title: Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory.
abstract: OBJECTIVE
  Sanger sequencing is currently considered the gold standard methodology for clinical molecular diagnostic testing. However, next-generation sequencing has already emerged as a much more efficient means to identify genetic variants within gene panels, the exome, or the genome. We sought to assess the accuracy of next-generation sequencing variant identification in our clinical genomics laboratory with the goal of establishing a quality score threshold for confirmatory Sanger-based testing.
  
  
  METHODS
  Confirmation data for reported results from 144 sequential clinical exome-sequencing cases (94 unique variants) and an additional set of 16 variants from comparable research samples were analyzed.
  
  
  RESULTS
  Of the 110 total single-nucleotide variants analyzed, 103 variants had a quality score ≥Q500, 103 (100%) of which were confirmed by Sanger sequencing. Of the remaining seven variants with quality scores <Q500, six were confirmed by Sanger sequencing (85%).
  
  
  CONCLUSIONS
  For single-nucleotide variants, we predict that going forward, we will be able to reduce our Sanger confirmation workload by 70-80%. This serves as a proof of principle that as long as sufficient validation and quality control measures are implemented, the volume of Sanger confirmation can be reduced, alleviating a significant amount of the labor and cost burden on clinical laboratories wishing to use next-generation sequencing technology. However, Sanger confirmation of low-quality single-nucleotide variants and all insertions or deletions <10 bp remains necessary at this time in our laboratory.
authors.affiliation: 1] Department of Pathology and Laboratory Medicine¸ David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA [2] Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Samuel P
authors.initials: SP
authors.lastname: Strom
authors.affiliation: Department of Pathology and Laboratory Medicine¸ David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Hane
authors.initials: H
authors.lastname: Lee
authors.affiliation: Department of Pathology and Laboratory Medicine¸ David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Kingshuk
authors.initials: K
authors.lastname: Das
authors.affiliation: 1] Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA [2] Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Vilain
authors.affiliation: 1] Department of Pathology and Laboratory Medicine¸ David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA [2] Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Stanley F
authors.initials: SF
authors.lastname: Nelson
authors.affiliation: 1] Department of Pathology and Laboratory Medicine¸ David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA [2] Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA [3] Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Wayne W
authors.initials: WW
authors.lastname: Grody
authors.affiliation: Department of Pathology and Laboratory Medicine¸ David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Deignan
mesh_terms: D059472:Exome / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000592:standards*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D054928:Validation Studies as Topic