Exome Sequencing for Genetic Diagnosis

DNA isolation from peripheral blood of patient and parents was performed using the Chemagic DNA Blood 4 k Kit (PerkinElmer, Waltham, MA, United States). Three micrograms of double-stranded DNA were fragmented (Covaris, Woburn, MA, United States) and exonic sequences were captured using the Agilent SureSelect Clinical Research Exome V2 (Agilent, Santa Clara, CA, United States). Paired-end sequencing was performed on a HiSeq 4,000 platform (150bp paired end) and an average coverage of at least 50X. Reads were mapped against the human reference genome GRCh37/hg19 with the Burrows-Wheeler Aligner (Li and Durbin, 2009 (link)), and variants were called using the Genome Analysis toolkit (Broad Institute, Cambridge, MA, United States). Alissa Interpret software (Agilent, Santa Clara, CA, United States) was used to filter and prioritize variants. Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines for the interpretation of sequence variants (Richards et al., 2015 (link)).