Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory

Genet Med. 2014 Jul;16(7):510-5. doi: 10.1038/gim.2013.183. Epub 2014 Jan 9.


Purpose: 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%).

Conclusion: 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.

MeSH terms

  • Exome / genetics*
  • Genome, Human / genetics*
  • Genotype
  • High-Throughput Nucleotide Sequencing / standards*
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Polymorphism, Single Nucleotide / genetics*
  • Sequence Analysis, DNA / economics
  • Sequence Analysis, DNA / methods*
  • Validation Studies as Topic