Fig. 3.

Considerations for accurate quantification. All illustrative examples given here used the QX200™ Droplet Digital™ PCR System (Bio-Rad) using duplex reactions; however, all of these are relevant for higher order multiplexing strategies. (A) For the analysis of a probe-competing duplex reactions to quantify a transition mutation. Cross-hybridisation of the probes caused by either mismatched binding of the probes or filter bleed-though can be visualised as a ‘leaning’ (blue) or ‘lifting’ (green) of the single-positive clusters. This can impact on positioning the thresholds to separate the four clusters. (B) Analysis of a probe-competing duplex reaction to detect a 3 amino acid deletion mutation to illustrate that mismatched binding of the probes does not cause the ‘leaning’ or ‘lifting’ of the clusters compared with that observed in (A).