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

Genetics in Medicine

(2014)

doi:10.1038/gim.2013.183

Received: 10 July 2013
Accepted: 18 October 2013
Published online: 09 January 2014

Abstract

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.

Genet Med advance online publication 9 January 2014

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