Comprehensive screening of BRCA1/2 genes
on the Ion PGM™ system, using an Ion AmpliSeq™
community designed gene panel
Arjen Mensenkamp, PhD
Department of Human Genetics
Laboratory of Tumor Genetics (LTG)
Radboud University Nijmegen Medical Center
The Netherlands
BRCA1/2 testing
• 15.8 kb ORF
• Genes highly polymorphic, many homopolymer regions
• Mutations scattered throughout the genes
• Mostly truncating mutations
BRCA1 (on chromosome 17)
5.6 kb, 1863 aa
BRCA2 (on chromosome 13)
10.3 kb, 3418 aa
Why Ion Torrent approach
in combination with AmpliSeq technology?
Present:
• 84 amplicons + MLPA
• Fully automated PCR-robot, ABI 3700 sequencing
• 50 - 60 samples per month
• Efficient data analysis using JSI SEQPatient; TAT 3 - 5 weeks
• “Expensive” technique
Aim:
• 2 - 3 multiplex PCR’s
• Standardized Ion Torrent workflow
• Shorter TAT
• Reduction of costs
Validation experiments multiplex AmpliSeq design
BRCA1 and BRCA2 Global Consortia
Prof. Harriet Feilotter
Dr. Nicola Williams
Dr. Marjolijn J.L. Ligtenberg,
Dr. Arjen R. Mensenkamp
Department of Pathology at
Queen's University. Ontario Canada
Southern General Hospital
Glasgow, United Kingdom
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, United States
Institute of Human Genetics
Friedrich-Alexander-University of
Erlangen-Nürnberg, Germany
Dr. Alfredo Hidalgo Miranda,
National Institute of Genomic Medicine.
Mexico City, Mexico
Dr. José Louis Costa
Dr. José Carlos Machado
IPATIMUP Medical Faculty of Porto,
Portugal
Primer design
Coverage of targets:
• 100% coverage of all coding exons and exon-intron boundaries
(-20 to +20; at least -10 to +10)
• Amplicons covering exons are overlapping
Primers:
• Primers do not overlap
• No validated SNPs in the last five nucleotides of primer
• Max 4 validated SNPs per primer
F1
F2
R1
>1 nt
R2
+20
Ion AmpliSeq BRCA 1/2 Panel
• 3 primer pools, each with ~55 amplicons
• All amplicons are <200 bp, except for one (~300 bp)
Depth of coverage
Depth of coverage
Ion AmpliSeq BRCA1/2 Panel (8 samples on 316 chip)
BRCA1
cds
exon
BRCA2
cds
exon
Coverage:
• 100% ORF
• All, but 1 amplicon, >100x
• Mean per amplicon: 2100 ± 302
Examples of ‘difficult’ mutations
BRCA1: c.1961delA
BRCA1: c.3329dupA
BRCA2: c.1813dupA
BRCA1: c.670G>C
Data analysis in Nijmegen and Porto with exchange of fastQ files
False positive calls
• Limited to homopolymer stretches
• 1 to 4 per sample
• Similar for all samples within a run; variable between
runs
>> easily recognizable during data analysis
• Should disappear with optimization of run conditions and
newer version of the variant caller
Validation experiments multiplex Ion AmpliSeq design
✓
100 mutations
detected in Nijmegen and Porto
✓
in progress
BRCA1 and BRCA2 Global Consortia
Ion Reporter v1.6 - Preliminary Results
• Data: Nijmegen - Porto
• Analysis: Ion Reporter optimized BRCA Workflow
• Workflow contains modified parameters for calling homopolymers
• 0-3 false positive calls depending on the quality of the run
Type of Mutation
Unique Mutations
Samples
Sensitivity
In long homopolymer
Indel
point mutations
4
32
14
4/4
37/37
17/17
100%
100%
100%
50
58/58
Ion Reporter™ Software
BRCA1 and BRCA2 Global Consortia
Ion Reporter v1.6 - Preliminary Results
•
•
Data: Erlangen
Analysis: Torrent Suite Software – Torrent Variant Caller *modified parameters
Type of Mutation
Unique Mutations
Samples
Sensitivity
In long homopolymer
Indel
point mutations
2
9
18
3/3
9/9
18/18
100%
100%
100%
29
30/30
Ion Reporter™ Software
Review Richly Annotated Variant list
Sequence
Import
>
Analyze
>
Requirements of a routine molecular test
(multiplex) PCR
IonTorrent sequencing
✓
✓
✓
✓
• robust performance using limited amounts of (FFPE)material (3 x 10 ng)
• high accuracy
• clinically relevant sensitivity
• availability of results within days
Detection of copy number variations is currently being validated
Ongoing validation multiplex PCR approach
•
•
•
•
BRCA1 and BRCA2
Hereditary Paragangliomas /
Pheochromocytomas
Mismatch Repair Genes
…...........
•
•
•
•
Colon/Lung Cancer Panel
Melanoma panel
TP53
……….
Acknowledgements
Dr. Marjolijn Ligtenberg
Dr. Arjen Mensenkamp
Dr. Bastiaan Tops
Dr. Kornelia Neveling
Dr. Marcel Nelen
Dr. Arif B. Ekici
Institute of Human Genetics
Friedrich-Alexander-University of Erlangen
Nürnberg, Germany
Prof. Jeffrey N. Weitzel
Division of Clinical Cancer Genetics
City of Hope Cancer Center
Los Angeles, United States
Dr. José Luis Costa
Dr. José Carlos Machado
Dr. Nicola Williams
Southern General Hospital
Glasgow, United Kingdom
Dr. Rosella Petraroli
Dr. Alain Rico
Dr. Chrysanthi Ainali
Dr. Guoying Liu
Dr. Mark Andersen
Dr. Alexander Joyner
Prof. Harriet Feilotter
Department of Pathology at Queen's University
Ontario, Canada
Dr. Alfredo Hidalgo Miranda,
National Institute of Genomic Medicine
Mexico City, Mexico