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.