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BACKGROUND: Next-generation sequencing (NGS) has become an indispensable diagnostic tool across various diseases. However, sequencing and analysis errors remain major barriers to clinical implementation. In cancer diagnostics, detecting low-level somatic variants is particularly challenging due to tumor heterogeneity and contamination from normal cells.RESULTS: We assess targeted next-generation sequencing (T-NGS) performance using reference-standard DNA mixtures of homozygote hydatidiform mole and heterozygote blood DNA at varying ratios, analyzed by certified NGS providers. Analytical sensitivity differs by up to 13.9-fold, and false positive (FP) error rates vary up to 615-fold, depending on provider and pipeline. For identical raw data, DRAGEN and the in-house pipeline differ by up to 36.3-fold in FP error rates. Moderately recurrent FP-prone alleles, although representing only 5.37% of all FP sites, contribute to 36.7% of total FP errors in the Geninus in-house result. Among 22 discordant variant calls between DRAGEN and in-house analyses, more than half of them are not confirmed by single base extension assays, indicating likely false positives. Compared to DRAGEN, a conventional BWA + GATK Mutect2 pipeline maintains equivalent sensitivity but produces a 4-fold increase in FP errors, along with a notable enrichment of recurrent FP-prone alleles.CONCLUSIONS: T-NGS results from certified providers exhibit substantial variability in both sensitivity and FP error rates. Conventional pipelines not only increase FP errors but also accumulate recurrent FP-prone alleles. These findings underscore the urgent need for standardized pipelines and rigorous quality control measures to ensure the reliability of T-NGS in clinical diagnostics.