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Sanger Confirmation Is Required to Achieve Optimal Sensitivity and Specificity in Next-Generation Sequencing Panel
Wenbo Mu1, Hsiao-Mei Lu1, Jefferey Chen1
1Ambry Genetics, Aliso Viejo, California.
The Journal of Molecular Diagnostics : JMD
|October 11, 2016
Summary
Next-generation sequencing (NGS) is crucial for hereditary cancer gene panels. Sanger sequencing confirmation is necessary to maintain high sensitivity and detect all variants, especially in complex genomic regions.
Area of Science:
- Genomics
- Molecular Diagnostics
- Oncology
Background:
- Next-generation sequencing (NGS) is the standard for diagnostic gene-panel testing, particularly for hereditary cancer.
- Accurate variant detection is critical for clinical management and treatment decisions.
Purpose of the Study:
- To evaluate the necessity of Sanger sequencing confirmation for variants identified by NGS in hereditary cancer gene panels.
- To assess the impact of adjusting quality thresholds on NGS assay sensitivity and specificity.
Main Methods:
- Analysis of 20,000 hereditary-cancer NGS panels covering 47 genes.
- Sanger sequencing confirmation of all 7845 nonpolymorphic variants identified by NGS.
- Simulation of zero false-positive rates by adjusting quality thresholds.
Main Results:
- 98.7% concordance between NGS and Sanger sequencing.
- 1.3% of variants were identified as NGS false-positives, primarily in complex genomic regions.
- Adjusting thresholds to eliminate false-positives reduced NGS sensitivity to 97.8%, missing 176 variants, including mosaic mutations.
Conclusions:
- Sanger sequencing confirmation of NGS variants is essential for maintaining the highest sensitivity in hereditary cancer testing.
- Quality thresholds should be established after analyzing large datasets to balance sensitivity and specificity.
- Complex genomic regions and mosaic mutations pose challenges for accurate variant detection.
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