Owing to its unique genetic characteristics and inherent stability, circular multicopy mitochondrial DNA (mtDNA) is essential for maternal lineage tracing from degraded and trace samples, making it a key focus in forensic science and evolutionary anthropology. However, achieving accurate and reliable mtDNA sequencing from such samples remains technically challenging. In this study, we developed a whole-genome sequencing system for mtDNA using a short-overlapping amplification strategy on the DNB sequencing platform. For blood card samples—the most common sample type in forensic practice—the system yielded a mean Q30 score of 97.65% (SD = 0.24%), with 99.47% (SD = 0.16%) of reads mapping to the revised Cambridge Reference Sequence (rCRS), covering 99.64% of the full mtDNA genome. Of the 16,569 sites, 99.06% achieved a sequencing depth exceeding 10% of the mean depth. The system enabled accurate calling of both point variants and length variants, with average base error rates of 0.2009%, 0.0892%, 0.2155%, and 0.1174% for A, C, T, and G, respectively, and average error rates of 7.7075% and 1.0977% for insertions and deletions, respectively. Heteroplasmy detected at position 309—including the third most abundant allele by sequencing depth in the massively parallel sequencing (MPS) data—was validated by Sanger sequencing. Analysis of different hair segments, fingernails, and buccal swabs from three individuals revealed notable variation in mtDNA variants across hair segments in some individuals, with greater heteroplasmy observed in the distal hair shaft than in the hair follicle. The system also achieved balanced whole-genome mtDNA sequencing for samples with high-temperature degradation (degradation index > 60), with 95.0117% of sites showing depth > 10% of the mean. For extremely degraded samples precluding effective small-fragment recovery, 93.6930% of the mtDNA genome was still covered. Reliable and balanced whole-genome mtDNA sequencing data were consistently obtained from blood card samples degraded by immersion in tap water or extended storage at room temperature. Collectively, this system provides a balanced, accurate, and robust tool for whole-genome mtDNA sequencing in forensic applications, with promising utility in complex kinship analysis, maternal lineage screening, and population genetic studies.

Authors

• Man Chen (Academy of Forensic Science, Ministry of Justice, China)
• Weifen Sun (Academy of Forensic Science, Ministry of Justice, China)
• Xufeng Chu (Academy of Forensic Science, Ministry of Justice, China)
• Ya Li (Academy of Forensic Science, Ministry of Justice, China)
• Xiling Liu (Academy of Forensic Science, Ministry of Justice, China)