Precise and rapid mutation detection is critical to the advancement of cancer research. In addition, patient outcomes can depend on the rapid mutation identification workflows.
Compared to other methods, Droplet Digital™ PCR (ddPCR™) allows researchers to detect rare mutations at very low fractional abundance, with faster turnaround times and at lower costs.
This app note highlights the latest ddPCR solutions for advanced assay multiplexing. It demonstrates how researchers can get the most out of precious clinical samples, with time and cost-saving advantages over gene sequencing methods.
Download this app note to discover:
- The wider benefits of ddPCR over next-generation sequencing technology
- Mutation detection examples that can be applied to multiple cancer types
- An elegant 6-plex solution that leverages both sensitivity and accuracy
Mutation Detection Multiplexing Using
the QX600 Droplet Digital PCR System
Abiodun Bodunrin, Leisa Jackson," Sarah Kreston,7 and Aki Uehara"
"Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547, USA
Biodesix, Inc., 2970 Wilderness Place, Boulder, CO 80301, USA
Introduction
Mutation detection is vital in translational and clinical oncology
research, as cancers arise from germline and somatic genetic
mutations. Molecular profiling utilizing mutation detection can
help to inform early detection and diagnosis, treatment selection
and response, and monitoring of minimal residual disease
(MRD), ultimately resulting in improved survival rates and patient
outcomes. Mutation detection entails the detection of a variant
sequence present at a minute frequency in a wild-type (WT)
genetic background. The challenge, therefore, is the precise
discrimination between two highly similar sequences, one of
which is significantly more abundant than the other. The ability to
detect and quantify very rare events is particularly important in
noninvasive sample types like cell-free DNA (cfDNA) from plasma.
Droplet Digital PCR enables mutation detection with sensitivity
and precision levels beyond the capabilities of other methods. In
identifying mutations, ddPCR Systems enable researchers to find
"a needle in the haystack" by partitioning a sample into nanoliter-
sized droplets. Compared to next-generation sequencing (NGS),
ddPCR Systems have higher sensitivity and are capable of detecting
mutations at a minor allele frequency (MAF) rate as low as 0.001%.
Comparatively, most validated clinical NGS platforms have a lower
limit of detection between 2-15%, depending on the workflow and
mutation being detected (Singh 2020). Most NGS workflows are not
deally suited for specialized applications requiring detection of low-
evel mutations, such as monitoring therapy response and minimal
esidual disease by liquid biopsy. Other advantages of ddPCR
Abstract
Precise and rapid mutation detection is critical to the advancement of cancer research, as patient
outcomes depend on the early and accurate identification of these mutations. Compared to other methods
of mutation detection, Droplet Digital" PCR (ddPCR") allows researchers to detect rare mutations at very
ow fractional abundance, with faster turnaround times, and at lower costs. In this study, we demonstrate
how the QX600" ddPCR System can detect mutations commonly associated with non-small-cell lung
cancer (NSCLC) in a single-well, 6-plex reaction. While multiplexing cancer mutations is often challenging
due to the need to discriminate multiple sets of highly similar sequences in a single well, here we show the
capability of the QX600 ddPCR System to detect mutations with simple analysis.
Systems over NGS include faster turnaround time, less hands-
on-time, and lower cost. These combined benefits allow ddPCR
technology to advance discoveries and improve outcomes across
the patient journey. It is currently utilized in targeted mutational
screening and monitoring, therapy response tracking, and molecular
relapse monitoring, and appears in over 2,000 peer-reviewed
publications related to mutation detection and liquid biopsy
(bio-rad.com/ddPCR/publications).
Bio-Rad's QX600 Droplet Digital PCR System has six channels to
enable advanced multiplexing, which researchers and investigators
can utilize to obtain more answers from precious samples. Here,
we demonstrate an elegant 6-plex mutation detection example that
leverages the sensitivity and accuracy of the QX600 ddPCR System.
Materials and Methods
The objective of the experiment was to create a multiplex assay for
the quantification of multiple mutations and a WT control in a single
well on the Bio-Rad QX600 ddPCR System. Mutation targets that
were used for the assay design are shown in Table 1, and were
combined with the ddPCR EGFR Exon 19 Deletions Screening Kit
(Bio-Rad Laboratories, Inc., catalog #12002392) to detect the
EGFR/KRAS/BRAF mutant targets and EGFR WT. Assays were
combined to create a 1x solution and have been made available
as a ddPCR Expert Design Assay (catalog #12008212; assay
ID: dEXD95349967). Analytical materials used throughout the
experiment were synthetic DNA sequences (gBlocks Gene
Fragments, Integrated DNA Technologies, Inc. [IDT]) and Human
Table 1. Mutation detection assays and corresponding detection channels.
Assay
EGFR Exon 19 Deletions
EGFR WT
KRAS G12C
EGFR L858R
BRAF V600E
EGFR T790M
WT, wild type.
Table 2. ddPCR reaction setup.
Volume per Reaction, ul Final Concentration
900 nM primers/
250 nM probe
Detection Channel
FAM
HEX
Cy5
Cy5.5
ROX
ATTO 590
Component
ddPCR Multiplex Supermix
20x 4-Plex Assay (EGFR L858R,
KRAS G12C, BRAF V600E,
EGFR T790M;
(ddPCR Expert Design Assay,
catalog #12008212; assay
ID: dEXD95349967)
20x EGFR Exon 19 Deletions
Screening Assay (#12002392)
Target DNA sample and/or
DNase-free water
Total volume
* For the Automated Droplet Generator, prepare 22 pl per well.
