A large amount of data is available on the functional impact of missense mutations in TP53 tumor suppressor gene. and on mutation patterns in many different cancers. TP53 direct sequencing is a time-consuming method with limitations in detection level. Here we describe the development and verification of a TP53 Next Generation Sequencing method based on Ion AmpliSeq technology. Preliminary data demonstrate that the TP53 Ion AmpliSeq panel and Ion Reporter Software analysis solution meets the requirements of clinical research laboratories.

1. © 2012 Life Technologies
Development and verification of an Ion AmpliSeq™ TP53 Panel
Langerød1,2, L. Silwal-Pandit1,2, E. Rødland1,2, A. Dahlsgaard1,2, I. Bergheim1,2, C. Ainali3, C. Scafe3, F. Hyland3, G. Liu3, C. Van Loy3, M. Andersen3, H. Viernes3, R. Petraroli3, A. Børresen-Dale1,2;
1Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway, 2The K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical
Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway, 3Thermo Fisher Scientific - Life Sciences Solutions, South San Francisco, CA, United States.
Abstract
A large amount of data is available on the functional impact
of missense mutations in TP53 tumor suppressor gene. and
on mutation patterns in many different cancers. TP53 direct
sequencing is a time-consuming method with limitations in
detection level. Here we describe the development and
verification of a TP53 Next Generation Sequencing method
based on Ion AmpliSeqTM technology. Preliminary data
demonstrate that the TP53 Ion AmpliSeqTM panel and Ion
ReporterTM Software analysis solution meets the
requirements of clinical research laboratories.
Introduction
The tumor suppressor gene TP53 is altered in more than
half of all cancer types and encodes a transcription factor
which is activated in response to several forms of cellular
stress. It is a challenging gene with high homology to other
genes and Sanger Sequencing methods currently available
are tedious and time-consuming.Here we present the
results of a collaborative study for the development and
verification of a TP53 gene Ion AmpliseqTM Panel to cover
exonic regions of TP53 gene. The verification of the panel
has been performed in two phases. (Fig. 1). Here we present
the results of Phase 1.
Sequencing
q Sequencing reactions were carried out following standard
protocol with the Ion PGMTM Sequencer on Ion TM 318 chip.
Data analysis was performed on Ion Reporter TM Software 4.0
using the optimized TP53panel workflows.
q Ion Reporter™ Software was further used for optimization of
the algorithm, variant detection, annotation and interpretation,
with the aim of developing a variant knowledgebase specific to
P53 (Fig. 3).
Table 1. Performance sensitivity achieved in Phase 1 (30 samples)
Conclusion
The Ion AmpliSeq™ TP53 Panel enables the rapid
identification of disease-causing variants in your research
with the simplicity of PCR, flexible throughput, and a fully
integrated data analysis pipeline. The results obtained
during the verification (Table1) demonstrated that the panel
may be used in the clinical research setting.
Ø Cover TP53 coding regions comprehensively
Optimized primers sets to minimize off-target sequencing
Ø Enrich TP53 gene coding regions with simplicity and
speed of Ion AmpliSeq™ workflow
Only 30 minutes hands-on time
Ø Achieve flexible throughput
4 to 30 samples per chip to fit your experimental needs
Ø Filter and annotate your relevant variants
Integrated and optimized TP53-panel workflows
For more information on Ion AmpliSeqTM products, visit www.ampliseq.com
For Ion Reporter, visit ww.ionreporter.lifetechnologies.com/
For more information on Ion Torrent Systems, visit www.iontorrent.com
For Research Use Only. Not For Use in Diagnostic Procedures.
© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of
Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
Figure 4. Evaluation Metrics for the Reproducibility Study
Mutation type
# mutations
# miscalls
# no calls
% sensitivity
SNV
10
0
0
100%
Deletions
6
0
0
100%
Insertions
5
1
0
83.33%
TOTAL
21
1
0
95.23%
Results
The Ion AmpliSeq™ TP53 Panel covers TP53 coding regions
comprehensively using optimized primers to minimize off-
target sequencing. It is comprised of 24 primer pairs across 2
pools requiring only 20 ng of DNA. The design of short
amplicons of 125 to 175 bp permits the amplification of DNA
starting from formalin-fixed paraffin embedded material.
During Phase 1 all the expected somatic mutations including
single base substitutions, insertions and deletion were
identified by Ion ReporterTM Software, only a complex
duplication of 18bp was not detected. The overall analytical
sensitivity was 95,23% (Table.1). Average coverage
uniformity was over 87.5% for all the samples, the per base
accuracy was over 99% and the percentage of mapped reads
on the target over 96.8%. (Fig. 4). We also identified
additional mutations and we are now in the process of
verification . Ion Reporter was further used to develop a
systematic analytical pipeline for the variant calling, filtering,
annotation and reporting (Fig. 5).
1"
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On"Target"
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Figure 5. Simple workflow, simple reports, easy to filter variants of
interest
Figure 1. Verification Study
• Same 30 archived tumor
samples with know mutation
status
• 2 labs
Phase 1
Analysis parameter
optimization
Reproducibility study
• 200 archived tumor samples
Phase 2
Extended performance
verification
Material and Methods
Design
Non-overlapping primers were designed to provide
q 100% coverage of all coding exons and exon-intron
boundaries (+/- 30bp padding)
q overlapping amplicons covering exons
q minimal off-target sequencing
q no SNPs in the last five nucleotides of primer
q To permit successful amplification of DNA extracted
from FFPE material, amplicons were designed with a
length of 125-175bp (Fig. 2).
Figure 3. Ion Reporter Software Workflow
Figure 2. Ion AmpliSeq™ Community TP53 Panel: Design
(left y axis: coverage Uniformity and percent of mapped reads on target; right y axis: mean
depth, log transformed; x axis: samples)
Step1
Step2: Results and Filtering
Step3: Report