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Table 1 Correlation of FFPE DNA ‘functional’ quality (QFI) with the incidence of false positives in targeted NGS

From: Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies

Sample

Quality by QFI-PCR

QFI (%)

Percentage functional (Qubit)

Total number of mutations from AmpliSeq

Overlapping variants in 16 gene panel

Overlapping variants in 16 gene panel (PIK3CA false positives removeda)

Overlapping variants in 16 gene panel (PIK3CA false positives removedaand >5%)

Variant confirmed with independent run (16 gene panel)

True positives (%)

Number of germline SNPs annotated in dbSNP

Confirmed COSMIC mutation

RS00863

Low

0.5

4

451

48

44

28

3

10.7

2

BRAF V600E 23.8%

RS00856

Low

0.7

5

352

42

36

26

3

11.5

2

KRAS G13C 29.0%

RS01279

Low

1.1

4

123

23

19

8

3

37.5

3

None

RS00865

Low

2.2

4

222

33

30

21

3

14.3

2

NRAS G12R 23.0%

RS01283

Low

2.3

0

31

9

6

3

3

100.0

3

None

RS01282

Low

2.4

5

98

21

17

10

3

30.0

3

None

RS01289

Low

2.7

2

35

11

8

5

4

80.0

3

None

RS01274

Low

2.9

3

22

12

9

6

4

66.7

3

NRAS Q61R 42.6%

RS00866

Medium

6.3

7

24

11

8

5

5

100.0

3

NRAS Q61H 72.1%

PIK3CA H1047R 12.4%

RS00860

Medium

6.5

7

19

7

4

3

3

100.0

3

None

RS01294

Medium

6.6

8

8

3

3

3

3

100.0

3

None

RS00875

Medium

6.7

5

17

6

3

2

1

50.0

1

None

RS00855

Medium

6.8

11

16

8

5

3

3

100.0

2

KRAS G12D 37.8%

RS00876

High

16.5

12

15

6

4

4

3

75.0

2

PIK3CA H1047R 43.6%

RS01291

High

16.9

13

18

9

6

3

3

100.0

3

None

RS00871

High

17.8

16

9

4

2

2

2

100.0

2

None

RS00873

High

19.5

10

14

6

3

3

3

100.0

3

None

RS00857

High

20.5

13

15

5

4

4

4

100.0

3

KRAS G12V 29.8%

RS00877

High

23.9

20

12

7

4

4

4

100.0

3

KRAS G12S 48.0%

  1. aTo ensure maximum relevance in the final results, only variants with >5% mean allele frequency from AmpliSeq NGS were evaluated for the calculation of true positives, and systemic variants in the PIK3CA gene were excluded. True positives were confirmed from AmpliSeq NGS variant calls using an independently designed 16 gene confirmation panel [14].