1. 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
2. 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
2
3. Sample & Assay Technologies
What is copy number?
Copy Number changes are Genomic Structural Variations
3
4. Sample & Assay Technologies
What is copy number?
Copy Number changes are Genomic Structural Variations
4
5. 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 organisms
Humans, Mice, Chimpanzees, Rhesus macaques,
Cows, Chickens, Arabidopsis thaliana, Fruit flies,
C.elegans, Saccharomyces cerevisiae
5
6. Sample & Assay Technologies
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
7. Sample & Assay Technologies
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
8. 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
8
9. Sample & Assay Technologies
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
10. Sample & Assay Technologies
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
11. Sample & Assay Technologies
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
12. qPCR
Sample & Assay Technologies
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
13. Sample & Assay Technologies
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
14. 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
14
15. 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
16. Sample & Assay Technologies
Reference genome – current practice
Variations don’t discriminate based on you experiment.
TERT - not a reliable reference genome
16
17. Sample & Assay Technologies
Reference genome – current practice
RNase P also has documented Structural Variations
RNaseP - not a reliable reference genome
17
18. Sample & Assay Technologies
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
19. 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
20. Reference gene copy number has dramatic effects
Sample & Assay Technologies
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
21. 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
21
22. Sample & Assay Technologies
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
23. Genomic changes effect single gene references
Sample & Assay Technologies
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
25. Sample & Assay Technologies
Multicopy reference assay yields increased accuracy
CNA Experimental Setup
Genomic DNA Samples
Wildtype (Calibrator sample)
SKBR3 cells
Gene Of Interest (GOI)
GRB7 (qBiomarker Assay)
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
26. Sample & Assay Technologies
Multicopy reference assay yields increased accuracy
Using a Single Copy Gene as a Reference (RNase P)
Genomic DNA Samples
Wildtype (Calibrator sample)
SKBR3 cells
Gene Of Interest (GOI)
GRB7 (qBiomarker Assay)
Reference Genome
RNaseP or
Multicopy reference assay
Using Multicopy Reference Assay
Result
Calculated CNV ~ 2-fold different
Validation Experiment
Blend DNA samples at various ratios
26
27. Sample & Assay Technologies
Multicopy reference assay yields increased accuracy
Using a Single Copy Gene as a Reference (RNase P)
Typical CNA Experimental Setup:
Genomic DNA Samples
Wildtype (Calibrator sample)
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
Blend DNA samples at various ratios
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
28. 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.
28
29. 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
29
30. Sample & Assay Technologies
Agenda
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
Introduction
Product offering
Data Analysis
Application
Testing gene dosage with Aneuploidy samples
Copy Number Variations in Liposarcoma
Resources
Summary and Questions
30
31. Sample & Assay Technologies
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
32. Sample & Assay Technologies
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
33. 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
33
34. Sample & Assay Technologies
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
35. Sample & Assay Technologies
qBiomarker copy number arrays
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
36. Sample & Assay Technologies
Example qBiomarker copy number PCR array
Description for the content of each array
36
37. Sample & Assay Technologies
Example qBiomarker copy number PCR array
Technical Assay Details for each array are provided in Gene Table
37
38. Sample & Assay Technologies
Make it your own… with custom PCR arrays
38
39. Sample & Assay Technologies
qBiomarker copy number arrays
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
41. 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
41
42. Sample & Assay Technologies
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
43. 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
43
44. Sample & Assay Technologies
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
45. Copy number assay in liposarcoma
Sample & Assay Technologies
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)
46. 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
46
47. Sample & Assay Technologies
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
48. Sample & Assay Technologies
Search for qBiomarker copy number assays
Use SABiosciences.com
48
49. 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
49
50. Sample & Assay Technologies
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
51. Sample & Assay Technologies
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!
52. Sample & Assay Technologies
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