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

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

Development of Ion AmpliSeq™ Community PanelsFALCON Global Consortia

Nathalie Bernard, Market Development Manager, Inherited Disease

Page 2: 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

Page 3: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

BRCA1 and BRCA2 Global [email protected]

Prof. Jeffrey N. Weitzel Division of Clinical Cancer GeneticsCity 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 HospitalGlasgow

Dr. Jose Louis Costa andDr Jose Carlos MachadoIPATIMUP Medical Faculty of Porto. Portugal

Marjolijn J.L. Ligtenberg, Arjen R. MensenkampRadboud University NijmegenMedical Centre, The Netherlands

Dr. Arif B. Ekici Institute of Human GeneticsFriedrich-Alexander-University of Erlangen-Nürnberg

Page 4: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 5: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 6: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ BRCA1 & BRCA2 PanelProject 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 reproducibilityPanel launch

• Global verification will be performed in 8 labs on additional 200 archived samples with known variant status

Phase 3:Global consortium

verification

Page 7: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ BRCA1 & BRCA2 Panel50 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 reproducibilityPanel launch

• Global verification will be performed in 8 labs on additional 200 archived samples with known variant status

Phase 3:Global consortium

verification

Page 8: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ BRCA1 & BRCA2 PanelMetrics

– Average coverage uniformity: 98.8%– Average on-target specificity: 97.4%

http://ioncommunity.lifetechnologies.com/docs/DOC-7184

Sample 1 Sample 20.00%

20.00%40.00%60.00%80.00%

100.00%

On-target Specificity

Sample 1 Sample 20.00%

40.00%

80.00%

Coverage Uni-formity

Page 9: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ BRCA1 & BRCA2 PanelProject 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 reproducibilityPanel 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

Page 10: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Bioinformatics pipeline

• 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

Read Generation Read Mapping

Variant Calling and Variant Annotation

Variant confirmation

and Interpretive

Report

Ion Reporter™ Software

Report

FilterAnalyze

Ion Reporter pre-configured

workflow

Page 11: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion Reporter™ Software Review Richly Annotated Variant list

>

Analyze

>

Sequence Import

Page 12: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

BRCA 1 and BRCA2 Global ConsortiumPreliminary 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 67/67 100%

point mutations 51 55/55 100%

123 134/134 100% 

Ion Reporter™ Software

Page 13: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Example of FP detection rate in one lab

Ion Reporter™ SoftwareBRCA1/2 single sample workflow

TP FP Sensitivity PPV

Run1 (10 samples) 109 3 100% 97.32%

Run2 (10 samples) 75 5 100% 96.15%

Run3 (10 samples) 55 3 100% 94.8%

Run4 (10 samples) 67 2 100% 97%

Page 14: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Coverage Analysis per Lab Across Runs

10

100

1000

10000

100000

lab1lab2lab3lab4lab5

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™

Page 15: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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 runrun1 run2 run3 run4

10

100

1000

10000

100000

lab1lab2lab3lab4lab5

Page 16: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ BRCA1 & BRCA2 Panel303-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

Page 17: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Molecular subsets of lung and colon adenocarcinoma

Pao & Hutchinson et al. Nature 2012

Page 18: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

OncoNetwork [email protected]

Dr. Nicola NormannoCentro Ricerche Oncologiche

Mercogliano, Italy

Prof. Orla SheilsTrinity College Dublin, Ireland

Dr. Marjolijn Ligtenberg & Dr. Bastiaan TopsRadboud University

Nijmegen Medical CentreThe Netherlands

Prof. Ian CreeWarwick Medical School United Kingdom

Prof. Pierre Laurent PuigUniversité Paris Descartes, France

Dr. Ludovic LacroixInstitut Gustave Roussy Paris, France

Prof. Aldo ScarpaARC-NET University of Verona,

Italy

Dr. Cristoph Noppen & Dr. Henriette Kurth

VIOLLIER AG Basel, Switzerland

8 labs experienced in colon & lung cancer research

Page 19: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 20: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ Colon and Lung Cancer PanelPanel 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 469

22 genes – 90 Amplicons- more than 500 variantsReceptor 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

Page 21: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 22: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ Colon and Lung Cancer Panel v1Amplicon Coverage

Sensitivity too low Loss of chip capacity

Page 23: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ Colon and Lung Cancer Panel v2Amplicon Coverage

Further optimization of primer set

More equal coverage, novel verification

8 instead of 5 samples on Ion 316 ™ chip

Page 24: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 25: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Phase 1: Ion AmpliSeq™ Colon and Lung Cancer Panel v2100% 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 ✓ ✓ ✓ ✓ ✓ ✓ ✓

1- Xenograft KRAS G12D ✓ ✓ ✓ ✓ ✓ ✓ ✓

1- Xenograft TP53 G244D ✓ ✓ ✓ ✓ ✓ ✓ ✓

2- Xenograft PIK3CA E545K ✓ ✓ ✓ ✓ ✓ ✓ ✓

2- Xenograft KRAS G12D ✓ ✓ ✓ ✓ ✓ ✓ ✓

2- Xenograft FBXW7 R465H ✓ ✓ ✓ ✓ ✓ ✓ ✓

1- Lung KRAS G12C ✓ ✓ ✓ ✓ ✓ ✓ ✓

1- AcroMetrix® KRAS G13D ✓ ✓ ✓ ✓ ✓ ✓ ✓

2- AcroMetrix® KRAS G12A ✓ ✓ ✓ ✓ ✓ ✓ ✓

W5 EGFR Deletion 19 ✓ ✓ ✓ ✓ ✓ ✓ ✓

W3 EGFR L858R ✓ ✓ ✓ ✓ ✓ ✓ ✓

Page 26: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

   

