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Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease.

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Presentation on theme: "Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease."— Presentation transcript:

1 Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

2 Ion AmpliSeq Community Panels FALCON Leadership Consortia Your workflow with your own content Clinical Research Verification Enabled by Life through leadership networks Sign in to share your work with your peers Check what is available on the Ion Community Colon & lung CFTR - panel Cardio Genes panel In this consortia we are sharing knowledge, technology, this is the main point, Dr. Scarpa, University of Verona BRCA1 and BRCA2 BRCA1 and BRCA2

3 BRCA1 and BRCA2 Global Consortium Prof. Jeffrey N. Weitzel Division of Clinical Cancer Genetics City of Hope Cancer Center. Los Angeles Prof. Harriet Feilotter Department of Pathology at Queen's University. Ontario Canada Dr. Alfredo Hidalgo Miranda, National Institute of Genomic Medicine. Mexico City, Mexico Dr. Nicola Williams Southern General Hospital Glasgow Dr. Jose Louis Costa and Dr Jose Carlos Machado IPATIMUP Medical Faculty of Porto. Portugal Marjolijn J.L. Ligtenberg, Arjen R. Mensenkamp Radboud University Nijmegen Medical Centre, The Netherlands Dr. Arif B. Ekici Institute of Human Genetics Friedrich-Alexander-University of Erlangen-Nürnberg

4 BRCA1 and BRCA2 Global Consortium Goal: Develop a BRCA1 and BRCA2 NGS panel with Ion AmpliSeq technology and Ion PGM Sequencer 1.Coverage of targets: –100% coverage of all coding exons and exon-intron boundaries (-20 to +20) –Amplicons covering exons are overlapping 2.European Molecular Genetics Quality Network Guidelines –Primers do not overlap –No validated SNPs in the last five nucleotides of primer –Max 3 validated SNPs per primer 3.Adoptable by other research labs - accurate, affordable & easy –Single day workflow –Multiplex at least 6 samples per chip (316) –Reliable and easy data analysis – Ion Reporter Software

5 Ion AmpliSeq BRCA1 & BRCA2 Panel Resulting design meets requirements –167 amplicons across 3 primer pools (30 ng of DNA) –200 bp design (single exception 349 bp) FFPE samples with lower performance –No SNP in the 3 end of the primer –EMQN Best Practices Guidelines Care must be taken when designing PCR primers to avoid sequence variants (e.g. SNPs) in primer binding sites that could result in allele- biased amplification European Molecular Genetics Quality Network

6 Ion AmpliSeq BRCA1 & BRCA2 Panel Project Design

7 Ion AmpliSeq BRCA1 & BRCA2 Panel 50 archived samples verified at Nijmegen and IPATIMUP

8 Ion AmpliSeq BRCA1 & BRCA2 Panel Metrics –Average coverage uniformity: 98.8% –Average on-target specificity: 97.4% 7184

9 Ion AmpliSeq BRCA1 & BRCA2 Panel Project Design

10 Bioinformatics pipeline Ion Reporter Software Ion Reporter pre-configured workflow

11 Ion Reporter Software Review Richly Annotated Variant list > Analyze > SequenceImport

12 BRCA 1 and BRCA2 Global Consortium Preliminary Results from five labs (Phase 3 verification) Data: Nijmegen – Porto – Erlangen – Glasgow – Canada Analysis: Ion Reporter pre-configured BRCA Workflow Workflow contains modified parameters for calling homopolymers Not including in the sensitivity the samples with large exon deletions Type of MutationUnique MutationsSamplesSensitivity In long homopolymer1112/12100% Indel6167/67100% point mutations5155/55100% /134100% Ion Reporter Software

13 Example of FP detection rate in one lab Ion Reporter Software BRCA1/2 single sample workflow TPFPSensitivityPPV Run1 (10 samples) %97.32% Run2 (10 samples)755100%96.15% Run3 (10 samples)553100%94.8% Run4 (10 samples)672100%97%

14 Coverage Analysis per Lab Across Runs Amplicon in exon 23 of BRCA2 Take home message: Minimum coverage 100x. However, amplicon in exon 23 of BRCA2 might exhibit low coverage ( >~60x) in some runs. Even in that case, variants can be detected in this exon in this region with the current workflow in Ion Reporter

