Pedigree-Based comparison of short-read and long-read sequencing for forensic mitochondrial genome analysis.

The choice between short-read and long-read sequencing represents a critical trade-off in forensic mitochondrial DNA (mtDNA) analysis. In this study, Illumina short-read sequencing (SRS) and QitanTech Nanopore long-read sequencing (LRS) platforms were systematically compared using data from 177 individuals across 39 maternal pedigrees. The results demonstrated that LRS, which utilized a single-amplicon enrichment strategy, delivered superior coverage uniformity (median 100%), effectively reducing the coverage variability typically associated with SRS multiplex PCR. Moreover, LRS improved the discrimination of interference from nuclear mitochondrial DNA segments (NUMTs) by spanning homologous regions with longer reads. In contrast, the SRS data showed pronounced off-target alignment, which was notably enriched on chromosomes 17 and 2 because of spurious sequence homology. However, although both platforms produced concordant consensus haplotypes, SRS exhibited higher accuracy for low-level heteroplasmy detection. Pedigree-based validation revealed that the current LRS workflow produced background-noise signals that were misclassified as heteroplasmy at a 10% allele frequency threshold. Consequently, we propose a complementary workflow: employing LRS for structural characterization and NUMT exclusion while relying on SRS for high-fidelity quantification of heteroplasmy. This integrated strategy offers a robust solution for forensic casework involving complex or low-input samples.