Table 3. Recommended thermal cycling conditions for the Bio-Rad C1000
Touch Thermal Cycler .*
Cycling Step
Enzyme
activation
Denaturation
Annealing/
extension
Enzyme
deactivation
Hold (optional)
* The same protocol can be used for the Bio-Rad PTC Tempo Deepwell
Thermal Cycler (#12015392; see bulletin 3530).
Genomic DNA (Female) from Promega Corporation (catalog
#G1521) for the WT quantification. No template controls (NTCs)
were performed using nuclease-free water in place of gBlocks
Gene Fragments. Full assay concentrations for the components
of a 1x assay were 900 nM of each primer and 250 nM of each
probe. ddPCR Multiplex Supermix (Bio-Rad, #12005909) was
used. Table 2 shows the reaction components, including the
master mix and all other reagents.
Droplet generation was performed on the Automated Droplet
Generator (Bio-Rad, #1864101) using DG32 Automated Droplet
Generator Cartridges (Bio-Rad, #1864108) and Automated Droplet
Generation Oil for Probes (Bio-Rad, #1864110). Thermal cycling
was performed on the C1000 Touch Thermal Cycler with 96-
Deep Well Reaction Module (Bio-Rad, #1851197) with the thermal
cycling protocol listed in Table 3. Detection was performed on
the QX600 Droplet Reader (Bio-Rad, #12013328) using ddPCR
Droplet Reader Oil (Bio-Rad, #1863004).
1%
1x
Variable
20*
Results
These data show that the QX600 System and Bio-Rad multiplex
assay enable multiplex mutation detection with simple analysis.
The NTC samples are clean, with no positive clusters detected
in the NTC control wells, as shown in Figure 1. In Figure 2, the
two-dimensional fluorescence amplitude plots that were generated
after performing the multiplex ddPCR reaction demonstrate that
the distinct targets form unique clusters in a single well. The
FAM (mutant)/HEX (WT) channels are derived from the ddPCR
EGFR Exon 19 Deletions Screening Kit, a nondiscriminatory assay
for 15 different EGFR mutations on exon 19 of the EGFR gene. The
remaining four assays belong to different cancer mutation assays:
KRAS G12C, EGFR T790M and L858R, and BRAF V600E. These
mutations are commonly associated with NSCLC samples.
Beyond the mutations shown here, the QX600 System can be used
for many types of cancer assays. The Bio-Rad assay catalog and
design engine are available to support numerous additional mutation
detection assays. An expert design service is also available for
advanced design needs and other applications.
Temperature, C
95
94
55
98
Time
10 min
30 sec
1 min
10 min
Infinite
Ramp Rate,
"C/sec
2
Number of
Cycles
40
2
2
1
1
Mutation Detection Multiplexing Using the QX600 Droplet Digital PCR System
A
C
9.000
8,000
FAM amplitude
8.000
7,000
6,000
5,000
4,000
3,000
2.000
1,000
4.000
3.500
3,000
Cy5 amplitude
6.000
5.000
4,000
3,000
2,000
1,000
ROX amplitude
2,500
2,000
1,500
1.000
500
-1,000
0
2,000
4.000
HEX amplitude
6.000
8.000
1.000
5.000
1.000
ATTO 590 amplitude
3.000
5.000
A
3.000
Cy5.5 amplitude
Fig. 1. Multiplex ddPCR analysis of no template control (NTC) samples with a 6-plex mutation detection assay. The two-dimensional plots show no amplification in any
of the NTC wells. The assay mix shows a clean assay and environment with no false positives, nonspecific binding, or primer interactions. A, data for EGFR exon 19 deletions
(FAM) and wild-type EGFR (HEX). B, data for KRAS G12C (Cy5) and EGFR L858R (Cy5.5). C, data for BRAF V600E (ROX) and EGFR T790M (ATTO 590).
E
-1,000
-1,000
-500-
-1,000
C
EGFR AE746-A750
KRAS G12C
BRAF V600E
EGFR T790M
FAM amplitude
8.000
7,000
8.000
5,000
4,000
3.000
2,000
1,000
Cy5 amplitude
ROX amplitude
EGFR WT
8.000
7,000
6,000
5,000-
4,000
3.000
2,000
1,000
EGFR L858R
3.500
3,000
2 500
2,000
1,500
1,000
500
-1,000
-500
Fig. 2. The two-dimensional fluorescence amplitude plots show a single well of five different mutant targets and a single wild-type (WT) EGFR target. A, data for the
FAM and HEX channels, showing EGFR exon 19 deletion-positive mutants (blue) and EGFR WT positive droplets (green). The black cluster represents droplets negative for both
targets. B, The Cy5/Cy5.5 channels, with KRAS G12C positive droplets (light red) and EGFR L858R positive droplets (purple). The black cluster represents negative droplets.
C, The ROX/ATTO 590 channels, with BRAF V600E positive droplets (dark red) and EGFR T790M positive droplets (cyan). The black cluster represents negative droplets.
Conclusions
Multiplexing allows for the detection of multiple targets from a small
sample, saving costs and increasing sample throughput. However,
multiplexing in mutation detection is challenging due to the need to
discriminate multiple sets of highly similar sequences in a single well.
This study demonstrates that the QX600 Droplet Digital PCR System
can detect a 6-plex mutation detection assay with simple analysis,
which is currently unique among similar systems on the market.
The advanced 6-color detection capabilities of the QX600 ddPCR
System set a new standard in user-friendliness when developing
highly multiplexed assays.
References
Singh RR (2020). Next-generation sequencing in high-sensitive detection of mutations
in tumors: Challenges, advances, and applications. J Mol Diagn 22, 994-1007.
-1,000 -
-500
2.000
4.000
HEX amplitude
6,00
500
1.500
Cy5.5 amplitude
2,50
3,500
2.000
4.000
ATTO 590 amplitude
6.000