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Supplementary Information
Microdroplet Digital PCR
Principles of mdPCR
These have been discussed in detail elsewhere 1, 2. In brief, DNA is diluted with
buffer and primers/probes. The reaction mixture is partitioned into
microdroplets prior to PCR. In most experiments, each microdroplet contains on
average less than a haploid genome per aliquot. The primer/probe sets are
designed to test the presence or absence of a specific locus in a microdroplet. The
number of droplets that are positive for a given locus can then be counted and
compared.
Reactions may be a single assay with a single fluorophore-labelled (usually FAM)
probe or a dual assay in which both the wild type allele (usually HEX-labelled)
and the mutant allele usually (FAM-labelled) can be tested for simultaneously.
Validation experiments
Wild type DNA derived from cell lines was used in each experiment to
demonstrate the potential for mdPCR to be used to detect rare mutational
variants in a background of excess wild type alleles.
mdPCR gives an absolute estimate of the number of copies of a locus of interest
in template DNA. For example, in the case of a cell line that is wild type for KRAS
(H1975) the absolute number of copies of KRAS in a DNA extract were estimated
using the QX100 protocol described in the methods section. From this DNA was
diluted so that the equivalent of 3000 copies of wt KRAS were loaded.
Details of primers/probesets
All of these reagents were purchased directly from BioRAD or Life Technology,
so the specific sequences used are not available.
Life Technology
RNaseP (RPPH1)
FTH1
EGFR Exon 19 deletion
EGFR Exon 19 deletion
EGFR T790Mmut
EGFR T790Mwt
Catalogue No. 4403328
Catalogue No. 4331182; Hs01694011_s1
Catalogue No. 4465805; Hs00000228_mu
Catalogue No. 4465805; Hs00000177_wt
Catalogue No. 4465804; Hs00000106_mut*
Catalogue No. 4465804; Hs00000105_wt*
*only used for the experiments in Figure 3b; otherwise the BioRAD assay was
used for this locus
BioRAD
EGFR T790Mmut
EGFR T790Mwt
dHsaCP2000019
dHsaCP2000020
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EGFR L858Rmut
EGFR L858Rwt
KRAS G12R mut
KRAS G12R wt
KRAS G12C mut
KRAS G12C wt
dHsaCP2000021
dHsaCP2000022
dHsaCP2000009
dHsaCP2000010
dHsaCP2000007
dHsaCP2000008
Droplet generation and cycling conditions
The droplet generation was performed exactly as per manufacturer’s
instructions.
Final reactions of 20µl consisted of 1xSupermix, 2µl probe and 1-7µl of DNA with
the remainder nuclease-free water. Microdroplets were generated using the
QX100 droplet generator (BioRad) prior to cycling on a T100 Touch™ screen
thermal cycler gradient according to one of two protocols:
1) For BioRAD probes
95C for 10min, 49x (95C for 10s, 60C for 30s), 98C for 12min.
2) For LifeTechnology probes
95C for 10min, 40x (95C for 10s, 50C for 30s), 98C for 12min.
The cycling conditions for Life Technology and BioRAD were optimised
separately. In pilot experiments for the BioRAD probesets the amplitude of
fluorescent signaling was found to improve with 9 extra cycles and this was
adopted as our standard protocol. The cycling conditions for the BioRAD probes
are otherwise as per the manufacturer’s recommendations.
Results analysis
Approximate thresholds were established based on analysis of cell line DNA of
known genotypes or commercial reference standards (Horizon Diagnostics).
Each experiment was controlled with a positive (cell line or reference standard
DNA) and negative (cell line or reference standard guaranteed wild type DNA).
ctDNA
For single-label analysis of ctDNA, microdroplets over a threshold (1000) in the
FAM channel were called as positive. For dual labeled analysis of ctDNA (Rare
variants), microdroplets in the left upper quadrant were called as mutation
positive (FAM+, HEX negative) and in the right lower quadrant (HEX+, FAM
negative). Threshold values for a positive microdroplet for a mutation was 3000.
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Supplementary References
1.
Day E, Dear PH, McCaughan F. Digital PCR strategies in the development
and analysis of molecular biomarkers for personalized medicine. Methods
2013;59:101-107.
2.
Hindson BJ, Ness KD, Masquelier DA, et al. High-throughput droplet digital
PCR system for absolute quantitation of DNA copy number. Anal Chem
2011;83:8604-8610.