Fig. 3

Examples of the results of uniplex (A), duplex (B) and discrimination tests (C). In a typical one-dimensional diagram for uniplex assays (A), the positive droplets (for fluorescence with a single fluorophore, e.g. FAM) are separated from the negative ones by an adjustable threshold (here a black line set at 3000 AU), while all droplets obtained per sample-containing well (e.g. A01) are separated from the others (i.e. B01) by vertical yellow dashed lines. Both the positive and negative controls and the amplitude of fluorescence (on the y-axis; usually expressed in AU) are used to distinguish the droplet populations into positive and negative. In a two-dimensional diagram (B), typically used in the analysis of fluorescence emitted by two (or more) fluorophores simultaneously, the amplitudes for the four droplet clusters (double negative—grey, FAM-positive—blue, HEX-positive—green and double positive—orange) and thus the composition of each sample in relation to the two DNA targets are determined by simultaneously setting thresholds on the x and y axes. In discrimination tests (C), which are duplex tests by default, the two-dimensional plots resemble those of other duplex tests. However, droplet clusters tend to align more closely (especially in tests based on SNP frequency estimation) due to the generally low, indiscriminate binding of probes that are similar in nucleotide sequence. For this reason, discrimination tests based on competitive binding of probes can usually only be used for relative frequency estimation of a variant and not for absolute quantification