Whole-Exome Sequencing for Genetic Diagnosis

Whole-exome sequencing (WES) was performed on genomic DNA obtained from the patient’s peripheral blood as previously described [15 (link)]. The DNA library was prepared using the Illumina’s TruSeq DNA Exome Kit. Sequencing was performed on the NextSeq-500 platform using the NextSeq 550 High Output kit (150 cycles) according to the manufacturer protocol (Illumina, California, USA). Raw sequence reads were aligned to the human genome reference sequence hg19 using FastQC 0.11.7 and BWA (Aligner) 0.7.15. Imported variants were annotated, filtered, and classified using VariantStudio (Illumina, California, USA). The variants were further filtered using the dbSNP (https://www.ncbi.nlm.nih.gov/snp/) and the Genome Aggregation Database (https://gnomad.broadinstitute.org/). Sanger sequencing was performed to confirm the presence of the NGS-derived variant in the patient. Familial segregation analysis by Sanger sequencing was limited to the patient’s non-affected brother due to the unavailability of parents or other family members.