User manual

78 | Droplet Digital
™
PCR Applications Guide
9 Droplet Digital
™
PCR Tips,
Assay Considerations,
and Troubleshooting
Assay-Dependent Cluster Shifts
As with any PCR-based technology, assay design and sample preparation are important for
obtaining good quality data. Before running a Droplet Digital PCR (ddPCR
™
) experiment,
know the goal or possible expected outcomes of the experiment because different types
of experiments require different controls, sample preparation, amounts of DNA or RNA,
and data analysis.
Shifted Clusters Due to Probe Cross-Reactivity
If you see a shift inwards or upwards on the 2-D plot, this is most likely probe cross-
reactivity. Probe cross-reactivity occurs when a probe binds to a nonperfect sequence and
undergoes cleavage (Figure 9.1). This is more common in rare mutation detection (RMD)
assays (for example, single nucleotide polymorphism [SNP] assays), where the two probes
differ by only one base. The amount of nonspecific probe cleavage is a function of how
close the melting temperature (T
m
) of the mismatched probe is to the annealing/extension
temperature used for the assay. For this reason, RMD assays are designed to maximize
the T
m
difference between a perfectly matched and a mismatched target. A cross-reacting
probe causes single positive clusters (Ch1+/Ch2– and/or Ch1–/Ch2+) to migrate toward
the axis of the other channel, which is most easily viewed in the 2-D amplitude plots
(see Figure 9.1A). Figure 9.1 exemplifies this phenomenon where the FAM probe cross-
reacts to a PCR product perfectly complemented by the VIC probe, resulting in mild to
moderate FAM probe cleavage in the droplets. This causes an upward shift (or migration) in
the location of the droplets in 2-D space. A similar shift is observed for the FAM+ droplets.
These shifts are clearly seen in the 2-D amplitude plots. By contrast, the 1-D amplitude
plots and histograms of these same data are difficult to interpret (Figures 9.1B–E). To avoid