Linearity was determined using mixtures of plasmids carrying the wild type or mutant sequence for the mutation V600E (GTG->GAG) in codon 600 of the BRAF gene (see figure " Linearity of V600E mutation"). The plasmids were mixed in proportions to give four levels of mutation (5, 10, 30, and 50%). Each mixture was analyzed with three different lots of the therascreen BRAF Pyro Kit in three Pyrosequencing runs with three replicates each.
The results were linear within an allowable nonlinearity of 5 % units in the tested range of 5 to 50% mutation level.
The precision data allows the determination of the total variability of the assays and was obtained at three different levels by analysis of the above mentioned plasmid mixtures with three replicates each.
Repeatability (intra-assay and inter-batch variability) was calculated based on the data for determination of linearity (three runs on the same day using varying lots of the therascreen BRAF Pyro Kit). Intermediate precision (intra-laboratory variability) was determined in three runs within one laboratory on three different days with varying operators, PyroMark Q24 systems, and lots of the therascreen BRAF Pyro Kit. Reproducibility (inter-laboratory variability) was calculated from two runs each in an internal and external laboratory and using varying lots of the therascreen BRAF Pyro Kit.
Precision estimates are expressed as standard deviation of the measured mutation frequencies in % units. The repeatability, intermediate precision, and reproducibility for the mutation V600E (GTG->GAG) in codon 600 was 0.6–2.1, 0.7–1.8, and 0.8–2.1 % units, respectively, in the measured range of 5 to 50% mutation level.
Precision values for the mutation V600E (GTG->GAG) in codon 600
% mutated plasmid
Repeatability (Mean, SD)
Intermediate precision (Mean, SD)
Reproducibility (Mean, SD)
| 5.2, 0.6 || 4.4, 0.7 || 5.1, 0.8 |
| 10 || |
| 9.6, 1.0 || 9.6, 1.3 |
| 30 || 28.1, 2.1 || 27.9, 1.8 || 28.3, 2.1 |
| 50 || 46.9, 1.2 || 46.3, 1.5 || 47.9, 1.7 |