Analysis of NCF1 by ddPCR. Two distinct probes (one that recognizes the GTGT [blue] found in NCF1 and a second that recognizes the ΔGT [green] found in NCF1B and NCF1C) were added to diluted genomic DNA. A 20-µL PCR mixture was dispersed into 20 000 oil-coated nanodrops, and PCR was performed. The droplets were analyzed to determine the number of “Blue” and “Green” nanodrops. The image at the bottom left depicts the bead distribution of the ddPCR reaction. The clear beads represent beads without GTGT or ΔGT sequences, and therefore have no amplified probe or color. The blue beads represent beads containing a GTGT sequence and PCR amplification of the GTGT-specific probe. The green beads represent beads containing a ΔGT sequence and PCR amplification of the ΔGT-specific probe. Although the DNA has been diluted to minimize the number of double-colored droplets, they do occur and are included in the analysis. However, they are detected in independent channels and, therefore, do not affect the results. The text at the bottom right depicts the possible sequences in the example in the top of the panel. Because a total of 6 alleles were expected, normal subjects were predicted to yield 2 GTGT/4 ΔGT, p47^phox carriers were predicted to yield 1 GTGT/5 ΔGT, and patients with p47^phox CGD were predicted to yield 0 GTGT/6 ΔGT.