Accurate and objective copy number profiling using real-time PCR

Transcription

1 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

2 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

3 Copy number changes

4 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)

5 qpcr based copy number screening Advantages of qpcr Sensitive Accurate Open format Flexible Fast Affordable

6 qpcr based copy number screening D haene et al., 2010, Methods

7 qpcr workflow Experiment design Sample prep Assay design qpcr reactions Cq values Data processing Statistical analysis & interpretations

8 qpcr workflow experiment design Experiment design # reactions per run # amplicons # replicates # samples # controls # reference genes Pipetting strategy Sample maximization strategy Experiment design Sample prep Assay design qpcr reactions Cq values Data processing Statistical analysis & interpretations

9 qpcr workflow sample preparation Sample preparation Nucleic acid extraction EDTA blood samples <> heparin Experiment design Sample prep Assay design qpcr reactions Cq values Sample quality control Concentration A260/A230 SPUD assay. Data processing Statistical analysis & interpretations

10 qpcr workflow assay design Primer design Primer3Plus, PrimerQuest In silico validation Specificity BLAST Secondary structures Mfold SNPs in silico PCR in UCSC Experiment design Sample prep Assay design qpcr reactions Cq values Data processing Statistical analysis & interpretations

11 qpcr workflow validation of assays Empirical validation Melt curve analysis specificity Gel electrophoresis specificity Standard dilution series efficiency Assessment the normal variation Experiment design Sample prep Assay design qpcr reactions Cq values Data processing Statistical analysis & interpretations 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

12 qpcr workflow assay design E = E = 3.408

13 qpcr workflow validation of assays Empirical validation Melt curve analysis Gel electrophoresis Standard dilution series Assessment the normal variation Experiment design Sample prep Assay design qpcr reactions Cq values Data processing Statistical analysis & interpretations 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

14 qpcr workflow validation of assays 95% confidence intervals

15 qpcr workflow validation of assays Cq = 0.6 Cq = 1 CN: 1 2 3

16 qpcr workflow qpcr reactions qpcr reactions Sample maximization Sample prep Experiment design Assay design qpcr reactions Cq QC Melting curves Technical replicates Positive/negative controls Cq values Data processing Statistical analysis & interpretations

17 qpcr workflow qpcr reactions gene maximization sample maximization REF1 REF2 REF3 GOI1 GOI2 GOI3 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 NTC S1 S2 S3 S4 S5 S6 S7 NTC REF1 REF2 REF3 GOI1 REF1 REF2 REF3 GOI1 GOI2 GOI3 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 NTC S1 S2 S3 S8 S9 S10 S11 NTC GOI2 GOI3

18 qpcr workflow qpcr reactions qpcr reactions Sample maximization Sample prep Experiment design Assay design qpcr reactions Cq QC Melting curves Technical replicates Positive/negative controls Cq values Data processing Statistical analysis & interpretations

19 qpcr workflow qpcr reactions Data processing and quality control Normalization factors Relative quantification Amplification efficiency correction Experiment design Sample prep Assay design qpcr reactions Cq values Data processing Statistical analysis & interpretations qbase - Hellemans, 2007, Genome Biol genorm - Vandesompele, 2002, Genome Biol

20 qpcr workflow data processing Relative quantification Normalization with >1 reference assay Amplification efficiency correction Error propagation Quality control Inter run calibration genorm PLUS qbase PLUS 2.0 September 2010 New calculation engine Statistical package Copy number analysis Numerous other features

21 Quality control using qbase PLUS

22 Quality control using qbase PLUS

23 Quality control using qbase PLUS

24 Quality control using qbase PLUS

25 qpcr workflow qpcr reactions Statistical analysis and interpretation Calibration Calibrate with more than 1 sample Allow samples to have different copy numbers Experiment design Sample prep Assay design qpcr reactions Cq values CF n NRQi i CNi 1 NRQ CN n CF Data processing Statistical analysis & interpretations Example: calibration with normal sample & sample with deletion CF NRQ norm NRQ del Calculation of Z-scores

26 qpcr workflow statistical analysis & interpretations % 95% 99.7%

27 qpcr workflow statistical analysis & interpretations Normal control Sample with partial deletion

28 qpcr based copy number screening in a clinical context Hoebeeck et al., 2005 Laboratory Investigation D haene et al., 2010 J Clin Endocrinol Metab

29 qpcr based copy number screening in a clinical context - SHOX 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 Blaschke and Rappold, 2000 Disease gene: SHOX

30 qpcr based copy number screening in a clinical context - SHOX 15% 39% 8% 38% Diagnosis of ISS or LWD SHOX deletion screening PAR1 deletion screening SHOX sequencing

31 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 +

32 qpcr based copy number screening in a clinical context - SHOX Thirteen qpcr amplicons were designed based upon:

33 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 qbase PLUS 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

34 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

35 qpcr based copy number screening in a clinical context - SHOX Plate lay-out 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 S1 S2 S3 S4 S5 S6 S7 S8 Positive N1 N2 NTC

36 qpcr based copy number screening in a clinical context

37 qpcr based copy number screening in a clinical context 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

38 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

39 Acknowledgements Jan Hellemans Jo Vandesompele Elfride De Baere