Supplementary Materials and Methods s8

Supplementary Materials and Methods

Sample collection

Genomic DNA extraction from formalin-fixed, paraffin-embedded (FFPE) specimens of surgically resected tissue was performed in an independent clinical laboratory (SRL, Tokyo,

JAPAN). Genomic DNA mass was measured using the PicoGreen dsDNA quantitation assay (Life

Technologies) as per the manufacturer’s recommendation. Fluorescent intensity from double strand

DNA was measured by GloMax-Multi Microplate Multimode Reader (Progega, Madison, WI).

Probes and primers for digital PCR

Primers and probes were procured from MBL-IDT K.K. (Nagoya, Japan). Fluorescent probes targeting wild-type and mutant sequences were respectively conjugated to tetrachlorofluorescein

(TET, λex 522 nm/λem 539 nm) or 6-carboxyfluorescein (FAM, λex 494 nm/λem 522 nm) fluorophores with ZEN/IABkFQ double quencher. The sequences of primers and probes for detection of the T790M mutation are given in Supplementary Table S1.

DNA controls

Positive and negative control plasmids for the EGFR assay were prepared by cloning DNA fragments containing wild-type or the EGFR T790M mutation were using a TOPO TA Cloning Kit

(Life Technologies, Carlsbad, CA). The appropriate concentration of plasmid DNA was determined

pg. 1 empirically to yield a mixture in which the number of copies of mutant DNA was ca. 0.001–1.000% of the number of wild-type EGFR fragments.

Human wild-type genomic DNA were purchased from Clontech (Mountain View, CA). The tumor cell line A549 was obtained from the American Type Culture Collection (ATCC; Lockville, MD).

Genomic DNA was extracted using a QIAamp DNA mini Kit (QIAGEN, Hilden, Germany). Human wild-type and A549 genomic DNA was digested with CviQ1 (NEB, Ipswich, MA) and 400 ng of digested DNA was used for control experiments to determine the limit of blank (LOB).

A549 cell block was prepared using sodium alginate method. A549 cells were collected in 1.5 mL tube and fixed by 1 mL of 10% formalin solution (Muto chemicals, Tokyo, Japan) for 24 hours at room temperature. Fixed cells were subsequently centrifuged at 300 × g for 5 min. After removed formalin solution, cells were suspended in 1% sodium alginate solution (Wako, Osaka, Japan). Then

1 M calcium chloride was added to cell suspension for making gel. The gel including cell pellet were embedded into paraffin by regular procedure. Thin sections (8 µm) were cut from paraffin-embedded cell blocks, and then gDNA was isolated from the sections using QIAamp DNA FFPE Tissue Kit

(QIAGEN).

Genomic DNA extraction from adjacent normal tissue samples of EGFR-activating mutation positive adenocarcinoma and EGFR wild-type squamous cell carcinoma was performed in an independent clinical laboratory (SRL). Genomic DNA mass was measured using the Qubit 2.0 (Life

Technologies) as per the manufacturer’s recommendation.

pg. 2 Droplet Digital PCR

The duplex assay is based on the parallel amplification of wild-type and specific mutant sequences. In a pre-PCR environment, 20.0 μL TaqMan Genotyping Master Mix (Life Technologies) was mixed with the assay solution containing 2.0 μL 10 μM forward and reverse primers, 2.0 μL 10

μM of FAM and TET labeled-probes, 4.0 μL Droplet Stabilizer (RainDance Technologies,

Lexington, MA), 4.0 μL sterile DNase- and RNase-free water (Life Technologies), and 4 μL or 8 μL genomic DNA from patients (9.3–392.3 ng) to a final reaction volume of 40 μL.

A collection of uniformly sized aqueous droplets was produced by hydrodynamic flow-focusing with a droplet-generating microfluidic chip (Souse chip, RainDance Technologies) following the manufacturer’s instructions. The resulting emulsion was collected in a PCR tube strip comprising eight 0.2-mL conical-bottom PCR tubes (Axygen, Tewksbury, MA). The PCR tube strip, containing a total of 75 μL of droplets and carrier oil, was tightly capped with an 8-Strip Dome Cap (Axygen), and then placed in a thermal cycler with a hot lid (Proflex PCR system, Life Technologies). The emulsion was thermal cycled, starting with a hot start denaturation step of 10 min at 95 °C, followed by 45 cycles of: 95 °C, 15 sec; 58 °C, 15 sec; 60 °C, 45 sec (using a 0.5 °C/min ramp rate). These cycles were followed by a final step at 98 °C for 10 min. The emulsions were processed immediately to measure the endpoint fluorescence signal from each droplet.

The thermal-cycled emulsion was transferred into a second microfluidic chip (Sense chip,

pg. 3 RainDance Technologies), and then endpoint fluorescence signals were measured following the manufacturer’s instructions.

