Validation
To estimate the false discovery rate of our CNV-calling algorithm, CNVs were validated by genomic real-time qPCR. For this purpose, we randomly selected 25 CNV loci and performed genomic qPCR using DNAs from study subjects who showed corresponding CNVs on that loci. The PCR primers used in this study were designed using the PrimerQuest program (http://www.idtdna.com/Scitools/Applications/Primerquest). To verify the specificity of the PCR reactions under the unified denaturation temperature (60℃), we performed PCR and agarose gel electrophoresis for each primer set. We also screened the University of California Santa Cruz (UCSC) database (http://genome.ucsc.edu/) to confirm the unique sequence without any repeat sequences in the primers. Sequence information of the primers for genomic qPCR validation is listed in Table 1.
Ten microliters of the reaction mixture contained 10 ng of genomic DNA, SYBR Premix Ex Taq TM II (TaKaRaBio, Shiga, Japan), 1×ROX (Toyobo, Osaka, Japan), and 10 pmol of primers. Thermal cycling conditions consisted of 1 cycle of 10 s at 95℃, followed by 40 cycles of 5 s at 95℃, 10 s at 61℃, and 20 s at 72℃. All PCR experiments were repeated twice, and amplification efficiencies for both target and reference genes were evaluated using a standard curve over 1:5 serial dilutions. The copy number of each target was defined as 2-ΔΔCT, where ΔCt is the difference in threshold cycles for the sample in question normalized against the reference gene (RNaseP) and expressed relative to the value obtained by calibrator DNA (NA10851 and Promega DNA), as described elsewhere [28].