RainDrop assay using 4 μL of genomic DNA

Concentration of amplifiable DNA ≥ 50 ng?

Copies/assay ≥ 10? T790M positive

T790M negative

reRainDrop assay using 8 μL of genomic DNA

Yes

No

Yes

No

Copies/assay ≥ 10? T790M positive

Yes

T790M negative

No

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).

Supplementary Figure S1 Watanabe et al.

Supplementary 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.

WT plasmid: 2 × 105 2 × 105 2 × 105 2 × 105 MT plasmid: 2 × 103 2 × 102 2 × 101 -

EG

FR

_w

t-T

ET

EGFR_T790M-FAM

T790M

WT

T790M

WT

T790M

WT

T790M

WT

A

B C

0 500 1000 1500 2000 2500

0

500

1000

1500

2000

2500

3000

f(x) = 1.282585273498 x − 33.14903544067R² = 0.998202877243584

Ob

serv

ed

co

py#

(co

pie

s/sa

mp

le)

0 10 20 30 40 50

0

10

20

30

40

50

0.001 0.01 0.1 1 10

0.01

0.1

1

10

R² = 0.980745068094275

Expected copy# (copies/sample)

Ob

serv

ed

mu

tan

t%

Expected mutant%

Watanabe et al.

Supplementary 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.

A

B C

WT plasmid: 2 × 106 2 × 106

MT plasmid: - 2 × 101

EG

FR

_w

t-T

ET

T790M

WT

EGFR_T790M-FAM

T790M

WT

WT plasmid: 2 ×106

MT plasmid: -WT plasmid: 2 ×106

MT plasmid: 2 ×101

Wild-typecopies

T790Mcopies

Wild-type copies

T790M copies

#1 1,996,515 4.6 1,995,756 23.6

#2 1,996,589 8.5 1,996,196 13.7

#3 1,995,765 6.3 1,996,609 15.8

#4 2,254,353 9.8 2,167,051 18.2

#5 2,214,362 4.4 2,242,653 16.2

#6 2,237,715 7.2 2,306,536 19.5

Mean 6.8 17.8

95% CI 1.5 2.4

WT plasmid: 2 × 106 2 × 106

MT plasmid: - 2 × 101

T79

0M c

opie

s/as

say

p < 0.01 (Wilcoxon test)

Watanabe et al.

EG

FR

_w

t-T

ET

EGFR_T790M -FAM

Wild-type control plasmid Human genomic DNA

T790M

WT

T790M

WT

A549 genomic DNA

T790M

WT

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).

Supplementary Figure S4 Watanabe et al.

Input A549 genomic DNA from FFPE cell block (400 ng)

Sample #Amplifiable

DNA (ng)Wild-type

eventsFalse positive

events

#1 102.0 63162.5 2.8

#2 107.3 66417.5 3.1

#3 106.1 65719.9 4.3

#4 104.7 64840.0 2.5

#5 104.8 64871.9 2.7

#6 103.2 63892.8 2.7

#7 103.7 64207.9 6.1

#8 103.7 64214.5 2.6

Event 26.8

Mean 3.35

Std. 1.3

95% Upper Limit

(One-tail Poisson

Dist.)

7

Watanabe et al.

EG

FR

_wt-

TE

T

EGFR_T790M -FAM

T790M

WT

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.

Supplementary Figure S5

A BA549 gDNA from FFPE cell block

EG

FR

_w

t-T

ET

EGFR_T790M-FAM

JME-013140.8 ng

JME-082156.8 ng

JME- 69572.8 ng

JME- 823151.2 ng

JME- 885216.4 ng

98.8%

98.7%

99.3%98.9%

98.5%

T790M

WT

T790M

WT

T790M

WT

T790M

WT

T790M

WT

Supplementary Figure S6 Watanabe et al.

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.

Operator 2 (% of MT allele)

Op

era

tor

1 (

% o

f M

T a

llele

)

0% 5% 10% 15% 20% 25% 30%0%

5%

10%

15%

20%

25%

30%

f(x) = 1.03529747960339 x − 0.00148762359650487R² = 0.998745972627605

Supplementary Figure S7 Watanabe et al.

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.

Operator 2 (% of MT allele)

Op

era

tor

1 (

% o

f M

T a

llele

)

Supplementary Figure S8 Watanabe et al.

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.

0 1 2 3 4 5 6 7 8 90

1

2

3

4

5

6

7

8

9

f(x) = 1.02110869002281 x − 0.0181507320732282R² = 0.997984387913243

-2.7

7555

7561

5628

9E-1

70.

05 0.1

0.15 0.

20

0.050.1

0.150.2