Fig. 1.

Graphical outputs for duplexing strategies. All illustrative examples given here used the QX200™ Droplet Digital™ PCR System (Bio-Rad). A schematic is given for the primer and probe hybridisation arrangements with an example of the configuration of clusters in the 2D plot underneath. For each plot, the amplitude in channel 1 (ch1) is represented on the y-axis with the amplitude in channel 2 (ch2) represented on the x-axis. Four clusters are identified as single-positive for Ch1 (blue) and Ch2 (green), double-positive (orange) and double-negative (grey). (A) For non-competing duplex reactions, a rectangular conformation is observed between the four clusters. The amplitude of the double-positive cluster is approximately equal to that of the two single-positive clusters. (B) For competing duplex reactions, the four clusters have been pulled out of the rectangular conformation observed in (A). The double-positive cluster has dropped inwards and formed an arc across the scatter plot. (C) For non-competing (hybrid) duplex reactions, only three clusters are visible. Discrimination of the variant and double-positive clusters is not possible (all partitions are coloured orange).