accelerating your analysis Accurate and objective copy number profiling using real-time PCR Barbara D’haene, PhD 3rd qPCR Meeting and Course on Quantitative Real-Time PCR June 25, 2010, Siena, Italy
accelerating your analysis
Accurate and objective copy number profiling using real-time PCR
Barbara D’haene, PhD
3rd qPCR Meeting and Course on Quantitative Real-Time PCR
June 25, 2010, Siena, Italy
accelerating your analysis
Outline
Copy number screening
qPCR based copy number screening
Experiment design
Assay design and validation
Data processing
Data interpretation
qPCR based copy number screening in a clinical context
accelerating your analysis
Copy number changes
accelerating your analysis
Copy number screening
Techniques for copy number screening
Karyotyping
Fluorescent in situ hybridization (FISH)
Microarray-based copy number screening
Multiplex Ligation Dependent Probe Amplification (MLPA)
Next-generation sequencing
Quantitative PCR (qPCR)
accelerating your analysis
qPCR based copy number screening
Advantages of qPCR
Sensitive
Accurate
Open format
Flexible
Fast
Affordable
accelerating your analysis
qPCR based copy number screening
D’haene et al., 2010, Methods
accelerating your analysis
qPCR workflow
Cq values
Data processing
Statistical analysis & interpretations
Experiment design
Sample prep Assay design
qPCR reactions
accelerating your analysis
qPCR workflow – experiment design
Experiment design
# reactions per run
# amplicons
# replicates
# samples
# controls
# reference genes
Pipetting strategy
Sample maximization strategy
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – sample preparation
Sample preparation
Nucleic acid extraction
EDTA blood samples <> heparin
Sample quality control
Concentration
A260/A230
SPUD assay
.
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – assay design
Primer design
Primer3Plus, PrimerQuest
In silico validation
Specificity BLAST
Secondary structures Mfold
SNPs in silico PCR in UCSC
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – validation of assays
Empirical validation
Melt curve analysis specificity
Gel electrophoresis specificity
Standard dilution series efficiency
Assessment the normal variation
1. Normal controls (>24 samples)
2. Log transformation
3. Calculate SD
4. Calculate 95% confidence intervals for CN=2
5. Deduce 95% confidence intervals for CN=1 and CN=3
6. Anti-log transformation
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – assay design
E = 1.941 E = 3.408
accelerating your analysis
qPCR workflow – validation of assays
Empirical validation
Melt curve analysis
Gel electrophoresis
Standard dilution series
Assessment the normal variation
1. Normal controls (>24 samples)
2. Log transformation
3. Calculate SD
4. Calculate 95% confidence intervals for CN=2
5. Deduce 95% confidence intervals for CN=1 and CN=3
6. Anti-log transformation
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – validation of assays
0.000
1.000
2.000
3.000
4.000
1 2 3 4 5 6 7 8 9 10 11 12 13 13-2
1.414
2.449
95% confidence intervals
accelerating your analysis
qPCR workflow – validation of assays
CN: 1 2 3
1
2
3
∆Cq = 1
∆Cq = 0.6
accelerating your analysis
qPCR workflow – qPCR reactions
qPCR reactions
Sample maximization
Cq QC
Melting curves
Technical replicates
Positive/negative controls
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – qPCR reactions
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11NTC
S1
S2
S3
S4
S5
S6
S7
NTC
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11NTC
S1
S2
S3
S8
S9
S10
S11
NTC
sample maximization
GOI2
GOI3
REF1
REF2
REF3
GOI1
gene maximization
REF1 REF2 REF3 GOI1 GOI2 GOI3
GOI2 GOI3REF1 REF2 REF3 GOI1
accelerating your analysis
qPCR workflow – qPCR reactions
qPCR reactions
Sample maximization
Cq QC
Melting curves
Technical replicates
Positive/negative controls
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – qPCR reactions
Data processing and quality control
Normalization factors
Relative quantification
Amplification efficiency correction
qBase - Hellemans, 2007, Genome Biol
geNorm - Vandesompele, 2002, Genome Biol
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
accelerating your analysis
qPCR workflow – data processing
Relative quantification
Normalization with >1 reference assay
Amplification efficiency correction
