Goal - Life Technologies

Report
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
BRCA1
and
BRCA2
‘In this consortia we are sharing
knowledge, technology, this is
the main point’, Dr. Scarpa,
University of Verona
Colon
& lung
Clinical Research Verification
CFTR panel
Enabled by Life through
leadership networks
Cardio
Genes
panel
Check what is available
on the Ion Community
Sign in to share
your work with
your peers
BRCA1 and BRCA2 Global Consortium
[email protected]
Prof. Harriet Feilotter
Dr. Nicola Williams
Department of Pathology at
Queen's University. Ontario Canada
Southern General Hospital
Glasgow
Marjolijn J.L. Ligtenberg,
Arjen R. Mensenkamp
Radboud University Nijmegen
Medical Centre, The Netherlands
Prof. Jeffrey N. Weitzel
Dr. Arif B. Ekici
Division of Clinical Cancer Genetics
City of Hope Cancer Center. Los
Angeles
Institute of Human Genetics
Friedrich-Alexander-University of
Erlangen-Nürnberg
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
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
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 allelebiased amplification”
European Molecular Genetics Quality Network
Ion AmpliSeq™ BRCA1 & BRCA2 Panel
Project Design
Phase 1:
Design and test
Phase 2:
Analytical verification
and reproducibility
Panel launch
Phase 3:
Global consortium
verification
• Design following collaborators’ requirements
• First analytical verification on 20 archived samples
• 9-mer homopolymer variants and MLPA variants
• 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
• Global verification will be performed in 8 labs on
additional 200 archived samples with known
variant status
Ion AmpliSeq™ BRCA1 & BRCA2 Panel
50 archived samples verified at Nijmegen and IPATIMUP
✓
Phase 1:
Design and test
Phase 2:
Analytical verification
and reproducibility
Panel launch
Phase 3:
Global consortium
verification
• Design following collaborators’ requirements
• First analytical verification on 20 samples
• 9-mer homopolymer variants and MLPA variants
• 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
• Global verification will be performed in 8 labs on
additional 200 archived samples with known variant
status
Ion AmpliSeq™ BRCA1 & BRCA2 Panel
Metrics
– Average coverage uniformity: 98.8%
– Average on-target specificity: 97.4%
http://ioncommunity.lifetechnologies.com/docs/DOC7184
On-target Specificity
100.00%
50.00%
0.00%
Sample 1 Sample 2
Coverage Uniformity
100.00%
50.00%
0.00%
Sample 1 Sample 2
Ion AmpliSeq™ BRCA1 & BRCA2 Panel
Project Design
✓
✓
Phase 1:
Design and test
Phase 2:
Analytical verification
and reproducibility
Panel launch
Phase 3:
Global consortium
verification
• Design following collaborators’ requirements
• First analytical verification on 20 samples
• 9-mer homopolymer variants and MLPA variants
• 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
• Global verification will be performed in 8 labs on
additional 200 samples with known variant status
• Ion Reporter Analysis workflow Optimization
Analyze Filter
Bioinformatics pipeline
Report
Read
Generation
•
•
•
Trim adapter
sequences
Remove poor signal
reads
Split reads per
barcode
Variant
confirmation
and Interpretive
Report
Variant Calling
and Variant
Annotation
Read Mapping
•
•
•
Assembly
Allignment
•
•
•
Coverage Analysis
SNP/Indel
Detection
Annotate Variants
Ion Reporter
pre-configured
workflow
Ion Reporter™ Software
•
•
•
Identify pathogenic
variants
Identify known
polymorphisms
Verify variants found
Extract Report
Ion Reporter™ Software
Review Richly Annotated Variant list
Sequence
Import
>
Analyze
>
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
100%
point mutations
51
55/55
100%
123
134/134
100%
Ion Reporter™ Software
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
100%
96.15%
Run3 (10 samples)
55
3
100%
94.8%
Run4 (10 samples)
67
2
100%
97%
Ion Reporter™ Software
BRCA1/2 single sample workflow
Coverage Analysis per Lab Across Runs
100000
10000
lab1
1000
lab2
lab3
lab4
100
lab5
Amplicon in exon 23 of BRCA2
10
1
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
100000
10000
lab1
1000
lab2
lab3
100
lab4
lab5
10
Low high-throughput run
1
run1
run2
run3
run4
• 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
Molecular subsets of lung and colon adenocarcinoma
Pao & Hutchinson et al. Nature 2012
OncoNetwork Consortium
[email protected]
8 labs experienced in colon & lung cancer research
Prof. Ian Cree
Warwick Medical School
United Kingdom
Prof. Orla Sheils
Trinity College Dublin, Ireland
Dr. Ludovic Lacroix
Institut Gustave Roussy
Paris, France
Prof. Pierre Laurent Puig
Université Paris Descartes, France
Dr. Marjolijn Ligtenberg & Dr. Bastiaan Tops
Radboud University
Nijmegen Medical Centre
The Netherlands
Dr. Cristoph Noppen &
Dr. Henriette Kurth
VIOLLIER AG Basel, Switzerland
Prof. Aldo Scarpa
ARC-NET University of Verona,
Italy
Dr. Nicola Normanno
Centro Ricerche Oncologiche
Mercogliano, Italy
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
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
Verification Workplan
155 archived FFPE Samples by 7 laboratories
Phase 1:
Reproducibility
Accuracy
Phase 2:
Concordance
Phase 3:
Analytical
Sensitivity
• 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
• 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
• 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)
Ion AmpliSeq™ Colon and Lung Cancer Panel v1
Amplicon Coverage
Sensitivity too low
Loss of chip capacity
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
Verification Workplan 89 archived FFPE samples
Ion AmpliSeq™ Colon and Lung Cancer Panel v2
Phase 1:
Reproducibility and
Accuracy
• Same 7 control FFPE samples across 7 labs
• 2 KRAS AcroMetrix® cell line controls, 2 xenograft colon ,
3 lung tumor research samples
Phase 2:
Concordance
• 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 3:
Analytical
Sensitivity
• 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 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
✓
✓
✓
✓
✓
✓
✓
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
Phase 3: Ion AmpliSeq™ Colon and Lung Cancer Panel v2
100% Genotyping Sensitivity - 75 FFPE difficult samples
KRAS EGFR BRAF TP53 PTEN
Lab 1
LAB 2
LAB 3
LAB 4
LAB 5
3
-
-
ERBB2
-
-
11
FOUND
✓
SNVs
6
Indel
-
1
-
1
-
-
-
2
✓
SNVs
5
1
-
-
-
-
-
6
✓
indel
-
2
-
-
-
-
-
2
✓
SNVs
6
2
3
-
-
-
-
11
✓
indel
-
5
-
-
-
-
1
4
✓
SNVs
2
2
-
-
-
-
-
4
✓
indel
-
2
-
-
-
-
-
2
✓
SNVs
6
1 (dupl)
2
-
1
1
-
11
✓
indel
-
-
2
STK11
Expected
Variants
-
-
-
-
-
-
Detection
Rate
%
100
100
100
100
100
✓
** 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.
EGFR
ErbB-2
BRAF
PIK3CA
AKT1
MAP2K1
FISH,
Immunohistochemistry
EGFR
ErbB-2
MET
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
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
Dr. Ludovic Lacroix
Institut Gustave Roussy
Paris, France
Marjolijn J.L. Ligtenberg,
Arjen R. Mensenkamp
Radboud University Nijmegen
Medical Centre, The Netherlands
Prof. Aldo Scarpa
ARC-NET University of
Verona Italy
Dr. Jose Costa
IPATIMUP Medical Faculty of Porto.
Portugal
Dr. Nicola Normanno
Centro Ricerche
Oncologiche Mercogliano,
Italy
Ion AmpliSeq™ Colon and Lung Panel – Redesign
[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
Lung Fusion Panel
Goal: Develop a lung tumor fusion panel based on
Ion AmpliSeq™ RNA technology
1.
Selective gene content related to Lung tumor