Deep sequencing

Deep sequencing was performed as previously described (1). Briefly, a total of 72 genes frequently found to be mutated in cancer according to the COSMIC database (Catalogue Of Somatic

Mutations In Cancer), were sequenced using a TruSeq Amplicon Cancer Panel (TSACP, Illumina,

San Diego, CA) and a second panel kit consisted of an additional 24 cancer-related genes following the manufacturer’s instructions. Variant call and Coverage analysis were performed using the CLC genomics Workbench 6.0 (CLC Bio, Aarhus, Denmark).

Data analysis

The droplet event data were analyzed with the RainDrop Analyst software (RainDance

Technologies) following the manufacturer’s instructions. Briefly, sample data was loaded with a drop size gating template (RainDance Technologies). Data from the positive control sample was used to create the compensation matrix in the RainDrop Analyst software. The compensation matrix was applied to the data from each sample to eliminate the crosstalk fluorescence signals from the

TET and FAM fluorophores. The sizes and locations of the wild-type and the mutant gates were established by manual selection of the area containing wild-type or mutant clusters in the positive

pg. 4 control.

For each unknown sample, the number of PCR-positive droplet events was counted within each gate. The number of events within each gate was converted to the number of events per assay using the total number of intact drops.

References

1. Ito N, Kawaguchi T, Koh Y, Isa S, Shimizu S, Takeo S, et al. Driver mutations associated with

smoking and other environmental factors: Prospective and integrative genomic analysis from

the Japan Molecular epidemiology for Lung Cancer Study (JME). J Clin Oncol 32:5s, 2014

(suppl; abstr 7516).

pg. 5 Supplementary Figure Legends

Figure S1. Flowchart for mutation call. If the measured event in the T790M gate was ≥ 10 events/assay, the assay was considered to be “positive”. If the event within a gated region was < 10 events/assay, the assay was considered to be “negative”. An assay was inconclusive if the amount of amplifiable DNA was less than 50 ng, at which point then a further assay was performed using 8 μL of DNA (double the volume of the initial assay).

Figure S2. Quantification of performance of ddPCR analysis for EGFR T790M mutation.

Plasmid containing the EGFR T790M mutation (MT plasmid: 2,000, 200, 20, or 0 copy) were serially diluted in 200,000 copies of wild-type EGFR sequence-containing plasmid (WT plasmid).

Plasmids were encapsulated into droplets and subjected to the procedure described in Fig. 1. (A)

Two-dimensional histogram of ddPCR assay. FAM, 6-carboxyfluorescein; TET, tetrachlorofluorescein. (B) After ddPCR assay, the copy numbers of spiked T790M mutant plasmid were counted using RainDrop Analyst software. Each filled circle represents an individual data point

(n = 3). The dotted lines above and below the regression line (straight black line) display the 95% confidence interval. (C) Identical serial dilutions ranging from 0.01–1.00% T790M mutation copies per reaction were assayed in triplicates. The correlation coefficient (R2) is given on the graph. Data shown here are representative of two independent experiments for each assay.

pg. 6 Figure S3. Analytical sensitivity of 0.001% for the ddPCR assay. Twenty (2 × 101) copies of mutant plasmid were spiked into 2 × 106 copies of wild-type plasmid, resulting in 0.001% of the mutational fractional abundance. Spiked and non-spiked samples analyzed using the ddPCR assay.

(A) Two-dimensional histogram of the non-spiked (left) and the spiked (right) assays. (B) Table showing wild-type and mutant events in both samples. (C) Mutant event is shown in box plot for spiked and non-spiked samples. Box indicates the range of the 95% confidence interval, and the line inside the box indicates the mean. Lines above and below the box denote maximum and minimum events, respectively.

Figure S4. Determination of false-positive events from wild-type control DNA and normal human genomic DNA. Two-dimensional histogram of ddPCR assay with 2 × 105 wild-type control plasmid DNA (left), 400 ng human genomic DNA (middle), and 400 ng A549 genomic DNA (right).

Figure S5. Determination of false-positive events in genomic DNA from formalin-fixed paraffin-embedded (FFPE) A549 cells. (A) Two-dimensional histogram of ddPCR assay with 400 ng genomic DNA form FFPE A549 cell block. (B) Determination of the limit of blank (LOB) from the 95% confidence interval of the Poisson model fit.

pg. 7 Figure S6. Detection of EGFR T790M mutant alleles in patients with EGFR T790M-containing primary tumors. Two-dimensional histogram of ddPCR assay with genomic DNA from FFPE samples. Sample ID and amount of input DNA are indicated on plots. Percentages of empty drops are also displayed on the plot, indicating that > 98% of drops are empty.

Figure S7. Comparison of T790M mutation detection in 6 samples by different operators.

Operator-to-operator variation of T790M mutant events in DNA samples from 6 T790M-positive cases. The slope and correlation coefficient are given in the graph.

Figure S8. Reproducibility of ddPCR analysis from 16 T790M-positive samples. ddPCR assay was performed on different days by two different operators to confirm the reproducibility of the percentage of mutant allele. The slope and correlation coefficient are given in the graph.