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

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

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 Legal approval number 08455 Cardio was not approved but we added cardio genes panel that was agreed with legal to be acceptable in another call

BRCA1 and BRCA2 Global Consortium Rosella.Petraroli@lifetech.com
Prof. Harriet Feilotter Department of Pathology at Queen's University. Ontario Canada Dr. Nicola Williams Southern General Hospital Glasgow Marjolijn J.L. Ligtenberg, Arjen R. Mensenkamp Radboud University Nijmegen Medical Centre, The Netherlands Prof. Jeffrey N. Weitzel Division of Clinical Cancer Genetics City of Hope Cancer Center. Los Angeles Dr. Arif B. Ekici Institute of Human Genetics Friedrich-Alexander-University of Erlangen-Nürnberg Legal approval number 08455 Dr. Alfredo Hidalgo Miranda, National Institute of Genomic Medicine. Mexico City, Mexico Dr. Jose Louis Costa and Dr Jose Carlos Machado IPATIMUP Medical Faculty of Porto. Portugal

BRCA1 and BRCA2 Global Consortium Goal: Develop a BRCA1 and BRCA2 NGS panel with Ion AmpliSeq™ technology and Ion PGM™ Sequencer Coverage of targets: 100% coverage of all coding exons and exon-intron boundaries (-20 to +20) Amplicons covering exons are overlapping 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 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 Legal approval number 08455 1) It is the 100% coverage and not up so we can demonstrate that and we should promote If we say up to meant that we are not able to reach the 100% and it is not true

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 Legal approval number 08455 These are not goals but what we have achieved and we can demonstrate that Lower performance of FFPE in higher off-target reads, lower uniformity European Molecular Genetics Quality Network : extracted from “Best Practice Guidelines for Molecular Genetic in Hereditary Breast/Ovarian Cancer”

Ion AmpliSeq™ BRCA1 & BRCA2 Panel Project Design
Design following collaborators’ requirements First analytical verification on 20 archived samples 9-mer homopolymer variants and MLPA variants Phase 1: Design and test 30 archived samples with 65 known different variants tested and exchanged across 2 labs: Homopolymer stretches ✓ Deletions/insertions ✓ Point mutations ✓ Exon deletions ✓ Multiplex 8 samples per Ion 316™ Chip Phase 2: Analytical verification and reproducibility Panel launch Global verification will be performed in 8 labs on additional 200 archived samples with known variant status Phase 3: Global consortium verification Legal approval number 08455

Ion AmpliSeq™ BRCA1 & BRCA2 Panel 50 archived samples verified at Nijmegen and IPATIMUP
Design following collaborators’ requirements First analytical verification on 20 samples 9-mer homopolymer variants and MLPA variants Phase 1: Design and test 30 archived samples with 65 known different variants tested and exchanged across 2 labs: Homopolymer stretches ✓ Deletions/insertions ✓ Point mutations ✓ Exon deletions ✓ Multiplex 8 samples per Ion 316™ Chip Phase 2: Analytical verification and reproducibility Panel launch Global verification will be performed in 8 labs on additional 200 archived samples with known variant status Phase 3: Global consortium verification ✓ Legal approval number 08455

Ion AmpliSeq™ BRCA1 & BRCA2 Panel Metrics
Average coverage uniformity: 98.8% Average on-target specificity: 97.4% Legal approval number 08455 Data confirmed we have these data in house

Ion AmpliSeq™ BRCA1 & BRCA2 Panel Project Design
Design following collaborators’ requirements First analytical verification on 20 samples 9-mer homopolymer variants and MLPA variants Phase 1: Design and test 30 samples with 65 known different variants tested and exchanged across 2 labs: Homopolymer stretches ✓ Deletions/insertions ✓ Point mutations ✓ Exon deletions ✓ Multiplex 8 samples per Ion 316™ Chip Phase 2: Analytical verification and reproducibility Panel launch Global verification will be performed in 8 labs on additional 200 samples with known variant status Ion Reporter Analysis workflow Optimization Phase 3: Global consortium verification ✓ ✓ Legal approval number 08455

