Validation of digital RMD assays with artificial DNA mixtures
| Mutation . | Fetal DNA proportion, %* . | Digital PCR result . | mr‡ . | SPRT classification§ . | ||
|---|---|---|---|---|---|---|
| Total wells . | Mutant count† . | Wild-type count† . | ||||
| F8 | 20 | 4590 | 1495 | 1721 | 0.47 | Wild-type |
| c.826G>A | 10 | 4590 | 2917 | 3006 | 1.06 | Wild-type |
| F8 | 20 | 4590 | 406 | 507 | 0.12 | Wild-type |
| c.1171C>T | 10 | 4590 | 435 | 483 | 0.11 | Wild-type |
| F8 | 20 | 4590 | 485 | 593 | 0.14 | Wild-type |
| c.6278A>G | 10 | 4590 | 857 | 892 | 0.22 | Wild-type |
| F9 | 20 | 4590 | 88 | 109 | 0.02 | Wild-type |
| c.802T>A | 10 | 4590 | 1640 | 1740 | 0.48 | Wild-type |
| F9 | 20 | 4590 | 375 | 463 | 0.11 | Wild-type |
| c.874delC | 10 | 4590 | 388 | 438 | 0.10 | Wild-type |
| F9 | 20 | 4590 | 308 | 365 | 0.05 | Wild-type |
| c.1144T>C | 10 | 4590 | 380 | 408 | 0.04 | Wild-type |
| F9 | 20 | 4590 | 673 | 835 | 0.20 | Wild-type |
| c.1069G>A | 10 | 4590 | 966 | 1046 | 0.26 | Wild-type |
| Mutation . | Fetal DNA proportion, %* . | Digital PCR result . | mr‡ . | SPRT classification§ . | ||
|---|---|---|---|---|---|---|
| Total wells . | Mutant count† . | Wild-type count† . | ||||
| F8 | 20 | 4590 | 1495 | 1721 | 0.47 | Wild-type |
| c.826G>A | 10 | 4590 | 2917 | 3006 | 1.06 | Wild-type |
| F8 | 20 | 4590 | 406 | 507 | 0.12 | Wild-type |
| c.1171C>T | 10 | 4590 | 435 | 483 | 0.11 | Wild-type |
| F8 | 20 | 4590 | 485 | 593 | 0.14 | Wild-type |
| c.6278A>G | 10 | 4590 | 857 | 892 | 0.22 | Wild-type |
| F9 | 20 | 4590 | 88 | 109 | 0.02 | Wild-type |
| c.802T>A | 10 | 4590 | 1640 | 1740 | 0.48 | Wild-type |
| F9 | 20 | 4590 | 375 | 463 | 0.11 | Wild-type |
| c.874delC | 10 | 4590 | 388 | 438 | 0.10 | Wild-type |
| F9 | 20 | 4590 | 308 | 365 | 0.05 | Wild-type |
| c.1144T>C | 10 | 4590 | 380 | 408 | 0.04 | Wild-type |
| F9 | 20 | 4590 | 673 | 835 | 0.20 | Wild-type |
| c.1069G>A | 10 | 4590 | 966 | 1046 | 0.26 | Wild-type |
The artificial mixtures were constructed to simulate the fetal and maternal DNA compositions in maternal plasma. Sequential probability ratio test (SPRT) classification of “fetal genotypes,” which was mimicked by the normal placental DNA in the artificial mixtures, was wild-type. SNP genotypes were determined by mass spectrometry.
PCR indicates polymerase chain reaction; and RMD, relative mutation dosage.
Fetal DNA was mixed in the specified proportions with maternal DNA. Fetal DNA was obtained from the placenta of a normal male fetus. Maternal DNA was obtained from the blood cells of pregnant women heterozygous for the corresponding mutations.
Mutant count indicates the number of wells positive for the mutant allele; and wild-type count, the number of wells positive for the wild-type allele.
Average reference template concentration per PCR well. The reference template referred to the allele with the lesser count in each sample.
SPRT classification of “fetal genotypes”, which was mimicked by the normal placental DNA in the artificial mixtures.