Whole Exome Sequencing Workflow

Whole exome sequencing was performed at the Hereditary Research Laboratory, Bethlehem University, using Illumina’s NextSeq^TM 500 Sequencing System. DNA libraries were prepared using two preparation kits: the Illumina^® TruSeq^TM DNA Sample Prep Kit, or Nextera^TM Flex for Enrichment Prep Kit. Following sequencing, reads were aligned to the reference human genome (hg19) using the Burrows-Wheeler (BWA) aligner. Prior to variant calling by the Genome Analysis Toolkit³, mapped reads (BAM format) were re-processed by removing PCR duplicates, realigning around indels, and recalibrating base quality. The final list of variants was annotated by ANNOVAR⁴ (Wang et al., 2010 (link)) using several databases of minor allele frequency including gnomAD⁵ and PopFreqMax, as well as variant effect predictors such as SIFT (Sorting Intolerant from Tolerant⁶), PolyPhen-2⁷ and REVEL (Ioannidis et al., 2016 (link)). Candidate variants were validated by Sanger sequencing and then tested for co-segregation with the phenotype in additional family members.