Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using a standardized methodology and quantified. Each DNA sample is sheared, adaptor ligated, PCR-amplified and incubated with the exome baits. Captured DNA is eluted, and PCR amplified. Final quantified libraries are seeded onto an Illumina flow cell and sequenced using paired-end, 150 cycle chemistry on the Illumina HiSeq or NextSeq. 

Coding exons plus at least 6 bases into the 5’ and 3’ ends of all the introns are analyzed and reported. Gross deletion/duplication analysis is assessed for all genes within the targeted exome using a custom pipeline based on coverage (>4 exons in size) and/or breakpoint analysis from NGS data and confirmed by targeted chromosomal microarray, SNP array or MLPA when applicable. CNVs detected by NGS pipeline for which no orthogonal method of confirmation is available will not be included. 

When familial samples are received, co-segregation analysis of potentially informative alterations will be performed, except for gross deletions/duplications which are confirmed in the proband only. Co-segregation results may be confounded by many factors which cannot be completely ruled out including reduced penetrance, age-of-onset, and/or variable expressivity. In most cases, phase cannot be determined.