Variant analysis
Variant calling was performed with the Variant Caller plugin configured
with somatic high stringency parameters. Variants were annotated using
the Ion Reporter 5.14.1.0 software
(https://ionreporter.lifetechnologies.com/ir/).
Common single nucleotide variants (minor allele frequency [MAF]
> 5%), exonic synonymous variants, and intronic variants
were removed from the analysis, while exonic nonsynonymous, splice site,
and loss-of-function variants were analyzed.
The sequence analysis software Alamut® v2.13 (Interactive Bio software)
was used to interpret variants.
Online databases, including dbSNP (database the single nucleotide
polymorphism database), 1000 Genomes, ClinVar, EXAC (exome aggregation
consortium), COSMIC (catalog of somatic mutations in cancer), ESP (exome
sequencing project) were used. The pathogenicity prediction programs
such as PolyPhen2, SIFT, Mutation Taster, and splice prediction programs
were used to evaluate variants not previously described.