Nathalie Bernard, Market Development Manager, Inherited Disease

BRCA1 and
BRCA2
‘In this consortia we are sharing knowledge, technology, this is
the main point’, Dr. Scarpa,
University of Verona Your workflow with your own content
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

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. 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
Dr. Arif B. Ekici
Institute of Human Genetics
Friedrich-Alexander-University of
ErlangenNürnberg

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

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

BRCA1
BRCA2
Project Design
Phase 1:
Design and test
• Design following collaborators’ requirements
• First analytical verification on 20 archived samples
• 9-mer homopolymer variants and MLPA variants
Phase 2:
Analytical verification and reproducibility
Panel launch
• 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 3:
Global consortium verification
• Global verification will be performed in 8 labs on
additional 200 archived samples with known variant status

BRCA1
BRCA2
50 archived samples verified at Nijmegen and IPATIMUP
✓
Phase 1:
Design and test
• Design following collaborators’ requirements
• First analytical verification on 20 samples
• 9-mer homopolymer variants and MLPA variants
Phase 2:
Analytical verification and reproducibility
Panel launch
Phase 3:
Global consortium verification
• 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

BRCA1
BRCA2
Metrics
– Average coverage uniformity: 98.8%
– Average on-target specificity: 97.4%
On-target Specificity
100,00% http://ioncommunity.lifetechnologies.com/docs/DOC-
7184
50,00%
0,00%
Sample 1 Sample 2
Coverage Uniformity
100,00%
50,00%
0,00%
Sample 1 Sample 2

BRCA1
BRCA2
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

Read
Generation
• Trim adapter sequences
• Remove poor signal reads
• Split reads per barcode
Analyze
Filter
Report
•
•
Read Mapping
Assembly
Allignment
Variant Calling and Variant
Annotation
• Coverage Analysis
• SNP/Indel
Detection
• Annotate Variants
Variant confirmation and Interpretive
Report
• Identify pathogenic variants
• Identify known polymorphisms
• Verify variants found
• Extract Report
Ion Reporter pre-configured workflow
Ion Reporter™ Software

Sequence
>
Import
>
Analyze

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
In long homopolymer
Indel point mutations
11
61
51
123
Ion Reporter™ Software
Samples
12/12
67/67
55/55
134/134
Sensitivity
100%
100%
100%
100%

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

100000
10000
1000
100
10
Amplicon in exon 23 of BRCA2 lab1 lab2 lab3 lab4 lab5
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
1000
100 lab1 lab2 lab3 lab4 lab5
10
1
Low high-throughput run run3 run4 run1 run2
• 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™

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

Pao & Hutchinson et al. Nature 2012

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. Ludovic Lacroix
Institut Gustave Roussy
Paris, France
Prof. Pierre Laurent Puig
Université Paris Descartes, France
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

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

Panel design and relevance
22 genes – 90 Amplicons- more than 500 variants
Receptor Tyrosine Kinases genes
Receptor tyrosine kinases
Pathway Genes
EGFR, ERBB2, ERBB4, MET, FGFR1, FGFR2, FGFR3, DDR2, ALK
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

155 archived FFPE Samples by 7 laboratories
Phase 1:
Reproducibility
Accuracy
• 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 2:
Concordance
Phase 3:
Analytical
Sensitivity
• 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)

Amplicon Coverage
Sensitivity too low Loss of chip capacity

Amplicon Coverage
Further optimization of primer set
More equal coverage, novel verification
8 instead of 5 samples on Ion 316 ™ chip

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
1- Xenograft PIK3CA E542K
1- Xenograft
1- Xenograft
KRAS
TP53
G12D
G244D
2- Xenograft PIK3CA E545K
2- Xenograft KRAS G12D
2- Xenograft FBXW7 R465H
G12C 1- Lung KRAS
1AcroMetrix® KRAS
2AcroMetrix® KRAS
G13D
G12A
W5
W3
EGFR Deletion 19
EGFR L858R lab1
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓ lab4
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓ lab3
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓ lab2
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓ lab5
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓ lab6
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓ lab7
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓

