Methodology

Method: Next Generation Sequencing technology using an Illumina NextSeq instrument. Target region includes coding exons and a minimum of 10 bp of flanking intron boundaries of the genes tested. Target enrichment performed using a custom Integrated DNA Technologies (IDT) Exome Hyb Panel v2.

Average coverage ~150x, depth of coverage for all target regions is at least 20x.

Limitations: 

This testing is performed on an exome backbone with analysis restricted to the panel genes. This method can detect single nucleotide variants (SNVs), small deletions, small insertions, and copy number variants in the regions targeted. Some regions cannot be efficiently captured due to sequence homology or sequence properties. This method will not detect large insertions and deletions, complex indels, structural variants (e.g. inversions, translocations), short tandem repeats, or other complex variants. Variants located outside of targeted regions will not be detected.

Based on validation studies, the bioinformatics pipeline showed precision and detection >99% for SNVs in regions with coverage greater than 20x and high mapping quality. Sensitivity for CNVs involving multiple genes is >99% and sensitivity for intragenic CNVs is >90%. Mosaic sequence variants present at <25% allele frequency may not be reliably detected, and detection sensitivity is dependent on the nature of the variant. The sensitivity of detection of mosaic copy number variants has not been evaluated.