Inversion characterization by ddPCR genotyping. (A) Main features of the studied polymorphic inversions, with the inverted region shown in gray and the IRs as dark boxes. Brown boxes denote deletions affecting inversion breakpoints. (B) Strategy for ddPCR genotyping of an inversion (light gray arrow) mediated by IRs at inversion breakpoints (dark gray arrows) using as an example the simultaneous triplex amplification of amplicons A, B, and C (shown on top), although the same procedure was used for other amplicon combinations (BCD, ABD, or ACD). O1 linkage (AB, left) and O2 linkage (AC, right) were calculated separately from the triplex ddPCR results, ignoring, respectively, amplicons C (left graph) or B (right graph). Colors of droplet clusters indicate if they are used to estimate the molecules containing only amplicon A (green), only the B or C internal amplicon (blue), two amplicons at the same time, either A and B or A and C (orange), or if they are considered as negative (gray). (C) Plots of O1 linkage ratios obtained from two different triplex ddPCR reactions (indicated in each axis) interrogating both breakpoints of four inversions in 95 analyzed individuals and the correlation between them (R), which show clearly differentiated genotype groups (O1/O1, red; O1/O2, blue; O2/O2, yellow). HsInv0241, where DNA was digested to separate both breakpoints with a restriction enzyme (RE, red arrow in B), shows the expected linkage ratios, but homozygote and heterozygote clusters are closer in HsInv0390, HsInv1111, and HsInv0382 analyzed using undigested DNA. The effect of a large deletion within breakpoint AB in HsInv0382 can be seen as higher O1 linkage ratio values in the horizontal axis. The gray dot is a sample with altered amplicon ratios in HsInv1111 ABC breakpoint.
