Validating NGS Variant Calls with Sanger Sequencing

The poorly covered regions from NGS were amplified and verified by Sanger sequencing. The variants identified by multiplex genetic sequencing were also validated with Sanger sequencing. In brief, primers were designed using Primer3 software [16 (link)]. Primer sequences for validation are listed in S2 Table. Identified variants, including WRN (c.4108DelA), DICER1 (c.A3334G), and ELAC2 (c.A248G), were amplified in duplicate from genomic DNA of the six family members by using Hot FirePol DNA polymerase (Solis BioDyne, Tartu, Estonia). Sanger sequencing was performed using the Big Dye Terminator Cycle v1.1 Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) and ABI Prism 3130 Genetic Analyzer (Applied Biosystems).