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Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Project (1000G). Recurrent variants in unrelated samples resulted in 919 comparisons between NGS and Sanger, with 100% concordance. In a second comparison, 762 unique NGS results (736 SNVs, 26 indels) from seven 1000G samples were found to have 97.1% concordance with 1000G phase 1 data. Sanger sequencing and 1000G phase 3 data confirmed the accuracy of the NGS results for all 1000G phase 1 discrepancies. In all samples, the depth of coverage exceeded 100× in >99.7% of bases in the target regions. In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes.

Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patient's electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1) to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied.

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Original research article introduction FBN1 mutations are most commonly associated with Marfan syndrome (MFS), an autosomal dominant connective tissue disorder typically involving the ocular, skeletal, and cardiovascular systems; MFS less frequently involves the skin, integument, lung, muscle, and adipose tissue. Cardiovascular manifestations, which are the major cause of morbidity and early mortality in MFS, include aortic dilatation at the level of the sinus of Valsalva, predisposition for aortic dissection, mitral valve and tricuspid valve prolapse, and enlargement of the proximal pulmonary artery.In MFS, an age-dependent association with the occurrence of an aortic event (aortic dissection or prophylactic aortic surgery) has been demonstrated in 16% of 30-year-olds and 74% of 60-year-olds having an aortic event.1 A sex-dependent association has also been observed: aortic events occur at an earlier age in males than in females. 1Hundreds of mutations have been identified in FBN1-many of them unique to individual families. Missense mutations are the most common type of FBN1 mutation, the majority of which are cysteine substitutions. FBN1 mutations have been shown to occur across the gene with limited genotypephenotype correlations, with the exception of the association of early onset, severe (previously termed "neonatal") MFS and mutations in exons 24 through 32, as well as the association of ectopia lentis with missense mutations. A study investigating genotype-phenotype correlations with cardiovascular features did not observe significant differences with mutation type (e.g., frameshift versus missense) but did observe a higher probability of ascending aortic dilatation, aortic event, and mitral valve prolapse in patients with mutations altering a cysteine residue. 1The type of FBN1 mutation identified and its likelihood of being pathogenic are recognized as important factors when making a diagnosis of MFS and later clinical decisions. Some have argued that truncating and splicing mutations may be associated with a milder disease course. Accordingly, we evaluated 179 consecutive probands with FBN1 mutations to evaluate this important clinical issue. MAtEriALS And MEtHodS Study populationProband samples (n = 179) with a pathogenic or likely pathogenic FBN1 variant and detailed clinical information received over a 4.25-year period were included in this study. Each patient was examined by his or her referring physician. For Mayo Clinic patients, phenotypic information was extracted from the patients' electronic medical record. For patients external to the Mayo Clinic, phenotypic information was provided by the referring provider via a requisition form specific to FBN1, which included age, sex, suspected diagnosis, family history, and phenotypic features, including those related to the Ghent (2010 revised) nosology criteria. Purpose: Marfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype-phenotype correlations with aortic eve...