15 Coverage of Amplicon in Exon 23 - BRCA2 Gene within the labs Most of the runs in all the labs have coverage over 100x for this amplicon Low coverage is run-specific. Even low coverage ( > 60x), variants can be detected in this exon in this region with the current workflow in Ion Reporter Low high-throughput run

16 Ion AmpliSeq BRCA1 & BRCA2 Panel 303-bp deletion in IGV 303-bp deletion beyond scope of panel design and variant caller Heterozygous deletion initially detected by MLPA Deletion can be observed from coverage

17 Molecular subsets of lung and colon adenocarcinoma Pao & Hutchinson et al. Nature 2012

18 OncoNetwork Consortium Dr. Nicola Normanno Centro Ricerche Oncologiche Mercogliano, Italy Prof. Orla Sheils Trinity College Dublin, Ireland Dr. Marjolijn Ligtenberg & Dr. Bastiaan Tops Radboud University Nijmegen Medical Centre The Netherlands Prof. Ian Cree Warwick Medical School United Kingdom Prof. Pierre Laurent Puig Université Paris Descartes, France Dr. Ludovic Lacroix Institut Gustave Roussy Paris, France Prof. Aldo Scarpa ARC-NET University of Verona, Italy Dr. Cristoph Noppen & Dr. Henriette Kurth VIOLLIER AG Basel, Switzerland 8 labs experienced in colon & lung cancer research

19 OncoNetwork Consortium Goal: Develop a colon and lung tumor NGS panel with Ion AmpliSeq technology and Ion PGM Sequencer 22 selective gene content for colon and lung cancer research Markers in the receptor tyrosine kinase (RTK) pathway Include genes that might serve in the near future, AKT1, DDR2 and ERBB2 Selection of the genes regions based on mutation frequencies Use low amount of input DNA Single primer pool requiring only 10 ng of DNA Adoptable by other research labs Verified on archived FFPE samples Single day workflow Easy data analysis – Ion Reporter Software

20 Ion AmpliSeq Colon and Lung Cancer Panel Panel design and relevance –New genes DDR2 and MEK1 –KRAS exon4 to include codons 117 to 146 –EGFR exon12 to include codon 492 –BRAF exon11 to include codons 466 and genes – 90 Amplicons- more than 500 variants Receptor Tyrosine Kinases genes EGFR, ERBB2, ERBB4, MET, FGFR1, FGFR2, FGFR3, DDR2, ALK Receptor tyrosine kinases Pathway Genes KRAS, NRAS,PIK3CA, BRAF, PTEN, MAP2K1, AKT1 Cancer-related genes TP53, STK11, CTNNB1, SMAD4, FBXW7, NOTCH1

21 Verification Workplan 155 archived FFPE Samples by 7 laboratories

22 Ion AmpliSeq Colon and Lung Cancer Panel v1 Amplicon Coverage Sensitivity too low Loss of chip capacity

23 Ion AmpliSeq Colon and Lung Cancer Panel v2 Amplicon Coverage Further optimization of primer set More equal coverage, novel verification 8 instead of 5 samples on Ion 316 chip

24 Verification Workplan 89 archived FFPE samples Ion AmpliSeq Colon and Lung Cancer Panel v2

25 Phase 1: Ion AmpliSeq Colon and Lung Cancer Panel v2 100% Reproducibility - 7 FFPE samples - 7 labs Ion Reporter Software FFPE Sample type GeneProteinlab1lab2lab3lab4lab5lab6 lab7 1- XenograftPIK3CAE542K 1- XenograftKRASG12D 1- XenograftTP53G244D 2- XenograftPIK3CAE545K 2- XenograftKRASG12D 2- XenograftFBXW7R465H 1- LungKRASG12C 1- AcroMetrix®KRASG13D 2- AcroMetrix®KRASG12A W5EGFRDeletion 19 W3EGFRL858R

