Figure 5

Results from optimisation of BRAF V600E (AC > TT) ddPCR assay for frequency of false positive droplets, assay sensitivity and XenT compatibility optimisation steps. (a) 84 wells of WT DNA were run. At the previously determined thresholds, 1 well had 1 FAM false positive droplet, 83 wells had 0 FAM false positive droplets. The red circle highlights the false positive droplet. (b) At the previously determined thresholds, the BRAF V600E AC > TT ddPCR assay showed high sensitivity and detected very low levels of V600E AC > TT mutation (5 copies) even in the presence of high numbers of WT copies (up to 5000 copies WT). Two spurious HEX positive droplets were observed in the absence of WT DNA potentially due to polymerase error. Full details reported as copies/20 μl as well as measurement summaries are presented in the table below the respective 2D plots. (c) The XenT assay is fully compatible with the V600E AC > TT assay. The XenT gBlock was not recognised by the V600E AC > TT assay, and vice versa, the XenT/RPP30 assay did not bind to the V600E AC > TT gBlock. The table below the 2D plots demonstrates that presence of the XenT gBlock did not significantly affect the detection of very low copies of mutant V600E AC > TT. All measurements are reported as copies/20 μl using the V600E AC > TT assay with either a mixture of 5 copies of V600E AC > TT gBlock in a background of 500 copies WT DNA (WT + Mut) or this mixture additionally containing the XenT gBlock (WT + Mut + XenT). Cps – copies.