Additional quantification of the level of LOY for validation was performed using previously described procedure [21]. Briefly, extracted DNA was digested for 15 min with HindIII enzyme (Thermo Fischer) in 37 °C. After this, 50 ng of DNA was added together with PCR primers and probes targeting a known difference between the AMELX and AMELY gene assay C_990000001_10 (Thermo Fisher). Droplets were then generated using an automated droplet generator (Bio-Rad) and amplified using PCR. Droplets fluorescence intensity in two channels FAM and VIC was measured using the Bio-Rad’s QX200 Droplet Reader. The data were analyzed in Bio-Rad’s software QuantaSoft (version 1.7.4.0917) as described elsewhere [21]. The LOY quantification was performed by calculating the ratio between the amounts of AMELY and AMELX in each sample.
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