Figure 2

Detection of chromosome 15 AC216176 L1Hs by the 5’ junction droplet digital PCR (ddPCR) assay. Each panel represents a single ddPCR experiment whereby a DNA sample (defined below) is segregated into individual droplets and assessed for the presence of the L1 locus (FAM) and RPP30 locus (VIC) using two different fluorophores in Taqman™ assays (see Figure 1). The FAM and VIC fluorescence for each droplet is plotted as a data point on each graph. FAM fluorescent signal (Channel 1) is plotted on the y-axis and VIC fluorescent signal (Channel 2) is plotted on the x-axis. The droplet threshold for each fluorophore used is indicated by the magenta lines, determining whether a droplet is considered positive or negative for either FAM or VIC fluorescence. The positive or negative fluorescence assessment for each quadrant is labeled accordingly for the plot describing the experiment with 100% GM01632 DNA. The blue dots represent individual droplets that contain at least one copy of the L1 locus tested but not the RPP30 locus (FAM positive, VIC negative), the green dots represent droplets that contain at least one copy of the RPP30 gene and not the L1 locus (VIC positive, FAM negative), and the orange dots represent droplets that contain at least one copy of both the RPP30 gene DNA and the L1 locus tested (positive for both FAM and VIC). We tested 160 ng of BsaJI-digested genomic DNA from GM01632 cells, which are homozygous for the polymorphic L1 element (100%), and tenfold dilutions of this same sample as a mixture with BsaJI-digested genomic DNA from GM01631 cells, which do not have this polymorphic L1 insertion (10%-0.01%), thus keeping the total input genomic DNA constant for each ddPCR. Additionally, as a negative control, 160 ng of BsaJI-digested genomic DNA from GM01631 cells was tested (0%).