The forensic efficiency evaluation of degradation samples with a novel mtDNA whole genome detecting system of short overlapping amplicons on DNB sequencing platform.

With unique genetic characteristics and inherent stability, circular multicopy mitochondrial DNA (mtDNA) is critical for maternal lineage tracing from degraded and trace samples, making it a sustained focus in forensic science for identifying degraded samples when nuclear DNA analysis fails, as well as in evolutionary anthropology. However, accurate and reliable mtDNA sequencing from such samples remains technically challenging. By employing rolling circle amplification (RCA) to achieve linear amplification, the DNB sequencing platform effectively avoids error accumulation, yielding sequencing results with high accuracy, low error rates, and improved uniformity even for complex sequences (e.g., GC-rich regions). 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 obtained sequencing reads showed a mean Q30 value of 97.65% (SD = 0.24%), with an average of 99.47% (SD = 0.16%) of the reads aligning to the mtDNA reference sequence rCRS, covering 99.64% of the entire mtDNA sequence. Out of the 16,569 positions, 99.06% achieved a sequencing depth greater than 10% of the mean depth. The system enables accurate calling of point-variants and length-variants, with average error rates of 0.20%, 0.09%, 0.22%, and 0.12% for the four bases A, C, T, and G, respectively; and average error rates of 7.71% and 1.10% for insertion and deletion variants, respectively. Furthermore, heteroplasmy detected at position 309 by the system, including even the third most abundant allele by sequencing depth in the MPS data, was validated by Sanger sequencing. Additionally, the system enabled balanced whole-genome mtDNA sequencing of samples subjected to high-temperature degradation with a degradation index exceeding 60, achieving a sequencing depth greater than 10% of the mean depth at 95.01% of the positions. In extremely degraded samples where effective recovery of small fragments was not possible, 93.69% of the mtDNA sequence was still obtained. The development of this system provides a balanced, accurate, and robust tool for whole-genome mtDNA sequencing in forensic practice, with potential applications in complex kinship identification, maternal lineage screening, and population genetic relationship analysis.