Seqscape analysis software v 2

DNA was extracted from the fibroblasts using QIAamp^® DNA Blood kit (Qiagen, Milan, Italy) according to the manufacturer’s protocol. PCR amplifications were performed using platinum-Taq DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA, USA) and specific primers for the 23 different NSD1 exons (see Table S3). The PCR conditions were a single denaturation step at 95 °C for 3 min followed by 40 cycles of 95 °C/30 s, 58 °C/30 s and 72 °C (1 min/kb), with a final extension step at 72 °C for 5 min. PCR products were sequenced using the ABI BigDye Terminator Ready Reaction Mix (Thermo Fisher Scientific, Foster City, CA, USA) and analyzed on an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The sequences were aligned with Seqscape analysis software V.2.5 (Thermo Fisher Scientific, Foster City, CA, USA). To verify variants with deletions of the 5q35.3 region, array-CGH was performed with CGH 8 × 60 K (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. The data were analyzed with the Agilent Cytogenomics 4.0.3.12 software (Agilent Technologies, Santa Clara, CA, USA). All genomic positions were reported according to the human genome assembly (GRCh37/hg19).

Polymerase chain reaction (PCR) was performed either for screening purposes or for confirmation of NGS results on cDNA after reverse transcription of the NF1 transcript, or on DNA samples, respectively. Selected exons of the NF1 (NM_00267.3) and SPRED1 (NM_152594.2) genes were amplified by PCR and sequenced as previously described in Sabbagh et al., 2013 [6 (link)]. Variants identified by cDNA or NGS approaches were confirmed using DNA sequencing analysis performed on the corresponding exon. Sequences were aligned on the reference sequence with Seqscape analysis software v2.5 (Thermo Fisher Scientific).