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

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

Outline accelerating your analysis 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 accelerating your analysis

Copy number screening accelerating your analysis 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)

qPCR based copy number screening accelerating your analysis Advantages of qPCR Sensitive Accurate Open format Flexible Fast Affordable

qPCR based copy number screening accelerating your analysis D’haene et al. , 2010, Methods

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

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

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

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

qPCR workflow – validation of assays accelerating your analysis Experiment design Empirical validation Melt curve analysis  specificity Sample prep Assay design Gel electrophoresis  specificity qPCR reactions Standard dilution series  efficiency Cq values Data processing Assessment the normal variation 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

qPCR workflow – assay design accelerating your analysis E = 1.941 E = 3.408

qPCR workflow – validation of assays accelerating your analysis Experiment design Empirical validation Melt curve analysis Sample prep Assay design Gel electrophoresis qPCR reactions Standard dilution series Cq values Data processing Assessment the normal variation 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

qPCR workflow – validation of assays accelerating your analysis 95% confidence intervals 4.000 3.000 2.449 2.000 1.414 1.000 0.000 1 2 3 4 5 6 7 8 9 10 11 12 13 13-2

qPCR workflow – validation of assays accelerating your analysis 3 ∆Cq = 0.6 2 ∆Cq = 1 1 CN: 1 2 3

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

qPCR workflow – qPCR reactions accelerating your analysis gene maximization sample maximization REF1 REF2 REF3 GOI1 GOI2 GOI3 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11NTC S1 REF1 S2 S3 REF2 S4 S5 REF3 S6 S7 GOI1 NTC REF1 REF2 REF3 GOI1 GOI2 GOI3 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11NTC S1 GOI2 S2 S3 GOI3 S8 S9 S10 S11 NTC

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

qPCR workflow – qPCR reactions accelerating your analysis Experiment design Data processing and quality control Normalization factors Sample prep Assay design Relative quantification qPCR reactions Amplification efficiency correction Cq values Data processing Statistical analysis & interpretations qBase - Hellemans, 2007, Genome Biol geNorm - Vandesompele, 2002, Genome Biol

qPCR workflow – data processing accelerating your analysis 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 www.biogazelle.com Statistical package Copy number analysis Numerous other features

Quality control using qbase PLUS accelerating your analysis

Quality control using qbase PLUS accelerating your analysis

Quality control using qbase PLUS accelerating your analysis

Quality control using qbase PLUS accelerating your analysis

qPCR workflow – qPCR reactions accelerating your analysis Experiment design Statistical analysis and interpretation Calibration Sample prep Assay design qPCR reactions Calibrate with more than 1 sample Cq values Allow samples to have different copy numbers Data processing n  NRQ i CN NRQ   Statistical analysis & interpretations CN  i 1 i CF n CF Example: calibration with normal sample & sample with deletion NRQ NRQ  norm del  2 1 CF 2 Calculation of Z-scores

qPCR workflow – statistical analysis & interpretations accelerating your analysis 3 2.449 68% 2 1.414 95% 1 99.7% -4 -3 -2 -1 0 1 2 3 4

qPCR workflow – statistical analysis & interpretations accelerating your analysis Normal control Sample with partial deletion

qPCR based copy number screening in a clinical context accelerating your analysis Hoebeeck et al. , 2005 Laboratory Investigation D’haene et al. , 2010 J Clin Endocrinol Metab

qPCR based copy number screening in a clinical context - SHOX accelerating your analysis 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 Blaschke and Rappold, 2000

qPCR based copy number screening in a clinical context - SHOX accelerating your analysis 15% 39% SHOX deletion screening 38% 8% Diagnosis of PAR1 ISS or LWD deletion screening SHOX sequencing

qPCR based copy number screening in a clinical context - SHOX accelerating your analysis Requirements qPCR SHOX Sensitive + deletion screening Copy number screening Accurate + Diagnosis of PAR1 Reliable + ISS or LWD deletion screening Objective + SHOX Precise + sequencing Affordable + Flexible + Fast +

qPCR based copy number screening in a clinical context - SHOX accelerating your analysis Thirteen qPCR amplicons were designed based upon:

qPCR based copy number screening in a clinical context - SHOX accelerating your analysis 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

qPCR based copy number screening in a clinical context - SHOX accelerating your analysis 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

qPCR based copy number screening in a clinical context - SHOX accelerating your analysis 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

qPCR based copy number screening in a clinical context 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