Error propagation
Quality control
Inter run calibration
genormPLUS
qbasePLUS 2.0 September 2010
New calculation engine
Statistical package
Copy number analysis
Numerous other features
www.biogazelle.com
accelerating your analysis
Quality control using qbasePLUS
accelerating your analysis
Quality control using qbasePLUS
accelerating your analysis
Quality control using qbasePLUS
accelerating your analysis
Quality control using qbasePLUS
accelerating your analysis
qPCR workflow – qPCR reactions
Statistical analysis and interpretation
Calibration
Calibrate with more than 1 sample
Allow samples to have different copy numbers
Example: calibration with normal sample & sample with deletion
Calculation of Z-scores
Cq values
Data processing
Statistical analysis & interpretations
Sample prep Assay design
qPCR reactions
Experiment design
n
CN
NRQ
CF
n
i i
i 1
CF
NRQCN
2
12delnorm NRQNRQ
CF
accelerating your analysis
qPCR workflow – statistical analysis & interpretations
1
2
3
2.449
1.414
-4 -3 -2 -1 0 1 2 3 4
68%
95%
99.7%
accelerating your analysis
qPCR workflow – statistical analysis & interpretations
Normal control Sample with partial deletion
accelerating your analysis
qPCR based copy number screening in a clinical context
Hoebeeck et al., 2005
Laboratory Investigation
D’haene et al., 2010
J Clin Endocrinol Metab
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Short stature
Incidence: 1 in 300 children
Significant impact on quality of life
Léri-Weill dyschondrosteosis
Skeletal dysplasia characterised by
Disproportionate short stature
Mesomelic limb shortening
Madelung deformity of the wrist
Disease gene: SHOX
qPCR based copy number screening in a clinical context - SHOX
Blaschke and Rappold, 2000
accelerating your analysis
qPCR based copy number screening in a clinical context - SHOX
Diagnosis of
ISS or LWD
SHOX
deletion screening
PAR1
deletion screening
SHOX
sequencing
15%
38%
8%
39%
accelerating your analysis
qPCR based copy number screening in a clinical context - SHOX
Diagnosis of
ISS or LWD
SHOX
deletion screening
PAR1
deletion screening
SHOX
sequencing
Copy number screening
Requirements qPCR
Sensitive +
Accurate +
Reliable +
Objective +
Precise +
Affordable +
Flexible +
Fast +
accelerating your analysis
qPCR based copy number screening in a clinical context - SHOX
Thirteen qPCR amplicons were designed based upon:
accelerating your analysis
qPCR based copy number screening in a clinical context - SHOX
Methods
Empirical validation of the primers
2/13 excluded 11 amplicons left
qPCR and data-analysis
384 real-time PCR instrument
qbasePLUS
Assessment of the variation
– Screening of 32 normal controls to assess the normal variation
– Amplicon specific 95% confidence intervals
Implementation of a rescaling factor for objective interpretation
– Based upon 2 normal and 1 deletion control
accelerating your analysis
qPCR based copy number screening in a clinical context - SHOX
Validation studyqPCR was successfully performed for 170 probands
72 out 170 were prescreened using MLPA
– 14 MLPA positive samples
– 58 MLPA negative samples
98 (170 – 72) new unique probands
– 4 with known copy numbers
– 94 with unknown copy numbers
accelerating your analysis
qPCR based copy number screening in a clinical context - SHOX
Plate lay-out
S1 S2 S3 S4 S5 S6 S7 S8 Positive N1 N2 NTC
Assay 1
Assay 2
Assay 4Assay 5
Assay 6
Assay 7
Assay 8
Assay 10Assay 11
Assay 12Assay 13Ref 1Ref 2
accelerating your analysis
qPCR based copy number screening in a clinical context
accelerating your analysis
Results
11 validated amplicons
Reliable results for 170 samples
18 samples with known CNVs
58 MLPA negative samples
94 new samples
Conclusion
Novel molecular test
Reliable
Affordable alternative strategy for the
identification of copy number changes in
the SHOX region
qPCR based copy number screening in a clinical context
accelerating your analysis
Conclusions
qPCR-based copy number screening
Fast
Affordable
Easy (PrimerQuest, qbasePLUS, ...)
Assay flexibility (add or remove loci)
Sample flexibility (few – hundreds)
Sensitive and accurate
Multiple PCR replicates, reference assays and calibrator samples
Quality control
Objective interpretation with Z-scores
accelerating your analysis
Acknowledgements
Jan Hellemans
Jo Vandesompele
Elfride De Baere