Exome sequencing is a robust technology that sequences all of the functionally relevant regions of essentially all 20,000 genes of the human genome and has the capacity to pinpoint rare genetic lesions in an unbiased and efficient way. Exome sequencing is a cost-effective and comprehensive method to rapidly detect the underlying etiology in patients with genetic disease.1-6
Reference 7,8
Sequence alignments to the reference human genome (GRCh37) and variant calls are generated using various bioinformatics tools (CASAVA, NovoAlign, Pindel, and GATK). The HGMD, the dbSNP database, the 1000 Genomes Project, HapMap data, and online search engines (e.g., PubMed), and Ambry's internal database are used to search for previously described gene mutations and polymorphisms. Data are annotated with the Ambry Variant Analyzer tool, including nucleotide and amino acid conservation, biochemical nature of amino acid substitutions, population frequency (Exome Variant Server http://evsgs.washington.edu/EVS/, Exome Aggregation Consortium http://exac.broadinstitute.org/, and the 1000 Genomes Project), and predicted functional impact (including PolyPhen and SIFT in silico prediction tools). Sequence alignments of the reads are viewed using IGV (Integrative Genomics Viewer) software. Stepwise filtering includes the removal of common SNPs, intergenic and 3’/5’ UTR variants, non–splice related intronic variants, and synonymous variants (except those at the first and last nucleotide position of an exon). The filtering pipeline protects all variants annotated within the HGMD and/or the OMIM databases.
Variants are then filtered further based on family history and possible inheritance models using the affected status of each family member whose exome was sequenced and compares the proband’s genotype of each detected alteration to the genotype of the family members (most commonly parents). Alterations survive the filtering if they are consistent with Mendelian inheritance models and the affected status of each family member. A minimum of four inheritance models are executed for each family (autosomal dominant (AD), autosomal recessive (AR), X-linked recessive, X-linked dominant, and Y-linked in male probands). For increased sensitivity of potential phenocopies and genes associated with reduced penetrance, autosomal and X-linked “proband-only” models are also generated for each proband that captured heterozygous, homozygous, compound heterozygous, and hemizygous alterations irrespective of cosegregation based on family members’ genotypes.
Exome is 2-3 times more likely to identify the underlying genetic defect than traditional tests