Phase 3: Ion AmpliSeq ™ Colon and Lung Cancer Panel v2
100% Genotyping Sensitivity - 75 FFPE difficult samples
KRAS EGFR BRAF TP53 PTEN STK11 ERBB2
Expected
Variants
FOUND
Detection
Rate
%
Lab 1
LAB 2
LAB 3
LAB 4
LAB 5
SNVs
Indel
SNVs indel
SNVs indel
SNVs indel
SNVs indel
-
5
6
-
3
1
1
2
6
-
2
2
5
2
2 -
6 1 (dupl) 2
-
3
-
-
-
-
2
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
11
2
6
2
11
4
4
2
11
-
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
100
100
100
100
100
** Lab 3 tested three different samples with the new panel with three different new mutations which were correctly detected

Sequencing,
Real Time PCR etc.
FISH,
Immunohistochemistry
EGFR
ErbB-2
BRAF
PIK3CA
AKT1
MAP2K1
EGFR
ErbB-2
MET
EML4-ALK
ROS-1
RET
Modified from McConaill - JCO 2010

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
Marjolijn J.L. Ligtenberg,
Arjen R. Mensenkamp
Radboud University Nijmegen
Medical Centre, The Netherlands
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
Dr. Jose Costa
IPATIMUP Medical Faculty of Porto.
Portugal
Prof. Aldo Scarpa
ARC-NET University of
Verona Italy
Dr. Nicola Normanno
Centro Ricerche
Oncologiche Mercogliano,
Italy

Rosella.Petraroli@lifetech.com
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

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

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

Design Life Technologies Customer Community
Verification Life Technologies
Kits in inventory?
Yes, ready-to-use
Customer
Made-to-order via ampliseq.com
Community
Made-to-order via ampliseq.com

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

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

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

• 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

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
100000
10000
1000
100
10
1
Amplicons
Lab1
Lab2

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.

Rosella.Petraroli@lifetech.com
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

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

• 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 Samples Genotyping Sensitivity
9 8/9 88%
11 11/11
95.00 %
100%

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

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

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
Phase 2:
Detection Sensitivity
Phase 3:
Analytical Sensitivity
• Ion lab: overall panel performance
• Ion lab: testing on archived clinical samples with selected variants
• 4 network labs: 160 archived samples

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

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

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

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

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

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

Melanoma
Research
Minimal
Residual
Disease
Research
Thyroid
Cancer
Research
Circulating
Tumor Cell
Research
Colon
Hereditary
Research
Hearing loss
Research

Alain Rico
FALCON team
Global Clinical Research Program Manager
Picture: www.bio-itworld.com

Alain.Rico@lifetech.com
< 24 hours workflow
– 15 hours (3 hours hands on)
– 3 hours total WGA
70 € per embryo
– 32 embryos per run
– The cost of reagents will reduce over time
Same run, more info
– Aneuploidy
– Mitochondrial genome
– Mutations
* user data. Wells et al., submitted

Picture: www.bio-itworld.com
* user data. Wells et al., submitted

Select Cell(s)
Ion OneTouch™ 2
Whole Genome Amplification used for
Fragment library (100 bp insert)
+
Target specific amplico n
Ion 316™ Chip
Up to 32 embryos
100 bp reads
Custom Script or
Ion Reporter™

Day 1
1pm
O/N
Day 2
1pm

Ion PGM™
150K reads
aCGH
Data courtesy Wells et al., submitted

Detect chromosomal aberrations with built-in informatics baseline controls
1
Low-pass whole-genome sequencing
2
Analyze with aneuploidy workflow
3
Visualize aneuploidy through customize IGV karyotype view
4 Create your interpretive report

Sample 1: -5, -22
Average of 0.01X coverage per sample
– 100% concordance with aCGH
Data courtesy Dr Fiorentino , 10plex on Ion 316™ chip

Sample 2 : -13, +14, +15
Average of 0.01X coverage per sample – 100% concordance with aCGH
Data courtesy Dr Fiorentino , 10plex on Ion 316™ chip

