After heat inactivation of the MDA reaction, each well was diluted 13.4-fold with water. An aliquot of each well was diluted a further 5-fold with water. 1 μL of this 67-fold diluted DNA was used as template for qPCR to identify wells containing SNP regions linked to the de novo deletion – one paternally inherited SNP located 12,785 bp upstream of the deletion, and three maternally inherited SNPs located 506 bp upstream, 422 bp upstream, and 8,032 bp downstream of the deletion. Primers used were pSNP-12785qPCRseqFw, pSNP-12785qPCRseqRe, mSNP-506-422qPCRseqFw, mSNP-506-422qPCRseqRe, mSNP+8032qPCRseqFw, and mSNP+8032qPCRseqRe (Supplementary Table 2). Fast SYBR Green Master Mix (Applied Biosystems, Grand Island, NY) was used for qPCR. qPCR products of flanking SNPs were used as template for PCR with PfuTurboCx polymerase (Agilent Technologies, Santa Clara, CA), because the presence of UTP and uracil-N-glycosylase in the SYBR Green Master mix makes the qPCR products unstable.
