A full-length mtDNA dataset for studying genetic variations across generations and complex family structures.

Mitochondrial DNA (mtDNA) mutations are critical to disease research, evolutionary studies, and lineage tracing but are challenging to analyze due to interference from nuclear mitochondrial sequences (NUMTs). Current high-throughput sequencing techniques rely on multiple primers or probes to amplify short mtDNA fragments, followed by alignment to a reference genome. However, this approach fails to mitigate NUMTs interference, leading to ambiguous results. In this study, we presented a nanopore-based third-generation sequencing (TGS) method using a single primer pair to amplify full-length mtDNA, effectively circumventing NUMTs artifacts. Sequencing was carried out on the QITAN TECH QNome-3841hex platform, generating complete mtDNA coverage for 106 samples from eight distinct family pedigrees, including complex familial structures such as half-siblings and multi-generational households. The sequencing achieved 100% genome coverage with an average mapping rate of 99.96%, supporting comprehensive genome characterization. The resulting dataset offers valuable insights into mtDNA mutation detection, mitochondrial genetics, population genetics, ancestry tracing, and forensic identification, and may advance mtDNA sequencing technologies and intergenerational studies.