IGV data courtesy Wells et al., submitted

Ex: CFTR carrier typing
IGV data courtesy Wells et al., submitted

Listen to Prof. Wells www.youtube.com/iontorrent
Interview on BBC Radio 4 http://www.guardian.co.uk/science/
2013/jul/07/ivf-baby-born-geneticscreening
Mapping the genes of a baby before implantation
New Genetic Test May Boost IVF Results
First Child Born Following Embryo Screening With
New Genome Analysis Technique

Astrid.Ferlinz@lifetech.com
JCVI
Rockville
David
Wentworth
Picture
SMI
Stockholm
Steve
Glavas
Picture
SMI Smittskyddsinstitutet
ISS Istituto Superiore di Sanità
CDC Centers of Disease Control
JCVI J Craig Venter Institute
USDA – NVSL US Dep of
Agriculture
– National Veterinary
Service Labs
ISS Rome
Gabriele
Vaccari
Picture
USDA –
NVSL
Ames, IA
Mary Lea
Killian
Picture
CDC
Atlanta
Catherine
Smith
Picture
Goals & Objectives:
Evaluate & define protocol for Flu Typing
Share & discuss results
Make protocol/publication publicly available

TM
Influenza A Virus
RNA virus
Genome size: approx 13.5 kb
Comprised of 8 segments that encode up to 11 proteins
Transcriptase: cap binding
Transcriptase: elongation
Transcriptase: protease activity?
Haemagglutinin
Nucleoprotein: RNA binding – transport of vRNA
Neuraminidase: release of virus
Matrix protein 1: major component of virion
Matrix protein 2: Integral membrane protein – Ion Channel
Non-structural protein 1: RNA transport, translation, splicing
Non-structural protein 2: function not known

TM
Core consensus 5’ Core consensus 3’
PCR primer RT/PCR primer
Influenza A virus genomic RNAs
•Highly specific Influenza A primer set (RT primer and PCR primer)
•High-fidelity master mixes for Reverse Transcription and PCR amplification of all 8 segments in a single tube
•Whole genome amplification
•Generates fragments that range in size from 900 bp to 2.4 kbp

Throughput
Data
TAT
Ion Torrent™
10 samples/run (Ion 314™ chip)
Whole genome
18 hrs/10 samples
Current CE (Sanger)
1 sample/run
H and N genes only
2-3 Days
Use RUO, research, epidemiology, monitoring
Screening (subset of samples only)
Specificity
Cost
Sub-types, mixed infections
115 Euro/sample*
Main H/N variants only; single infections
> €200 (H/N genes only)
Ion PGM™ Sequencing allows:
Deeper understanding of genetic landscape and re-assortments within the
Influenza A genome

RNA Extraction
MagMax™-96 Viral
RNA Isolation Kit
60 min
Reverse Transcription
20 µl with up to 8 µl
Sample
3 hrs
PCR
50 µl with ALL RT product
Optional: Agarose gel analysis
8 segments from 2400 to 900 bp size
30 min
Amplicon clean up &
Quantitation
MagMax™ Viral RNA
Isolation Kit
Nanodrop
~6 hrs
DNA library prep
Ion Xpress™ Plus
5 hrs
Enrichment of library
Ion PGM™ 200
OneTouch™ Template Kit
3 hrs
Sequencing
Ion 314™/316™ Chip

Ion 316™ Chip
Data provided by S Glavas, SMI,
Stockholm
Decrease of segment lengths
Clinical isolates (H1N1, 2009 H1 pandemic, H3) run in 10-plex on Ion 316™ Chip simultaneously
Results were in 100% concordance with previous CE- data (H and N only) and were also confirmed by CE-sequencing of all 8 segments

70
Released April 2013
Collaborator & internal R&D data

Launched in IC in April 2013 by collaborators from SMI

Flu Season Winter/Spring 2013
PathAmp TM FluA Pre-
Amplification Reagents were used to sequence whole-genome of H7N9
Highly accurate and sensitive results from both swab samples and isolated virus samples
Detection of mixed infections
Publication submitted

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 nathalie.bernard@lifetech.com
!

Alexander
Simone Alain
Nathalie
Frank
Astrid
Rosella
Annelore
Melanie Chrysanthi

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.

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