Bioinformatics pipeline
Report Filter Analyze Bioinformatics pipeline Read Generation Read Mapping Variant Calling and Variant Annotation Variant confirmation and Interpretive Report Trim adapter sequences Remove poor signal reads Split reads per barcode Assembly Allignment Coverage Analysis SNP/Indel Detection Annotate Variants Identify pathogenic variants Identify known polymorphisms Verify variants found Extract Report The Ion sequencers output raw sequencing data in the form of DAT files. These raw data are transferred to the Torrent Server for analysis pipeline processing and read generation. First step in the analysis pipeline is the read filtering which involves generation of various quality metrics for each base call, quality metrics for each read, and using those metrics to filter out low quality reads and trim reads back to acceptable quality levels. Additionally, adapter trimming is performed on the ends of each read. Unmapped reads are produced and uploaded in Ion Reporter Software, where the alignment QC stage is first taking place. When reads will be mapped into the reference genome, variant calling algorithm will start. In this stage variants will be detected, coverage will be generated and then variants will be annotated to various databases (Pfam, dbSNP, 1000Genomes, 5000 Exomes, UCSC common SNPs, COSMIC, OMIM, Cytogenetic band, Gene ontology, DrugBank, ClinVar, etc). In Step 4, user has the option to confirm the variants that were detected, save and classify them and finally generate an interpretive report. Ion Reporter pre-configured workflow Ion Reporter™ Software

Ion Reporter™ Software Review Richly Annotated Variant list
Sequence Import Analyze > > Ion Reporter is a software that includes a suite of bioinformatics tools. After the analysis will finish, a variant table report is created, where the user can look at the identified variants, get information about the function, coding, amino acid change, variant effect, as well other annotations from different databases (e.g. dbSNP, Oncomine etc)

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 Mutation Unique Mutations Samples Sensitivity In long homopolymer 11 12/12 100% Indel 61 67/67 point mutations 51 55/55 123 134/134 100% Please note that large deletion variants have not been included in the results since these variants were not included in the original requirements of the design of the panel so the panel has not been designed for that scope since at the time of the start of the project we had no solution for CNV analysis. We are now investigating the possibility to detect it and consortia performed more than 10 samples with these variants . We will have more information in the next weeks The data are preliminary since we recently get a new run from ipatimub and we are working on that to optimize parameter and reanalyze all the data. the key message to provide in this slide is that we are still collecting data from the consortium and these are just preliminary data at the end our aim is to optimize the parameter on more than 250 samples Ion Reporter™ Software

BRCA1/2 single sample workflow
Example of FP detection rate in one lab TP FP Sensitivity PPV Run1 (10 samples) 109 3 100% 97.32% Run2 (10 samples) 75 5 96.15% Run3 (10 samples) 55 94.8% Run4 (10 samples) 67 2 97% Ion Reporter™ Software BRCA1/2 single sample workflow

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™

Coverage of Amplicon in Exon 23 - BRCA2 Gene within the labs
Low high-throughput run 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™

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 Not approved by legal

Molecular subsets of lung and colon adenocarcinoma
Legal approval number 08455 The number of predictive biomarkers that will be assessed in clinical practice will rapidly increase with the availability of drugs that target specific molecular alterations. In some tumor types, such as non-small-cell lung carcinoma (NSCLC) and colorectal carcinoma (CRC), a new classification based on the identification of specific predictive and/or prognostic molecular alterations has emerged in the past decade. However, molecular diagnostics is faced with its own specific challenges. Costs per test, turn-around-time and the amount of material required for testing are increasing with the number of tests performed on a sample. In particular, availability of tissue is limiting in NSCLC, which is often diagnosed on small histological or cytological samples yielding limited amounts of DNA. Pao & Hutchinson et al. Nature 2012

OncoNetwork Consortium Rosella.Petraroli@lifetech.com
8 labs experienced in colon & lung cancer research Prof. Ian Cree Warwick Medical School United Kingdom Dr. Marjolijn Ligtenberg & Dr. Bastiaan Tops Radboud University Nijmegen Medical Centre The Netherlands Prof. Orla Sheils Trinity College Dublin, Ireland Dr. Cristoph Noppen & Dr. Henriette Kurth VIOLLIER AG Basel, Switzerland Dr. Ludovic Lacroix Institut Gustave Roussy Paris, France Prof. Aldo Scarpa ARC-NET University of Verona, Italy Legal approval number 08455 Dr. Nicola Normanno Centro Ricerche Oncologiche Mercogliano, Italy Prof. Pierre Laurent Puig Université Paris Descartes, France

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 Legal approval number 08455

Ion AmpliSeq™ Colon and Lung Cancer Panel Panel design and relevance
22 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 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 469 Legal approval number 08455 Yes there are reference and these are the list of genes that our collaborators provided to us .

