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  1. Development of an Evidence-Based Algorithm that Optimizes Sensitivity and Specificity in ES-Based Diagnostics of a Clinically Heterogeneous Patient Population

Development of an Evidence-Based Algorithm that Optimizes Sensitivity and Specificity in ES-Based Diagnostics of a Clinically Heterogeneous Patient Population

Dr. Peter Bauer, MD 1 Krishna Kumar Kandaswamy, PhD 1 Maximilian E. R. Weiss 1 Omid Paknia, PhD 1 Martin Werber 1 Aida M. Bertoli-Avella, MD 1 Zafer Yüksel, MD 1 Malgorzata Bochinska 1 Gabriela-Elena Oprea, PhD 1 Shivendra Kishore, PhD 1 Volkmar Weckesser, Dr 1 Ellen Karges, Ms 1 Prof. Arndt Rolfs, MD 1
1 CENTOGENE AG
January 23, 2019

Genet Med. 2019 Jan;21(1):53-61. doi: 10.1038/s41436-018-0016-6. Epub 2018 Aug 13.

Abstract

Purpose

Next-generation sequencing (NGS) is rapidly replacing Sanger sequencing in genetic diagnostics. Sensitivity and specificity of NGS approaches are not well-defined, but can be estimated from applying NGS and Sanger sequencing in parallel. Utilizing this strategy, we aimed at optimizing exome sequencing (ES)–based diagnostics of a clinically diverse patient population.

Methods

Consecutive DNA samples from unrelated patients with suspected genetic disease were exome-sequenced; comparatively nonstringent criteria were applied in variant calling. One thousand forty-eight variants in genes compatible with the clinical diagnosis were followed up by Sanger sequencing. Based on a set of variant-specific features, predictors for true positives and true negatives were developed.

Results

Sanger sequencing confirmed 81.9% of ES-derived variants. Calls from the lower end of stringency accounted for the majority of the false positives, but also contained ~5% of the true positives. A predictor incorporating three variant-specific features classified 91.7% of variants with 100% specificity and 99.75% sensitivity. Confirmation status of the remaining variants (8.3%) was not predictable.

Conclusions

Criteria for variant calling in ES-based diagnostics impact on specificity and sensitivity. Confirmatory sequencing for a proportion of variants, therefore, remains a necessity. Our study exemplifies how these variants can be defined on an empirical basis.