<i>mtDNApipe</i> : A Pioneering Pipeline for High-Sensitivity Detection of Low-Frequency Mitochondrial DNA Mutations

Accurate detection of low-frequency mitochondrial DNA (mtDNA) mutations is essential for advancing molecular profiling, yet it is often confounded by sequencing artifacts, nuclear mitochondrial DNA (NUMTs), and oxidative damage-induced errors. Although unique molecular identifier (UMI)-based duplex sequencing can reduce such errors, its high cost and limited efficiency restrict its widespread use. Here, we present <i>mtDNApipe</i>, a bioinformatics pipeline tailored for capture-based mtDNA sequencing that integrates multiple layers of error suppression. The workflow combines stringent prealignment filtering to remove low-quality reads, overlap-based correction exploiting paired-end redundancy, and exclusion of soft-clipped reads to minimize NUMT interference. Additionally, an endogenous UMI (eUMI)-guided deduplication strategy corrects strand-specific damage, while terminal mutation filtering mitigates end-repair artifacts. When applied to technical replicates of peripheral blood mononuclear cells with low mtDNA copy numbers and paired fresh tumor tissues with high copy numbers, <i>mtDNApipe</i> reduced false positives by more than 80% in the low-frequency range while maintaining sensitivity. Notably, it achieved accuracy comparable to that of conventional UMI-based methods but without their cost and complexity. Compared with existing tools, <i>mtDNApipe</i> demonstrated superior robustness for detecting low-frequency heteroplasmy, offering a reliable and cost-effective solution for high-fidelity mtDNA mutation analysis with broad applications in biomarker discovery, molecular diagnostics, and analytical genomics.