26 Phase 3: Ion AmpliSeq Colon and Lung Cancer Panel v2 100% Genotyping Sensitivity - 75 FFPE difficult samples ** Lab 3 tested three different samples with the new panel with three different new mutations which were correctly detected KRASEGFRBRAFTP53 PTENSTK11 ERBB2 Expected Variants FOUND Detection Rate % Lab 1 SNVs Indel LAB 2 SNVs indel LAB 3 SNVs indel LAB 4 SNVs indel LAB 5 SNVs 61 (dupl) indel

27 The major classes of genomic alterations that give rise to cancer Modified from McConaill - JCO 2010 EGFR ErbB-2 BRAF PIK3CA AKT1 MAP2K1 EML4-ALK ROS-1 RET EGFR ErbB-2 MET Sequencing, Real Time PCR etc. FISH, Immunohistochemistry

28 OncoNetwork Global Consortium Prof. Harriet Feilotter Department of Pathology at Queen's University. Ontario Canada Dr. Jose Costa IPATIMUP Medical Faculty of Porto. Portugal Marjolijn J.L. Ligtenberg, Arjen R. Mensenkamp Radboud University Nijmegen Medical Centre, The Netherlands Dr. Nicola Normanno Centro Ricerche Oncologiche Mercogliano, Italy Prof. Orla Sheils Trinity College Dublin, Ireland Prof. Ian Cree Warwick Medical School United Kingdom Prof. Pierre Laurent Puig Université Paris Descartes, France Dr. Ludovic Lacroix Institut Gustave Roussy Paris, France Prof. Aldo Scarpa ARC-NET University of Verona Italy Dr. Cristoph Noppen & Dr. Henriette Kurth VIOLLIER AG Basel, Switzerland Prof. Kazuto Nishio, M.D. Kinki University School of Medicine, Osaka, Japan Cecily P. Vaughn ARUP Institute for Clinical and Experimental Pathology

29 Ion AmpliSeq Colon and Lung Panel – Redesign Goal: Redesign the Ion AmpliSeq Colon and Lung panel to include new biomarkers and copy number detection Include the same gene targets of the colon and lung panel Add NRAS exon 4 variants ( p.117, p.146) and more ALK variants Add Copy number detection for the genes MET, FGFR1,FGFR2, ERBB2, MEK1, EGFR, ALK, KRAS, PTEN. Do not change the primers design of the existing amplicons Use low amount of input DNA Single primer pool requiring only 10 ng of DNA Adoptable by other research labs Verified on archived FFPE samples Single day workflow Easy data analysis – Ion Reporter Software

30 Lung Fusion Panel Goal: Develop a lung tumor fusion panel based on Ion AmpliSeq RNA technology 1.Selective gene content related to Lung tumor Covers fusion variants of ALK, ROS and RET genes. 2.Use low amount of input RNA Single primer pool requiring only 10 ng of RNA 3.Internal positive control included Use ALK, ROS, RET gene expression targets 4.Panel Verified by the Consortium on FFPE archived samples: 200+ FFPE archived samples previously tested by FISH, ICH or qPCR for EML/ALK fusions High selection of positive samples from archived samples. 5.Adoptable by other research labs Single day workflow Multiplex at least 8 samples per Ion 316 chip Reliable and easy data analysis Provide the same level of information as FISH

31 FALCON Global Consortia Process Ion Community Panels Develop applications that satisfy customer needs Content and workflow defined by International Consortia Analytical verification part of the development process Panel tested on clinical research samples at collaborators lab Complete workflow including software solution Include collaborators need to use the panel in their settings Share experiences of it with other users Be part of a community

32 Ion AmpliSeq Portfolio Positioning Design Verification Kits in inventory? Life TechnologiesCustomerCommunity Life TechnologiesCustomerCommunity Yes, ready-to-use Made-to-order via ampliseq.com

33 Ion AmpliSeq Community Panels Design Roadmap Human Genetics and Cancer Research focus

34 CFTR Global Consortium Prof. Peter Ray Sick Kids Hospital, Toronto Ontario Canada Prof. Claude Ferec LGMH – CHU Brest Brest, France Prof. Martin Somerville Alberta Health Services Edmonton, AB, Canada Dr Roland Achmann GenteQ Hamburg, Germany Prof. Thierry Bienvenu Institut Cochin Paris, France Prof Karsten Tiemann LaborKrone Bad Salzuflen, Germany

