Accurate and objective copy number profiling using real-time PCR - - PowerPoint PPT Presentation

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Accurate and objective copy number profiling using real-time PCR - - PowerPoint PPT Presentation

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accelerating your analysis Accurate and objective copy number profiling using real-time PCR Barbara Dhaene, PhD 3rd qPCR Meeting and Course on Quantitative Real-Time PCR June 25, 2010, Siena, Italy Outline accelerating your analysis Copy

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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

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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

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Copy number changes

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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)

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qPCR based copy number screening

Advantages of qPCR

Sensitive Accurate Open format Flexible Fast Affordable

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qPCR based copy number screening

D’haene et al., 2010, Methods

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qPCR workflow

Cq values Data processing Statistical analysis & interpretations Experiment design Sample prep Assay design qPCR reactions

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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

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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

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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

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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

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qPCR workflow – assay design

E = 1.941 E = 3.408

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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

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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

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qPCR workflow – validation of assays

CN: 1 2 3 1 2 3 ∆Cq = 1 ∆Cq = 0.6

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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

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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 GOI3 REF1 REF2 REF3 GOI1

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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

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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

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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

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Quality control using qbasePLUS

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Quality control using qbasePLUS

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Quality control using qbasePLUS

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Quality control using qbasePLUS

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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 NRQ CN  2 1 2

del norm

NRQ NRQ CF  

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qPCR workflow – statistical analysis & interpretations

1 2 3 2.449 1.414

  • 4
  • 3
  • 2
  • 1

1 2 3 4

68% 95% 99.7%

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qPCR workflow – statistical analysis & interpretations

Normal control Sample with partial deletion

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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

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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%

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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 +

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qPCR based copy number screening in a clinical context - SHOX

Thirteen qPCR amplicons were designed based upon:

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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

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qPCR based copy number screening in a clinical context - SHOX

Validation study

qPCR 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

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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 4 Assay 5 Assay 6 Assay 7 Assay 8 Assay 10 Assay 11 Assay 12 Assay 13 Ref 1 Ref 2

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qPCR based copy number screening in a clinical context

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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

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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

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Acknowledgements

Jan Hellemans Jo Vandesompele Elfride De Baere