accurate and objective copy number profiling using real ... · 6/25/2010 · june 25, 2010, siena,...

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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


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


Copy number changes



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)



Advantages of qPCR

Sensitive

Accurate

Open format

Flexible

Fast

Affordable



D’haene et al., 2010, Methods


qPCR workflow

Cq values

Data processing

Statistical analysis & interpretations

Experiment design

Sample prep Assay design

qPCR reactions


qPCR workflow – experiment design

Experiment design

# reactions per run

# amplicons

# replicates

# samples

# controls

# reference genes

Pipetting strategy

Sample maximization strategy

Cq values

Data processing



qPCR reactions

Experiment design


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



qPCR reactions

Experiment design


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



qPCR reactions

Experiment design


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



qPCR reactions

Experiment design


qPCR workflow – assay design

E = 1.941 E = 3.408



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



qPCR reactions

Experiment design



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



CN: 1 2 3

1

2

3

∆Cq = 1

∆Cq = 0.6


qPCR workflow – qPCR reactions

qPCR reactions

Sample maximization

Cq QC

Melting curves

Technical replicates

Positive/negative controls

Cq values

Data processing



qPCR reactions

Experiment design



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



qPCR reactions

Sample maximization

Cq QC

Melting curves

Technical replicates

Positive/negative controls

Cq values

Data processing



qPCR reactions

Experiment design



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



qPCR reactions

Experiment design


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


Quality control using qbasePLUS



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



qPCR reactions

Experiment design

n

CN

NRQ

CF

n

i i

i 1

CF

NRQCN

2

12delnorm NRQNRQ

CF


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%


qPCR workflow – statistical analysis & interpretations

Normal control Sample with partial deletion



Hoebeeck et al., 2005

Laboratory Investigation

D’haene et al., 2010

J Clin Endocrinol Metab


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



Diagnosis of

ISS or LWD

SHOX

deletion screening

PAR1

deletion screening

SHOX

sequencing

15%

38%

8%

39%



Diagnosis of

ISS or LWD

SHOX

deletion screening

PAR1

deletion screening

SHOX

sequencing


Requirements qPCR

Sensitive +

Accurate +

Reliable +

Objective +

Precise +

Affordable +

Flexible +

Fast +



Thirteen qPCR amplicons were designed based upon:



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



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



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


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



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


Acknowledgements

Jan Hellemans

Jo Vandesompele

Elfride De Baere

accurate and objective copy number profiling using real ... · 6/25/2010 · june 25, 2010, siena,...

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