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This assay is performed using the PerkinElmer Sciclone® G3 Workstation combined with the Agilent SureSelect Clinical Research Exome capture kit (#G9496A 5190-7344; targeting coding regions (exons) and canonical splice sites) followed by sequencing on the Illumina NextSeq 550 (High-Output v2 kit). Reads are aligned to the GRCh37 reference sequence using the Burrows-Wheeler Aligner (BWA 0.7.17), and variant calls are made using the Genomic Analysis Tool Kit (GATK v4.0.3.0). Detection of copy number variants (CNVs) encompassing 2 or more exons is performed using next-generation sequencing read data and the VisCap algorithm. CNV analysis is only performed when data meets necessary quality standards and may not be available for all cases. Variant calls are limited bioinformatically to the associated region of interest for the assay (see above for details). Sanger sequencing is used for fill in when bases have <12x coverage. All clinically significant variants are confirmed by Sanger sequencing or droplet digital PCR; variants classified as likely benign or benign are not confirmed.

Variant classifications are based on ACMG/AMP criteria (Richards et al. 2015) with ClinGen rule specifications (https://www.clinicalgenome.org/working-groups/sequence-variant-interpretation/). Variants are reported according to HGVS nomenclature (www.hgvs.org/mutnomen). Likely benign and benign variants are not included in this report but are available upon request.

This test does not routinely detect variants in non-coding regions (aside from the canonical splice sites), triplet repeat expansions, translocations, inversions, and copy number variants encompassing less than 2 consecutive exons. There is reduced detection for larger indels, variants in low complexity regions, and variants in regions with high homology.

This test was developed, and its performance characteristics determined by the Laboratory for Molecular Medicine at Partners HealthCare Personalized Medicine (LMM, 65 Landsdowne St, Cambridge, MA 02139; 617-768-8500; CLIA#22D1005307). It has not been cleared or approved by the U.S. Food and Drug Administration (FDA). The FDA has determined that such clearance or approval is not necessary.