2.
Covers fusion variants of ALK, ROS and RET genes.
Use low amount of input RNA

3.
Internal positive control included

4.
Use ALK, ROS, RET gene expression targets
Panel Verified by the Consortium on FFPE archived samples:


5.
Single primer pool requiring only 10 ng of RNA
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
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
Ion AmpliSeq™ Portfolio Positioning
Design
Life Technologies
Verification
Life Technologies
Kits in
inventory?
Yes, ready-to-use
Customer
Customer
Made-to-order via
ampliseq.com
Community
Community
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
CFTR Global Consortium
[email protected]
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 Karsten
Tiemann
LaborKrone
Bad Salzuflen,
Germany
Prof. Thierry Bienvenu
Institut Cochin
Paris, France
CFTR Ion AmpliSeq™ Community panel
Goal: develop an NGS Panel for CFTR analysis
1.
Complete coverage of 160 frequent CFTR variants (cftr2.org)




–
2.
Use low amount of input DNA

3.
Works on DNA extracted from archived blood and Dried Blood Spot
Panel Verified by the global CFTR network on known samples:


4.
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
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
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
Indel
SNV
Sensitivity *
4/4
82/82
313/313
100 %
4/4
82/82
313/313
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)
•
•
•
100000
10000
1000
Lab1
Lab2
100
10
Amplicons
1
Evaluation Metrics*
100.00%
90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
Per Base Accuracy
Mapped reads on
target
Coverage Uniformity at
20x
* 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
[email protected]
Goals
1.
Complete coverage of the TP53 Gene Coding regions

2.
Analyzes exons, intron-exon boundaries.
Use low amount of input DNA

3.
Two primer pool requiring only 20 ng of DNA
Paraffin Embedded samples compatible

4.
Panel Verified on FFPE archived samples
Panel Verified by Prof Anne-Lise Borresen-Dale


5.
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
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
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
indel
point mutations
Overall sensitivity
Unique Positions
9
11
Samples
8/9
11/11
95.00 %
Genotyping Sensitivity
88%
100%
AML Gene Panel Consortium
[email protected]
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 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
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
Phase 1:
Performanc and Coverage
• Ion lab: overall panel performance
Phase 2:
Detection Sensitivity
• Ion lab: testing on archived clinical samples with
selected variants
Phase 3:
Analytical Sensitivity
• 4 network labs: 160 archived samples
CARDIO Network
[email protected]
Dr Zofia Miedzybrodzka
NHS Scotland
Aberdeen UK
Prof Gilles Millat
CHU Lyon
Lyon, France
Dr Nicola Marziliano
Ospedale Niguarda
Milan, Italy
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
1.
Selective gene content

2.
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:

3.
Access to more than 2000 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
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
[email protected]
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
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
Melanoma
Research
Minimal
Residual
Disease
Research
Thyroid
Cancer
Research
?
?
?
Circulating
Tumor Cell
Research
Colon
Hereditary
Research
Hearing
loss
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 [email protected] !
Alexander
Simone
Alain
Nathalie
Frank
Astrid
Rosella
Melanie
Chrysanthi
Annelore
Thank you !
Start sequencing now at
lifetechnologies.com/iontorrent
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.
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

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