Accurate and objective copy number profiling using real-time PCR. Jan Hellemans

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1 Accurate and objective copy number profiling using real-time PCR Jan Hellemans

2 Why qpcr based CNV analysis Flexibility samples loci Accurate results Affordable Infrastructure Consumables

nonsense, frameshift, splice site exon or gene

3 LEMD3 copy number profiling osteopoikilosis, BOS, (melorheostosis) loss-of-funtion mutations in LEMD3 nonsense, frameshift, splice site exon or gene deletions screening of 37 mutation negative patients for 14 LEMD3 assays

4 Requirements for qpcr based CNV analysis Accurate Precise Objective Quality control & assurance

5 Assay accuracy More is better Improve accuracy by including multiple... PCR replicates Reference assays for normalization Calibrator samples (Assays per locus)

6 Assay accuracy PCR replicates At least 2 Improved accuracy Detect pipetting errors Allows quality control (delta Cq) 2 is sufficient Low technical variability with good pipetting and instruments

22 23 24 25 26 27 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 0.25 0.

7 Assay accuracy PCR replicates Results for the LEMD3 study PCR replicate variability in 6 * 384-well plates % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% ΔCq < % ΔCq < % ΔCq < % ΔCq < %

8 Assay accuracy reference assays for normalization Normalization Correct for sample specific differences in DNA concentration based on results for reference assays At least 2 Improved accuracy Allows quality control (genorm M value & CV) 2 is sufficient gdna has little sample specific variation The math genorm Vandesompele 2002 n NF NRQ= RQ n RQ ref, i NF i 1

9 Assay accuracy reference assays for normalization Results for the LEMD3 study Reference assay variability in 6 * 384-well plates M CV M CV 0

10 Assay accuracy calibrator samples CN NRQ CN=1 CN=2 CN=3 NRQ CN

11 Assay accuracy calibrator samples At least 2 Improved accuracy Allows quality control (ratio control) The math calibrate with more than 1 sample allow samples to have different copy numbers CF i n 1 NRQ CN i n i CN NRQ CF example: calibration with normal sample & sample with deletion CF NRQ norm NRQ del 2 1 2

12 Assay accuracy calibrator samples normal deleted 1 2 CF CN CN normal deleted 1 2

13 Assay accuracy calibrator samples Results for the LEMD3 study Error rate after calibration with 1 or 2 samples 12% 10% 8% 6% 4% 2% Error rate 0% dup del -40% -80%

14 Assay accuracy summary 2 PCR replicates 2 reference assays 2 calibrator samples

15 Assay reliability Resolution: 1 assay per gene per exon? Assay evaluation In-silico: interfering SNPs & secondary structures In-vitro: specificity & amplification efficiency Assay variability: normal distribution

16 Assay reliability Resolution: 1 assay per gene per exon? Assay evaluation In-silico: interfering SNPs & secondary structures In-vitro: specificity & amplification efficiency Assay variability: normal distribution

17 Assay reliability Results for the LEMD3 study 22 normal controls Distribution interval containing 95%

18 Objective CN interpretation What is the most likely copy number? < 1,414 CN=1 1,414 2,449 CN=2 > 2,449 CN=

19 Objective CN interpretation Confidence in interpretation Calculate a Z-score Compare to predefined max Z-score (e.g. 1,96 or 2,58) 68% technical error 95% 99.7%

20 Quality control Technical replicates: Delta-Cq < 0,5 with majority <0,3 Reference assays: M < 0,5 preferably M < 0,25 CV < 25% preferably CV < 15% Calibrators: Expected CN ratio Z-score: Results within 95% or 99% of normal CN interval Z < 1,96 or Z < 2,58 Consecutive assays to improve confidence

21 LEMD3 copy number profiling - results 1 patient with a full LEMD3 deletion known microdeletion syndrome with osteopoikilosis, short stature and mental retardation Menten patient with a partial LEMD3 deletion osteopoikilosis patient with craniosynostosis no duplications

22 qpcr based copy number profiling ready for upscaling?

23 Sacaling up an overview Disorder Leri-Weill dischondrosteosis Idiopathic short stature Gene SHOX 13 assays Samples 95 patients 30 normal controls Disorder Stargardt disease Gene ABCA4 64 assays Samples 60 patients 30 normal controls Assays: = 79 qpcrs: 14,544 = 39*384-well plates

24 Assay design & quality control Automated primer design RTprimerDB Pattyn 2003, 2006; Lefever 2009

28 Assay design & quality control Automated primer design RTprimerDB Pattyn 2003, 2006; Lefever 2008 In-silico quality control SNP assessment of primer annealing regions BLAST/BiSearch specificity analysis amplicon secondary structures (UNAFold)

31 101% 920%

32 Assay design & quality control Automated primer design RTprimerDB Pattyn 2003, 2006; Lefever 2008 In-silico quality control SNP assessment of primer annealing regions BLAST/BiSearch specificity analysis amplicon secondary structures (UNAFold) Empirical validation Capillary gel electroforesis SYBR Green I melting curves PCR efficiency using standard curves or PCR miner

33 qpcr setup Two Technical replicates Reference assays (ZNF80 & GPR15) Calibrator samples 384-well plates Reduced PCR volume Increased throughput Allows full sample maximization Automated pipetting Tecan Freedom Evo 5 µl mastermix in multi-dispense 3 µl sample in free-dispense 7 minutes / plate Measurements LC480

34 Data processing qbaseplus qbase: Hellemans Relative quantification Normalization with 2 reference assays Error propagation Quality control

38 Data quality control PCR replicate variability Delta-Cq < 0,5: 99% Delta-Cq < 0,3: 95% Reference gene stability GeNorm M ~ 0,1 CV ~ 5% Normalization factors Fold change < 4

39 Results for SHOX & ABCA4 SHOX 11 entire gene deletions 5 partial gene deletions 2 single exon deletions 3 partial gene duplications Example: exon 10 ABCA4

40 Conclusions qpcr based copy number profiling Fast (< 4 hours) Cheap Easy (RTprimerDB, qbase PLUS,...) 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

41 Acknowledgements Jo Vandesompele Geert Mortier Barbara D'haene Frauke Coppieters

Accurate and objective copy number profiling using real-time PCR

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