We identified mutations in eleven cell free (cf) DNA samples by next generation sequencing (NGS) using the Ion AmpliSeq™ Colon & Lung Cancer Research Panel and the Ion PGM™ System. Since detection of low frequency mutant alleles may not always be called confidently in NGS, we verified results by rare mutation analysis using digital PCR on the QuantStudio™ 3D Digital PCR System as an independent method. We show that frequencies detected are consistent for both methods for low frequency mutant alleles at and below 1%.

1. Marion Laig1, Ana Justino2, Rosella Petraroli1, Le Lac1, Dieu Linh Vo1, Kamini Varma1, José Carlos Machado2, José Luis Costa2
1Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 2IPATIMUP, Porto, Portugal
RESULTS
Rare mutation analysis by digital PCR on
QuantStudio 3D confirmed the low mutation
frequency detected by NGS. Both
technologies showed matching results for all
eleven samples tested at mutation rates below
1%. Both technologies also detected expected
mutation rates based on the time points.
CONCLUSIONS
We confirmed the sensitivity and accuracy of
NGS for low frequency mutation detection.
Rare mutation analysis by digital PCR on
QuantStudio 3D is an excellent application to
verify NGS results in samples for cancer
research.
REFERENCES
Mills GB. 2012. An emerging toolkit for
targeted cancer therapies. Genome Res.
22(2):177-182
Kinde I. et al. 2011. Detection and
quantification of rare mutations with massively
parallel sequencing. PNAS 108(23):
9530-9535
Yates LR and Campbell PJ. 2012. Evolution of the
cancer genome. Nat Rev Gen 13:795-806.
Ma C et al. 2011. T790M and acquired resistance of
EGFR TKI: a literature review of clinical reports. J
Thorac Dis. 3(1): 10–18
Detection of Circulating Tumor DNA in Early- and
Late-Stage Human Malignancies. Bettegowda C et al.,
Sci Transl Med. 2014 February 19; 6(224)
ACKNOWLEDGEMENTS
We thank Alicia Burt and Rachel Formosa for
reviewing this document.
TRADEMARKS/LICENSING
© 2015 Thermo Fisher Scientific Inc. All rights
reserved
All trademarks are the property of Thermo Fisher
Scientific and its subsidiaries unless otherwise
specified. TaqMan is a registered trademark of Roche
Molecular Systems, Inc., used under permission and
license.
For Research Use Only. Not for use in diagnostic
procedures.
Rare Mutation Analysis Using Digital PCR on
QuantStudio™ 3D to Verify Ion AmpliSeq™ Next
Generation Sequencing for Cancer Research
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
ABSTRACT
We identified mutations in eleven cell free
(cf) DNA samples by next generation
sequencing (NGS) using the Ion AmpliSeq™
Colon & Lung Cancer Research Panel and
the Ion PGM™ System. Since detection of
low frequency mutant alleles may not always
be called confidently in NGS, we verified
results by rare mutation analysis using
digital PCR on the QuantStudio™ 3D Digital
PCR System as an independent method.
We show that frequencies detected are
consistent for both methods for low
frequency mutant alleles at and below 1%.
INTRODUCTION
Next generation sequencing (NGS) is
becoming an increasingly important analysis
tool for the discovery of biomarkers in
cancer research and to study tumor
heterogeneity in samples. Although NGS is
an excellent discovery tool, detection of low
frequency mutant alleles may not always be
called confidently due to an inherent low
error rate of the DNA polymerase used
(fidelity). It is therefore beneficial to verify
detection of rare alleles with an independent
method.
In this study, we set out to confirm NGS
sensitivity and accuracy detecting low
frequency mutations using rare mutation
analysis by digital PCR as an independent
technology. We present data showing
consistency in low level mutation detection
in cf DNA samples between two methods:
NGS and Rare Mutation Analysis using
digital PCR.
MATERIALS AND METHODS
Eleven cell free DNA samples isolated from
plasma were typed using the Ion AmpliSeq
panel and the Ion Proton PGM. The same
samples were then analyzed by digital PCR
to confirm the presence of the mutations
detected. Samples represented a mixture of
individual samples as well as the same
sample taken at different time points.
Mutations were identified and quantified by
digital PCR using TaqMan™ Rare Mutation
Assays (Applied Biosystems) for the
following mutations: EGFR T790M (COSMIC
ID 6240, Assay ID AHRSROS), L858R
(COSMIC ID 6224, Assay ID AHRSRSV),
and exon 19 deletions
E746_A750delELREA (COSMIC ID 6223,
Assay ID AHLJ0XO) and L747_P753>S
(COSMIC ID 12370, Assay ID AHZAGZG),
and KRAS mutations G12V (COSMIC ID
520, Assay ID AHX1IHY) and G12D
(COSMIC ID 521, Assay ID AH6R5PI).
Experiments were run according to the
TaqMan Rare Mutation Assays protocol.
Figure 3. Ion PGM System and QuantStudio 3D Rare Mutation Detection Solution
Table 1. Determination of Allelic Fractions in the EGFR and KRAS Genes in cell free DNA Samples
Allelic fractions of low frequency mutations in the EGFR and KRAS genes were determined in cell free (cf)DNA by
sequencing using the Ion AmpliSeq panel and verified by Rare Mutation Analysis using digital PCR. For digital
PCR, two chips were run for each mutation per sample. Allelic frequencies were consistent between the two
technologies at low percentages of 1% and below.
* One of two chips showed no mutation detection, the fraction represents the average of two chips ? Based on one positive read
Figure 2. Detection of EGFR T790M using the Ion AmpliSeq Colon & Lung Cancer Research Panel
Ion AmpliSeq results displayed in the Integrative Genomics Viewer (IGV, Broad Institute) Software detecting the
T790M mutation in the EGFR gene in the same sample as above (left), detection of wild-type only in the same
sample as above (right).
Mutant p.T790M Wild-type
Figure 1. Detection of the EGFR T790M Mutation by Digital PCR
Digital PCR results displayed in QuantStudio 3D AnalysisSuite™ Cloud Software showing the detection of the
T790M mutation in the EGFR gene in one sample (left, mutation in the outlined FAM cluster), detection of wild-type
only (VIC) in another sample (right).
Mutant p.T790M Wild-type
All samples were originally sequenced using the Ion AmpliSeq panel and the Ion PGM System (left) and verified
with Rare Mutation Assays by digital PCR using the QuantStudio 3D (right).
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