Verification Workplan 155 archived FFPE Samples by 7 laboratories
Same 5 FFPE control samples across 7 labs 2 KRAS AcroMetrix® cell line controls, 1 lung tumor research sample, 2 xenograft colon tumor research sample Phase 1: Reproducibility Accuracy 10 FFPE blind samples, 6 labs, 60 samples total 10 colon and lung tumor FFPE research samples Each lab sent in 10 previously tested samples & received back 10 blind samples for sequencing Phase 2: Concordance 15 FFPE samples, 6 labs 90 samples total Each lab sequences 10 lung & 5 colon tumor research samples Samples vary greatly in tumor content levels (heterogeneity) Phase 3: Analytical Sensitivity Legal approval number 08455

Ion AmpliSeq™ Colon and Lung Cancer Panel v1 Amplicon Coverage
Legal approval number 08455 Sensitivity too low Loss of chip capacity

Ion AmpliSeq™ Colon and Lung Cancer Panel v2 Amplicon Coverage
Legal approval number 08455 Further optimization of primer set More equal coverage, novel verification 8 instead of 5 samples on Ion 316 ™ chip

Verification Workplan 89 archived FFPE samples Ion AmpliSeq™ Colon and Lung Cancer Panel v2
Same 7 control FFPE samples across 7 labs 2 KRAS AcroMetrix® cell line controls, 2 xenograft colon , 3 lung tumor research samples Phase 1: Reproducibility and Accuracy 10 blind FFPE samples across 6 labs, 60 samples total 10 colon and lung tumor research Each lab sent in 10 previously tested samples & received back 10 blind samples for sequencing Phase 2: Concordance 15 FFPE samples in 5 labs, 75 samples total Each lab sequences 10 lung & 5 colon tumor research samples Samples vary greatly in tumor content levels (heterogeneity) Phase 3: Analytical Sensitivity Legal approval number 08455

Phase 1: Ion AmpliSeq™ Colon and Lung Cancer Panel v2
100% Reproducibility - 7 FFPE samples - 7 labs Ion Reporter ™ Software FFPE Sample type Gene Protein lab1 lab2 lab3 lab4 lab5 lab6 lab7 1- Xenograft PIK3CA E542K ✓ KRAS G12D TP53 G244D 2- Xenograft E545K FBXW7 R465H 1- Lung G12C 1- AcroMetrix® G13D 2- AcroMetrix® G12A W5 EGFR Deletion 19 W3 L858R Legal approval number 08455 It is not up to 100% but 100 % reproducibility has been demonstrated

Phase 3: Ion AmpliSeq™ Colon and Lung Cancer Panel v2 100% Genotyping Sensitivity FFPE difficult samples KRAS EGFR BRAF TP53 PTEN STK11 ERBB2 Expected Variants FOUND Detection Rate % Lab 1 SNVs 6 3 2 - 11 ✓ 100 Indel 1 LAB 2 5 - - indel LAB 3 4 LAB 4 LAB 5 1 (dupl) Legal approval number 08455 It is not up to 100% but 100 % reproducibility has been demonstrated ** Lab 3 tested three different samples with the new panel with three different new mutations which were correctly detected

The major classes of genomic alterations that give rise to cancer
Sequencing, Real Time PCR etc. FISH, Immunohistochemistry EGFR ErbB-2 BRAF PIK3CA AKT1 MAP2K1 EGFR ErbB-2 MET Legal approval number 08455 EML4-ALK ROS-1 RET Modified from McConaill - JCO 2010

OncoNetwork Global Consortium
Prof. Harriet Feilotter Department of Pathology at Queen's University. Ontario Canada Prof. Ian Cree Warwick Medical School United Kingdom Marjolijn J.L. Ligtenberg, Arjen R. Mensenkamp Radboud University Nijmegen Medical Centre, The Netherlands Cecily P. Vaughn ARUP Institute for Clinical and Experimental Pathology Dr. Cristoph Noppen & Dr. Henriette Kurth VIOLLIER AG Basel, Switzerland Prof. Orla Sheils Trinity College Dublin, Ireland Prof. Kazuto Nishio, M.D. Kinki University School of Medicine, Osaka, Japan Prof. Pierre Laurent Puig Université Paris Descartes, France Prof. Aldo Scarpa ARC-NET University of Verona Italy Legal approval number 08455 Dr. Ludovic Lacroix Institut Gustave Roussy Paris, France Dr. Nicola Normanno Centro Ricerche Oncologiche Mercogliano, Italy Dr. Jose Costa IPATIMUP Medical Faculty of Porto. Portugal