35 CFTR Ion AmpliSeq Community panel Goal: develop an NGS Panel for CFTR analysis 1.Complete coverage of 160 frequent CFTR variants (cftr2.org) Analyzes exons, intron-exon boundaries, and UTRs that contain common variants in the cystic fibrosis transmembrane regulator (CFTR) gene. No mutation in the 3 end of the primer Covers the common variants of the CFTR Gene as indicated by the CFTR2 database Detect Exon deletion to replace MLPA test – Feature nice to have –Inclusive of 23 CFTR mutations recommended by the American College of Medical Genetics (ACMG) ~85% of Caucasian CF carriers 2.Use low amount of input DNA Works on DNA extracted from archived blood and Dried Blood Spot 3.Panel Verified by the global CFTR network on known samples: More than 300 archived research samples previously tested by CE sequencing Access to a very large sample database through the network 4.Adoptable by other research labs Reliable and easy data analysis. Ion Reporter Software

36 Preliminary Results (146 samples) TypeVariants DetectedCorrect Genotype Long HP4/4 Indel82/82 SNV313/313 Sensitivity *100 % Ion Reporter 1.6 analysis CFTR Workflow with modified parameters for calling HP A new workflow will be developed for a correct genotype calling of a single difficult variant, not called automatically in IR 1.6 * Excluding difficult variant. Each position was considered as only one position, regardless of the number of samples at a particular position

37 Coverage Analysis For 2 Labs Preliminary results Amplicon Coverage (log10) Amplicons Final Panel design 102 amplicons One Lab on Ion 314 chip and the other lab on Ion 316 chip Up to 16 samples multiplexing is expected on Ion 314 chip, 48 on Ion 316 chip and 96 on Ion 318 chip Minimum 100x coverage – only one amplicon in one lab with low coverage but this is run specific

38 Evaluation Metrics* * Metrics have been calculated using 9 samples from one lab, GenteQ, ran in Life Tech laboratory in Darmstadt. These are preliminary results.

39 TP53 Ion AmpliSeq Community Panel Goals 1.Complete coverage of the TP53 Gene Coding regions Analyzes exons, intron-exon boundaries. 2.Use low amount of input DNA Two primer pool requiring only 20 ng of DNA 3.Paraffin Embedded samples compatible Panel Verified on FFPE archived samples 4.Panel Verified by Prof Anne-Lise Borresen-Dale More than 30 archived research samples previously tested by CE sequencing Access to a very large sample database through her network 5.Adoptable by other research labs Single day workflow Multiplex at least 8 samples per chip (Ion 316 chip) Reliable and easy data analysis using Ion Reporter Software

40 TP53 Ion AmpliSeq Community Panel Final Panel Design 24 amplicons across 2 pools – 20 ng DNA Compatible with DNA extracted by FFPE samples 100% coverage of CDS Recommended sample number to obtain 95% of amplicons at 500X Coverage: 2 (Ion 314 chip), 10 (Ion 316 chip), 20 (Ion 318 chip) 316 Chip: 10

41 TP53 Ion AmpliSeq Community Panel Ion Reporter Software Panel has been tested on 30 Samples previously genotyped by CE: 1 FN missed consistently due to complex mutation type and assembly New algorithm will further improve sensitivity in the next software release (~late Q3) Type of MutationUnique PositionsSamplesGenotyping Sensitivity indel98/988% point mutations1111/11100% Overall sensitivity %

42 First level: European expert network to develop AML gene panel –To propose list with significant targets –To test performance during design process by Ion Torrents specialists –To verify panel on archived samples –To demonstrate complete workflow –Members: Prof. Christian Thiede, Dresden Prof. Rosemary Gale, UCL Prof. Claude Preudhomme, CHRU, Lille Prof. Jacqueline Shoumans, CHUV Lausanne Second level: Extend network globally for panel review and feedback; project updates and early access option AML Gene Panel Consortium

