficiency and lead to altered CT values

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

18

Reference gene copy number has dramatic effects


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

20


Agenda

Introduction
Product offering
Data Analysis
Application
Resources

21


Multicopy reference assay (MRef)
The ideal reference assay should fulfill the following criteria:
■

Not be affected by a local change in the genome
‒

■

Copy number:
‒

■

Copy Number or SNP

>20 copies in a diploid genome

Location distribution:
‒
‒

≤ 10% copies concentrated on a single chromosome

‒
■

Located on different chromosomes

For copies on the same chromosome, preferably on different arms

Sequence:
‒

Sequence stable in human genome

Benefit: Superior Assay For Input Normalization

22

Genomic changes effect single gene references


Reference
Assay

GOI copy
number (real)

GOI copy
number
(calculated)

Copy Number
Call

0

2

2.00

No Change

2+1

- 0.58

2

1.33

Loss

2–1
Multicopy
Reference
Assay

CT change
(reference
assay)

2
RNase P

Copy Number
of Reference
Gene

+1

2

4.00

Gain

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

23


Multicopy reference assay yields increased accuracy
CNA Experimental Setup

Genomic DNA Samples
Wildtype (Calibrator sample)
SKBR3 cells
Gene Of Interest (GOI)
GRB7 (qBiomarker Assay)
Reference Genome
RNaseP or Multicopy Reference Assay

24


CNA Experimental Setup
Genomic DNA Samples
SKBR3 cells

Single Copy Gene as a Reference (RNase P)

Reference Genome
RNaseP or Multicopy Reference Assay

Result
Calculated CNV ~ 2-fold different

Validation Experiment
Blend DNA samples at various ratios
Multicopy Reference Assay
25



Using a Single Copy Gene as a Reference (RNase P)
Genomic DNA Samples
SKBR3 cells
Reference Genome
RNaseP or
Multicopy reference assay

Using Multicopy Reference Assay

Result
Calculated CNV ~ 2-fold different

Validation Experiment

26



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

Typical CNA Experimental Setup:
Genomic DNA Samples
SKBR3 cells
Measure GRB7 (qBiomarker Assay) and
either RNase P or Multicopy Reference
Assay
Result: Calculated Copy Number are
almost 2-fold different
Validation Experiment:

Using Multicopy Reference Assay
Calculate Expected Copy Number
Measure GRB7 (qBiomarker Assay) and
either RNase P or Multicopy Reference
Assay
Compare Expected Copy Number to
Observed Copy Number
27


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.

28


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

29


Agenda

‘Mref’ (Multi Copy Reference)- introduction and impact
Introduction
Product offering
Data Analysis
Application
Resources

30


qBiomarker copy number assays
Tiling the human genome at 200 base pair resolution

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

Gene B
2. Design a Primer assay within the tile
200 base pair Tile
3. Lab-test the Primer assay for performance

4. Available as single assays or array content

31


qBiomarker copy number array layout
■ 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

32


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

33


Search for qBiomarker copy number assays

Users can search by
1.Gene Symbol
TP53
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

34



Diseases/Disorders Diseases/Disorders Pathway-Focused
Associated with
Associated with
CNA
CNV

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

35


Example qBiomarker copy number PCR array
Description for the content of each array

36


Example qBiomarker copy number PCR array
Technical Assay Details for each array are provided in Gene Table

37


Make it your own… with custom PCR arrays

38


Profile disease or pathway-focused copy number profiling
Isolate genomic DNA from fresh, frozen
or FFPE samples using QIAamp or
DNeasy kits recommended in the handbook.

2

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

3

1 sample goes on 1 plate
Assays in technical quadruplicate

4

Standard 40 cycle PCR on most real-time
Thermocyclers.

5

Upload CT values to Data Analysis Webportal

39


Included data analysis


Agenda

Introduction
Product offering
Data Analysis
Application
Resources

41


Validated copy number assays yield accurate results
Testing Gene Dosage with Aneuploidy Samples

Aneuploidy samples that have different numbers of
X chromosomes
Samples acquired from Coriell Cell Repositories
Cell line
Copy number
Coriell NA 13619 (XY)
1
Coriell NA 0192 (XX)
2
Coriell NA 03623 (XXX)
3
Coriell NA11226 (XXXX)
4

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

42


Agenda

Introduction
Product offering
Data Analysis
Application
Resources

43


Liposarcoma collaboration
Initial Screen for Copy Number Changes

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

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)

44

Copy number assay in liposarcoma


Copy Number PCR Array Data for Sample T50
35

Copy Number

30

*

25
20
15
*

10

*

*
*

*

*

5
*

*

*

*

*

11

1

1

*

*

0

Chr

9

9

9

9

12 12

12 12 12

12 12

11 11 11

1

1

1

1

1 19

aCGH
qBiomarker
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)


Agenda

Introduction
Product offering
Data Analysis
Application
Resources

46


Protocol & product overview
What do you need to measure copy number by qPCR?
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


Search for qBiomarker copy number assays
Use SABiosciences.com

48


Agenda

Introduction
Product offering
Data Analysis
Application
Resources

49


Summary
From Discovery to Validation
Copy Number Alterations/Variations are important biological
changes with ramifications for human health
Experimental Solutions for Copy Number Determination
Discovery 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

50


Special offer for Webinar Attendees
Try any qBiomarker Copy Number Assay
Get 20% off + QIAGEN Watch
Promo Code: FDK-WN20WA3
Experience PCR Array Performance

Try them today!

Questions?
Contact Technical Support: 9 AM – 6 PM M – F ET
Contact: 1-800-742-4368 OR support@SABiosciences.com
Thousands of scientists have
discovered the power of PCR Arrays
Join them on the road to success!


Questions
Contact Technical Support
9 AM – 6 PM Eastern M – F
Telephone: 888-503-3187
Email: support@SABiosciences.com

Thank you!
Krishnan Allampallam
krishnan.allampallam@qiagen.com
QIAWEBINARS@QIAGEN.COM
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

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