Abstract 5058: Evaluation of false positive and false negative errors in targeted next generation sequencing

Abstract Background: Next-generation sequencing (NGS) has emerged as a vital diagnostic tool for various diseases; however, errors associated with NGS present significant challenges to its clinical implementation. Specifically, in cancer diagnostics, the analysis of low-level mutations is complicated by contamination from normal cells and tumor heterogeneity. Results: In targeted NGS (T-NGS) analyses involving reference-standard samples consisting of mixtures of homozygous Hydatidiform mole DNA and blood genomic DNA at varying ratios from four certified NGS service providers, we observed considerable variability in both the lower detection limits of variants (16.3-fold difference, ranging from 1.51% to 24.66%) and false positive (FP) error rates (4280-fold difference, ranging from 5.814 × 10^-4 to 1.359 × 10^-7). The commercially available Dragen system for bioinformatics analysis reduced FP errors in results from companies BB and CC; however, inherent errors from the raw NGS data remained. Adjustments to bioinformatics parameters aimed at increasing sensitivity (by less than two times) substantially elevated FP error rates (by 610 to 8200 times). Additionally, we identified issues such as biased base calling during bioinformatics analysis and a high frequency of false negative (FN) errors in HLA regions. Conclusion: T-NGS results from certified NGS service providers reveal considerable variability in sensitivity and FP error rates, underscoring the necessity for enhanced quality control measures in the clinical application of T-NGS. Furthermore, our study suggests that mixtures of homozygous and heterozygous DNA can serve as effective reference-standard materials for T-NGS quality control. Citation Format: Chunghwan Hong, Youngbeen Moon, Hye Won Choi, Eun-Kyung Kang, Jong-Kwang Kim, Young-Ho Kim, Dong-eun Lee, Tae-Min Kim, Seong Gu Heo, Namshik Han, Kyeong-Man Hong. Evaluation of false positive and false negative errors in targeted next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5058.