Ion AmpliSeq™ Colon and Lung Panel – Redesign Rosella
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 Not submitted to legal new slide

Lung Fusion Panel Goal: Develop a lung tumor fusion panel based on Ion AmpliSeq™ RNA technology
Selective gene content related to Lung tumor Covers fusion variants of ALK, ROS and RET genes. Use low amount of input RNA Single primer pool requiring only 10 ng of RNA Internal positive control included Use ALK, ROS, RET gene expression targets 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. 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 Legal approval number 08455

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 collaborator’s 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 Legal approval number 08455

Ion AmpliSeq™ Portfolio Positioning
Design Life Technologies Customer Community Verification Legal approval number 08455 Life Technologies Customer Community Kits in inventory? Yes, ready-to-use Made-to-order via ampliseq.com Made-to-order via ampliseq.com

Ion AmpliSeq™ Community Panels Design Roadmap Human Genetics and Cancer Research focus
Colon and Lung Panel BRCA1 - BRCA2 Panel CFTR Panel TP53 Panel AML Genes Panel Cardio Genes Panel Lung Fusion RNA Panel Colon and lung Panel new design Legal approval number 08455 We did not get approval to use cardio and AML but I hard that was accepted if we use cardio gene panel and AML gene panel AML and Cardio not approved by legal but this is the official name on our web site

CFTR Global Consortium Nathalie.Bernard@lifetech.com
Prof. Claude Ferec LGMH – CHU Brest Brest, France Prof. Peter Ray Sick Kids Hospital, Toronto Ontario Canada Dr Roland Achmann GenteQ Hamburg, Germany Prof. Martin Somerville Alberta Health Services Edmonton, AB, Canada Prof Karsten Tiemann LaborKrone Bad Salzuflen, Germany Peter Ray the owner of the largest CFTR database in north america and before cftr. Org was his site Martin sommerville head of all genetic testing in alberta –large ucranian population with cftr highly prevalence . Ambry variant GTyTx repeat Normal is 7 and can be 5 or 9 repeat Ferec breast france and istitute cochine – reference center for cftr – cochin first accepted dx test for NF1 lot of experience on dx Strong in implementing pgm in dx settings Laborkrone- contract for germany .. Region Achmann genetect – service company Prof. Thierry Bienvenu Institut Cochin Paris, France

CFTR Ion AmpliSeq™ Community panel
Goal: develop an NGS Panel for CFTR analysis 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 Use low amount of input DNA Works on DNA extracted from archived blood and Dried Blood Spot 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 Adoptable by other research labs Reliable and easy data analysis. Ion Reporter ™ Software Cf When baby is born blood stick and put on card and punch to do few test This is the first test – quite chip they can see if there are biological parameter that provide info on potential disease First is metabolite test Real test is a second tier.. If there is a suspicion they go ACMG mutation must be detected in this list is variants that are really important dollar if they do not find they go to sequence and if they do not find MLPA Replace OLA - capillary electrophoresis --- MLPA deletion or rearrangement UTR there are important variant Get knowledge about intron variant to include Cftr2 have also explanation if the mutation is potentially pathogenic or not . Not all the 160 variant are pathogenic but are the most frequent( cut off of 9 samples) comity decide if they accept the mutation- there should be a scientific publication ( NNH?). List of 162 normally but the 2 large deletions (CFTR Del 2,3 and CFTR Del 22,23 have full deletion of 2 exons and originally not included in the list of must-have ; 1.4 kb and 21 kb). One variant alone, Delta 508, represents 70% of cases. To be in that list 1 solution to replace OLA sequence -

Preliminary Results (146 samples)
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 Type Variants Detected Correct Genotype Long HP 4/4 Indel 82/82 SNV 313/313 Sensitivity * 100 % * Excluding difficult variant. Each position was considered as only one position, regardless of the number of samples at a particular position

Coverage Analysis For 2 Labs Preliminary results
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 Amplicon Coverage (log10) This amplicon covers exon 18, which seems that do not >>> include any important variant.. In this experiment, we had quite >>> high polyclonality (40%), which might be one reason for this >>> coverage performance Amplicons

