mtDNApipe: 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 mtDNApipe, 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, mtDNApipe 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, mtDNApipe 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.