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

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

Samuel P Strom  1 , Hane Lee  2 , Kingshuk Das  2 , Eric Vilain  3 , Stanley F Nelson  1 , Wayne W Grody  4 , Joshua L Deignan  2
Affiliations

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

Samuel P Strom et al. Genet Med. 2014 Jul.

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.

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Figures

Figure 1
Figure 1. Correlation between Quality Scores and Depth of Coverage
Individual quality scores are plotted against read depth for 110 SNV loci tested. Quality score threshold of Q500 is marked by a dashed grey vertical line. The correlation is positive and significant (Pearson Correlation Significance Test, P<10−13).
Figure 2
Figure 2. Validation results sorted by quality score
Each SNV tested is represented by a point, sorted by ascending quality score. Red points represent SNVs with quality scores
See this image and copyright information in PMC

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