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

TP53 Ion AmpliSeq™ Community Panel Rosella. Petraroli@lifetech
TP53 Ion AmpliSeq™ Community Panel Goals Complete coverage of the TP53 Gene Coding regions Analyzes exons, intron-exon boundaries. Use low amount of input DNA Two primer pool requiring only 20 ng of DNA Paraffin Embedded samples compatible Panel Verified on FFPE archived samples 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 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 Legal approval number 08455

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 New slide not yet reviewed by legal

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 Mutation Unique Positions Samples Genotyping Sensitivity indel 9 8/9 88% point mutations 11 11/11 100% Overall sensitivity 95.00 % New slide not yet reviewed by legal

AML Gene Panel Consortium alexander.sartori@lifetech.com
First level: European expert network to develop AML gene panel To propose list with significant targets To test performance during design process by Ion Torrent‘s 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 Add some to better present each center Thiede: World known and accepted expert on AML and influencing national gudelines, experienced on PGM Rosemary Gale: large AML sample database and expert on rare cases.Working with David Linch, past President President of the British Society of Hematology Claude Preudhomme, heading a large leulkemia networl in France and is accepted opinion leader in the field, running hemetologic service enter; huge expertise; experienced on PGM Jacqueline Schoumans PhD, Professeure Associée, Chef de service de Génétique Médicale ad interim Directeur de l'Unité de Cytogénétique du Cancer Departement de laboratoire, CHUV, Lausanne; experienced on PGM with AML custon panel Project started March 2013 Background: Last proposal for AML Panel does not fully match routine testing requirements in terms of performance, content and design Goal: Redevelopment of AML panel for Clinical routine with recognized experts in the European leukemia community

AML Ion AmpliSeq™ Community panel
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 FLT3-D835 FLT3-N676K

AML Ion AmpliSeq™ Community panel
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 Ion lab: overall panel performance Phase 1: Performanc and Coverage Ion lab: testing on archived clinical samples with selected variants Phase 2: Detection Sensitivity 4 network labs: 160 archived samples Phase 3: Analytical Sensitivity

CARDIO Network Nathalie.Bernard@lifetech.com
Dr Zofia Miedzybrodzka NHS Scotland Aberdeen UK Prof Gilles Millat CHU Lyon Lyon, France Dr Nicola Marziliano Ospedale Niguarda Milan, Italy Key opinion leader cardio myopathy in france Most of these collaborators 500 cardiomyophaty All specialized Silvia is also working in USA Different view respect the other bringing USA view Still looking to have people from Asia Dr Maria Iascone Ospedali Riuniti Bergamo, Italy Prof. Silvia Priori Foundation Silvio Maugeri Pavia, Italy

CARDIO Ion AmpliSeq™ Community Panel
Goal: develop a Pan CARDIO Gene Panel and a set of subpanels targeting genes involved in cardiomyopathy research 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) Pan CARDIO gene panel and subpanels to be validated by the CARDIO Network collaborators: Access to more than archived samples previously tested by other methods 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 Sequencing and 8 gene by CE- 50 % TIME FIND a mutation Idea to have larger panel Impossible to have a single panel decision – cover only known variants and not the variant non significant – What disease cover – idea to have 1 comprensive Pick and choose the subpanel Channelopaty silvia panel Hypertropic and dilated From research all the genes are ok Collectign feedback on the content of the two panel

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

HLA Global Consortium Frank.Opdam@lifetech.com
Goal: Develop NGS HLA high resolution assay on Ion Torrent™ PGM™ system including software solution 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 EFI: European federation of immunogenetics ASHI: American society of histo and immunogenetics Both organizations deal with HLA typing and accreditations in both continents In our network: EFI president: Gottfried Fischer, University Medical Hospital, Vienna Be The Match Foundation: American bone marrow registry for stem cell donors for leukemia patients British bone marrow registry Anthony Nolan Lab: holds the database for HLA alleles: archives, updates and protects from nonsense information And many others…

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

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 17th workshop training network meeting

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

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

Alexander Simone Alain Nathalie Frank Astrid Rosella Annelore Melanie Chrysanthi

Start sequencing now at lifetechnologies.com/iontorrent
Thank you ! Start sequencing now at lifetechnologies.com/iontorrent

Limitations and Disclaimers
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. © 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

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

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

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