KRAS EGFR BRAF TP53 PTEN STK11 ERBB2Expected Variants

FOUND Detection

Rate %

Lab 1SNVs 6 3 2 - - - - 11 ✓

100

Indel - 1 - 1 - - - 2 ✓

LAB 2 SNVs 5 1  - -  - - -  6 ✓

100

indel -  2 -  -  - - -  2 ✓

LAB 3 SNVs 6 2 3 - - - - 11 ✓

100

indel - 5 - - - - 1 4 ✓

LAB 4 SNVs 2 2  - -  - - -  4 ✓

100

indel  - 2 -  -  - - -  2 ✓

LAB 5 SNVs 6 1 (dupl) 2 - 1 1 - 11 ✓

100

indel - - - - - - - - ✓

Page 27: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

The major classes of genomic alterations that give rise to cancer

Modified from McConaill - JCO 2010

EGFRErbB-2BRAFPIK3CAAKT1MAP2K1 EML4-ALK

ROS-1RET

EGFRErbB-2MET

Sequencing,Real Time PCR etc.

FISH,Immunohistochemistry

Page 28: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

OncoNetwork Global Consortium

Prof. Harriet Feilotter Department of Pathology at Queen's University. Ontario Canada

Dr. Jose CostaIPATIMUP Medical Faculty of Porto. Portugal

Marjolijn J.L. Ligtenberg, Arjen R. Mensenkamp

Radboud University NijmegenMedical Centre, The Netherlands

Dr. Nicola NormannoCentro Ricerche

Oncologiche Mercogliano, Italy

Prof. Orla SheilsTrinity College Dublin, Ireland

Prof. Ian CreeWarwick Medical School United Kingdom

Prof. Pierre Laurent PuigUniversité Paris Descartes, France

Dr. Ludovic LacroixInstitut Gustave Roussy Paris, France

Prof. Aldo ScarpaARC-NET University of

Verona Italy

Dr. Cristoph Noppen & Dr. Henriette KurthVIOLLIER 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

Page 29: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ Colon and Lung Panel – [email protected]

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

Page 30: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 31: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

FALCON Global Consortia ProcessIon 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

Page 32: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Ion AmpliSeq™ Portfolio Positioning

Design

Verification

Kits in inventory?

Life Technologies Customer Community

Life Technologies Customer Community

Yes, ready-to-useMade-to-order via

ampliseq.comMade-to-order via

ampliseq.com

Page 33: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Colon and Lung PanelBRCA1 - BRCA2 Panel

CFTR PanelTP53 Panel

AML Genes PanelCardio Genes Panel

Lung Fusion RNA PanelColon and lung Panel new design

Ion AmpliSeq™ Community Panels Design RoadmapHuman Genetics and Cancer Research focus

Page 34: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

CFTR Global [email protected]

Prof. Peter RaySick Kids Hospital, Toronto Ontario Canada

Prof. Claude FerecLGMH – CHU BrestBrest, France

Prof. Martin SomervilleAlberta Health ServicesEdmonton, AB, Canada

Dr Roland AchmannGenteQHamburg, Germany

Prof. Thierry BienvenuInstitut CochinParis, France

Prof Karsten TiemannLaborKroneBad Salzuflen, Germany

Page 35: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 36: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Preliminary Results (146 samples)

Type Variants Detected Correct Genotype

Long HP 4/4 4/4Indel 82/82 82/82SNV 313/313 313/313

Sensitivity * 100 % 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

Page 37: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Coverage Analysis For 2 Labs Preliminary results

Am

plic

on

Co

vera

ge

(lo

g10

)

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

1

10

100

1000

10000

100000

Lab1Lab2

Page 38: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Evaluation Metrics*

20.00%40.00%60.00%80.00%

100.00%

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

Page 39: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

TP53 Ion AmpliSeq™ Community [email protected]

Goals1. 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

Page 40: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 41: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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 %  

Page 42: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

AML Gene Panel [email protected]

Page 43: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 44: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

• Ion lab: overall panel performancePhase 1:

Performanc and Coverage

• Ion lab: testing on archived clinical samples with selected variants

Phase 2:

Detection Sensitivity

• 4 network labs: 160 archived samplesPhase 3:

Analytical Sensitivity

Page 45: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

CARDIO [email protected]

Dr Zofia MiedzybrodzkaNHS ScotlandAberdeen UK

Prof. Silvia PrioriFoundation Silvio MaugeriPavia, Italy

Prof Gilles MillatCHU LyonLyon, France

Dr Maria IasconeOspedali RiunitiBergamo, Italy

Dr Nicola MarzilianoOspedale NiguardaMilan, Italy

Page 46: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 47: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Transplantation - HLA typing

Page 48: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

HLA Global [email protected]

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

Page 49: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 50: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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

Page 51: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Melanoma Research

New Panel Proposals

Colon Hereditary Research

Hearing loss

Research

?

??

Thyroid Cancer

Research

Circulating Tumor Cell Research

Minimal Residual Disease

Research

Page 52: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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 [email protected] !

Page 53: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

Frank

Astrid

Alexander

Rosella Annelore

SimoneNathalie

ChrysanthiMelanie

Alain

Page 54: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

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 atlifetechnologies.com/iontorrent

Thank you !

Page 55: Development of Ion AmpliSeq Community Panels FALCON Global Consortia Nathalie Bernard, Market Development Manager, Inherited Disease

© 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