Whole Exome Sequencing for Rare Disease Diagnosis

Whole exome sequencing (WES) was carried out on genomic DNA extracted from peripheral blood by the Nextera DNA Exome (Illumina, San Diego, CA, USA) on a NextSeq550Dx sequencer (Illumina). Sequencing reads were aligned to the human reference genome (UCSC hg19) by BWA (v0.7.7-isis-1.0.2) (Illumina). Variant calling was performed by GATK Variant Caller (v1.6-23-gf0210b3). DNA variants were annotated by eVai v2.5 (EnGenome). Variants mapping in genes associated to the following Human Phenotype Ontology (HPO) phenotypes were prioritized and then filtered by MAF < 0.01 (GnomAD v2.1): HP0001249, 0001256, 0002187, 0002342, 0006887, 0006889, 0010864, 0012759. Filtered variants were classified according to ACMG-AMP criteria [8 (link)]. The most phenotype-fitting variant was confirmed by Sanger sequencing. Genomic DNA from both parents was analyzed for segregation analysis of the variant.