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Comprehensive screening of BRCA1/2 genes

Comprehensive screening of BRCA1/2 genes on the Ion PGM™ system, using an Ion AmpliSeq™ community designed gene panel. Arjen Mensenkamp, PhD Department of Human Genetics Laboratory of Tumor Genetics (LTG) Radboud University Nijmegen Medical Center The Netherlands. BRCA1/2 testing.

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Comprehensive screening of BRCA1/2 genes

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  1. Comprehensive screening of BRCA1/2 genes on the Ion PGM™ system, using an Ion AmpliSeq™ community designed gene panel Arjen Mensenkamp, PhD Department of HumanGenetics Laboratory of Tumor Genetics (LTG) Radboud University Nijmegen Medical Center The Netherlands

  2. BRCA1/2 testing • 15.8 kb ORF • Genes highly polymorphic, many homopolymer regions • Mutations scattered throughout the genes • Mostly truncatingmutations 5.6 kb, 1863 aa 10.3 kb, 3418 aa BRCA1 (on chromosome 17) BRCA2 (on chromosome 13)

  3. Why Ion Torrent approachin combination with AmpliSeq technology? Present: • 84 amplicons + MLPA • Fully automated PCR-robot, ABI 3700 sequencing • 50 - 60 samples per month • Efficient data analysis using JSI SEQPatient; TAT 3 - 5 weeks • “Expensive” technique Aim: • 2 - 3 multiplex PCR’s • Standardized Ion Torrent workflow • Shorter TAT • Reduction of costs

  4. Validation experiments multiplex AmpliSeq design

  5. BRCA1 and BRCA2 Global Consortia Dr. Marjolijn J.L. Ligtenberg, Dr. Arjen R. Mensenkamp Radboud University Nijmegen Medical Centre, The Netherlands Prof. Harriet Feilotter Department of Pathology at Queen's University. OntarioCanada Dr. Nicola Williams Southern General Hospital Glasgow, United Kingdom Prof. Jeffrey N. WeitzelDivision of Clinical Cancer Genetics City of Hope Cancer Center. Los Angeles, United States Dr. Arif B. EkiciInstitute of Human Genetics Friedrich-Alexander-University of Erlangen-Nürnberg, Germany Dr. Alfredo Hidalgo Miranda, National Institute of Genomic Medicine. Mexico City, Mexico Dr. José Louis Costa Dr. José Carlos Machado IPATIMUP Medical Faculty of Porto, Portugal

  6. Primer design Coverage of targets: • 100% coverage of all coding exons and exon-intron boundaries (-20 to +20; at least -10 to +10) • Amplicons covering exons are overlapping Primers: • Primers do not overlap • No validated SNPs in the last five nucleotides of primer • Max 4 validated SNPs per primer F1 F2 R1 R2 >1 nt +20

  7. Ion AmpliSeq BRCA 1/2 Panel • 3 primer pools, each with ~55 amplicons • All amplicons are <200 bp, except for one (~300 bp)

  8. Ion AmpliSeqBRCA1/2 Panel (8 samples on 316 chip) BRCA1 Depth of coverage cds exon BRCA2 Depth of coverage cds exon • Coverage: • 100% ORF • All, but 1 amplicon, >100x • Mean per amplicon: 2100 ± 302

  9. Examples of ‘difficult’ mutations BRCA1: c.1961delA BRCA1: c.3329dupA BRCA2: c.1813dupA BRCA1: c.670G>C Data analysis in Nijmegen and Porto with exchange of fastQ files

  10. False positive calls • Limited to homopolymer stretches • 1 to 4 per sample • Similar for all samples within a run; variable between runs >> easily recognizable during data analysis • Should disappear with optimization of run conditions and newer version of the variant caller

  11. Validation experiments multiplex Ion AmpliSeq design ✓ 100 mutations detected in Nijmegen and Porto ✓ in progress

  12. BRCA1 and BRCA2 Global Consortia Ion Reporter v1.6 - Preliminary Results • Data: Nijmegen - Porto • Analysis: Ion Reporter optimized BRCA Workflow • Workflow contains modified parameters for calling homopolymers • 0-3 false positive calls depending on the quality of the run Ion Reporter™ Software

  13. BRCA1 and BRCA2 Global Consortia Ion Reporter v1.6 - Preliminary Results • Data: Erlangen • Analysis: Torrent Suite Software – Torrent Variant Caller *modified parameters

  14. Ion Reporter™ Software Review Richly Annotated Variant list Sequence Import Analyze > >

  15. Requirements of a routine molecular test (multiplex) PCR IonTorrent sequencing • robust performance using limited amounts of (FFPE)-material (3 x 10 ng) • high accuracy • clinically relevant sensitivity • availability of results within days ✓ ✓ ✓ ✓ Detection of copy number variations is currently being validated

  16. Ongoing validation multiplex PCR approach • BRCA1 and BRCA2 • Hereditary Paragangliomas / Pheochromocytomas • Mismatch Repair Genes • …........... • Colon/Lung Cancer Panel • Melanoma panel • TP53 • ……….

  17. Acknowledgements Dr. Arif B. Ekici Institute of Human Genetics Friedrich-Alexander-University of Erlangen Nürnberg, Germany Dr. Marjolijn Ligtenberg Dr. Arjen Mensenkamp Dr. Bastiaan Tops Dr. Kornelia Neveling Dr. Marcel Nelen Prof. Jeffrey N. Weitzel Division of Clinical Cancer Genetics City of Hope Cancer Center Los Angeles, United States Dr. José Luis Costa Dr. José Carlos Machado Dr. Nicola Williams Southern General Hospital Glasgow, United Kingdom Prof. Harriet Feilotter Department of Pathology at Queen's University Ontario, Canada Dr. Rosella Petraroli Dr. Alain Rico Dr. ChrysanthiAinali Dr. Guoying Liu Dr. Mark Andersen Dr. Alexander Joyner Dr. Alfredo Hidalgo Miranda, National Institute of Genomic Medicine Mexico City, Mexico

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