43 Goal: Develop an NGS panel for AML genetic analysis Markers in AML Core Panel: ASXL1, BRAF, CBL, CEBPA*, DNMT3A, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, TPN11, RUNX1, TET2, TP53, WT1 Target list confirmed by numerous experts around the world Design requirements/goals Hot spot on key variants and full exon coverage depending on the targets Allele frequency detection 5% Two pool design Panel development status Amplicon Design accepted and ready for synthesis (R&D) *in bold = all exons covered AML Ion AmpliSeq Community panel

44 Fast 1-day workflow –Adoptable by other research labs Reliable and easy data analysis using Ion Reporter Software Panel Verified by the European AML network on archived samples –Sequencing of 120+ mutated archived research samples (previously tested with other methods), plus 40 controls –Access to a large sample database through the network AML Ion AmpliSeq Community panel

45 CARDIO Network Dr Zofia Miedzybrodzka NHS Scotland Aberdeen UK Prof. Silvia Priori Foundation Silvio Maugeri Pavia, Italy Prof Gilles Millat CHU Lyon Lyon, France Dr Maria Iascone Ospedali Riuniti Bergamo, Italy Dr Nicola Marziliano Ospedale Niguarda Milan, Italy

46 CARDIO Ion AmpliSeq Community Panel Goal: develop a Pan CARDIO Gene Panel and a set of subpanels targeting genes involved in cardiomyopathy research 1.Selective gene content Gene content established by CARDIO Network collaborators. Decision to have one Pan-CARDIO gene panel and 3 smaller, targeted subpanels (focused on main genes involved in cardiomyopathies, ARVC and channelopathies) 2.Pan CARDIO gene panel and subpanels to be validated by the CARDIO Network collaborators: Access to more than 2000 archived samples previously tested by other methods 3.Adoptable by other research labs Single day workflow Multiplex possible (depending on the subpanel) Reliable and easy data analysis. Complete workflow using Ion Reporter Software

47 HLA typing is essential to match donor material with recipient for blood, bone marrow stem cell (leukemia) and organ transplantation Transplantation - HLA typing

48 HLA Global Consortium Network consists of 16 Organizations: 20 participants from Canada, US, Australia, Germany, Austria, Netherlands, UK, France registries, clinical research and research labs Alpha test in July 2013 in Darmstadt lab- European participants: British Bone Marrow Registry University of Maastricht University of Vienna; EFI President University of Tuebingen Goal: Develop NGS HLA high resolution assay on Ion Torrent PGM system including software solution

49 HLA Global Consortium High resolution analysis: 6 classical full HLA genes (class I: HLA-A,B,C and class II HLA-DRB, DQB, DPB) Tiled Multiplex SR-PCR design; 400bp chemistry to minimize ambiguities Software solution: HLA module Plugin software

50 HLA Global Consortium Next Steps: Now: availability HLA module plugin software v32 for Ion Suite -Evaluation and feedback Q4 2013: -Beta testing with consortium members -ASHI Chicago Nov 17 th workshop training network meeting

51 Melanoma Research New Panel Proposals Colon Hereditary Research Hearing loss Research ? ? ? Thyroid Cancer Research Circulating Tumor Cell Research Minimal Residual Disease Research

52 NEW!! User-shared panels on Ampliseq.com Well be launching soon a new page on the Ampliseq.com website to promote panels developed and validated by Ion users. Submit yours now to

53 Frank Astrid Alexander Rosella Annelore Simone Nathalie ChrysanthiMelanie Alain

54 For Research Use Only. Not for use in diagnostic procedures. © 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. Start sequencing now at lifetechnologies.com/iontorrent Thank you !

55 © 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners For Research Use Only. Not for use in diagnostic procedures. Limitations and Disclaimer: Life Technologies Corporation takes no corporate position on the use of selection methods in IVF and prenatal settings though we acknowledge that people disagree about its appropriate use and it should ALWAYS be provided with full and informed, non-coerced prior informed consent. The PGM System and equipment used herein is RUO marked and may not be GMP. The results shown may not represent actual performance in an IVF or any other setting. LTC does not assure or endorse the use of its methods in ANY clinical setting outside of those that have been reviewed by the FDA or similar oversight body. Limitations and Disclaimers


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