BRCA1 / 2 testing by massive sequencing highlights, shadows or pitfalls?

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1 BRCA1 / 2 testing by massive sequencing highlights, shadows or pitfalls? Giovanni Luca Scaglione, PhD Laboratory of Clinical Molecular Diagnostics and Personalized Medicine, Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Italy

2 Twenty years since the discovery of BRCA BRCA1 gene positionally cloned and sequenced 1, BRCA2 gene identified and sequenced 3, BRCA mutation life time cancer risks: breast (60 85%), ovarian (15 40%) Synthetic lethality of PARP inhibition in BRCA-deficient cell lines 8, Clinical antitumour activity of PARP inhibitor shown in BRCA-mutation associated cancer 10 Initiation of Phase III trials of PARP inhibitors in BRCA-mutant ovarian and breast cancers 2014: BRCA as PGx gene? 1. Friedman et al. Nat Genet 1994;8: ; 2. Miki et al. Science 1994;266:66 71; 3. Wooster et al. Nature 1995;378: ; 4. Tavtigian et al. Nat Genet 1996;12: ; 5. Brose et al. J Natl Cancer Inst 2002;94: ; 6. Thompson et al. J Natl Cancer Inst 2002;94: ; 7. Wooster, Weber. N Engl J Med 2003;348: ; 8. Farmer et al. Nature 2005;434: ; 9. Brody. N Engl J Med 2005;353: ; 10. Fong et al. N Engl J Med 2009;361:

3 Highlights Summary Working on 454 GS junior (Roche Diagnostics) 1. Up to 12 patients & multigenic profile within a run 2. Complete BRCA1/2 screening (100% coding regions) 3. Fast variant call report by means of bioinformatics tool: Amplicon Suite Shadows 1. Pre-NGS Quality Control >>> Fragment Analysis 2. NGS data robustness: Inter-run & Intra-run >>> Read coverage profile 3. Post-NGS Validation Experiments >>> Sanger Chemistry Pitfalls 1. PCR Bias (Primer Binding Energies, GC-content) 2. empcr errors 3. CNV Copy Number Variations

4 Summary Patients Sample 1. w/ breast cancer 2. and/or ovarian cancer 3. w/ a positive family history Collection whole blood sample (K 3 EDTA) 8-12 patients per run (3/4 day) DNA extraction

5 Complete BRCA1/2 screening (100% coding regions) Genomic DNA Fragment Library Barcoded Library Multiplex PCR, Amplification of Specific Targets 5 PCR multiplex 93 Amplicons Universal PCR, Addition of MIDs and 454 adaptor sequences

6 Complete BRCA1/2 screening (100% coding regions) Plex A B C D E empcr water-in-oil emulsion

7 Summary Gene Scan Profile Pre-emulsion QC step check-point Sanger Pre-NGS Quality Control (Fragment Analysis) Mut + or Re-Extraction Mut - NGS

8 Complete BRCA1/2 screening (100% coding regions) Pyrosequencing GS 454 Junior 454 GS Junior Clinical Lab Report Data Analysis

9 Summary Quantitative Analysis: Read Coverage > 38x NGS data robustness: Inter-run & Intra-run read cover profile

10 Case Report Read Coverage Warning!

11 Summary Fast variant call report by means of bioinformatics tool: Amplicon Suite

12 Summary QC Mut - NGS Mut + Sanger Post-NGS Validation Experiments (Sanger Chemistry)

13 Summary >Library Preparation< 1. Contamination (not completely excluded) 2. PCR Bias (Primer Binding Energies, GC-content) 3. Chimeras (up to 45%, high sequence similarity) 4. PCR errors (454 protocol TAQ error rate 1:9000 bases) >empcr< 1. empcr errors 2. Artificial Duplicates >Sequencing< Pitfalls Known Error Patterns 1. Well Cross-Talk Type I ( Ghost well ) 2. Well Cross-Talk Type II ( Optical Bleeding ) 3. Homopolymer Errors

14 Pitfalls Known Error Patterns >Library Preparation< 1. Contamination (not completely excluded) 2. PCR Bias (Primer Binding Energies, GC-content) Plex 3. Chimeras A B (up C D to 45%, E high sequence similarity) 4. PCR errors (454 protocol TAQ error rate 1:9000 bases) n=117 >empcr< 1. empcr errors 2. Artificial Duplicates >Sequencing< n of reads per plex 1. Well Cross-Talk Type [95% Conf. I ( Ghost Interval] well ) Variabl e (Mean ± SE) 2. Plex A Well Cross-Talk 120 ± 4 Type 107 II - ( Optical 124 Bleeding ) Plex B 180 ± Homopolymer Errors Plex C 130 ± Plex D 110 ± Plex E 150 ±

15 Case Report Read Coverage Warning! BRCA1 exon x MLPA Negative

16 Case Report Read Coverage Warning!

17 >Library Preparation< Pitfalls Known Error Patterns 1. Contamination (not completely excluded) 2. PCR Bias (Primer Binding Energies, GC-content) 3. Chimeras (up to 45%, high sequence similarity) 4. PCR errors (454 protocol TAQ error rate 1:9000 bases) >empcr< 1. empcr errors 2. Artificial Duplicates >Sequencing< 1. Well Cross-Talk Type I ( Ghost well ) 2. Well Cross-Talk Type II ( Optical Bleeding ) 3. Homopolymer Errors

18 Plex B - Amplicon Read Coverage PLEX B Variable n of reads per plex (Mean ± SE) [95% Conf. Interval ] Plex A 120 ± Plex B 180 ± Plex C 130 ± Plex D 110 ± Plex E 150 ±

19 Conclusions Amplicon Suite tool could be tuned for the Copy Number Variation (CNV) analysis in BRCA1/2 testing due to his highly flexible and fast capability of finding out data from GS Junior 454 runs The analysis performed not only in the inter-run series but also in intra-run series is the driving force in the setup of CNV analysis NGS data analysis could not be separated from amplicon library data analysis

20 Thank You

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