CNV Analysis by ddPCR

To confirm the CNVs identified by SNP array, Droplet Digital PCR (ddPCR) was performed. Before ddPCR, the genomic DNA of each family member was digested for 1 h at 37°C with the restriction enzyme Hind III. ddPCR was performed using the following set of primers: for DUP16q23.3 5′-TTGGTGTTTGACCCTGTGAA-3′ (Forward) and 5′-TGA GCTAGGGCTCCCACTTA-3′ (Reverse); for DUP3p26.3 5′-TCAGTGAAGTGCCTGGTTTG-3′ (Forward) and 5′-GGCT GTTCCATGAGGAATGT-3′) (Reverse). And the following 5′-FAM probes with a 3′-BHQ1 quencher: for DUP16q23.3 5′-FAM-TTTGGATTGCTTTGCCTACC-BHQ1-3′, and DUP3p 26.3 5′-FAM-CTAGGCTGGGCTCACTTGTC-BHQ1-3′. RPP30 was used as a reference control: 5′GATTTGGACCTG CGAGCG-3′ (Forward), 5′-GCGGCTGTCTCCACAAGT-3′ (Reverse), and the probe VIC-CTGACCTGAAGGCTCT-BHQ1 (Supplementary Table 3). The PCR was performed in a C1000 touch thermocycler (Bio-Rad, Hercules, CA, United States) with the following protocol: Initial denaturalization at 95°C for 10 min, followed by 39 cycles at 94°C for 30 s and an extension at 57°C for 1 min, with a final denaturing at 98°C for 10 min. Droplet analysis was performed using the Q200 Droplet Reader (Bio-Rad). CNVs were calculated using the QuantaSoft software (Bio-Rad)¹.