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

PURPOSE: Sanger sequencing is currently considered the gold standard methodology for clinical molecular diagnostic testing. However, next generation sequencing (NGS) 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 NGS 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: 103 of 110 total SNVs analyzed had a quality score ≥Q500, 103 (100%) of which were confirmed by Sanger sequencing. Of the remaining 7 variants with quality scores <Q500, 6 were confirmed by Sanger sequencing (85%). CONCLUSIONS: For single nucleotide variants, we predict we will be able to reduce our Sanger confirmation workload going forward 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 utilize NGS technology. However, Sanger confirmation of low quality single nucleotide variants and all indels (insertions or deletions less than 10 bp) remains necessary at this time in our laboratory.