Analysis of the sequencing quality of next-generation sequencing for the entire mitochondrial genome in decomposed human samples

Human body decomposition significantly damages DNA, particularly short tandem repeats used in DNA profiling. In degraded samples, mitochondrial DNA (mtDNA) is used for aiding identification, utilizing hard tissues such as bones as DNA sources. However, extracting DNA from these tissues is complex and time-consuming. This study explores soft tissues for mtDNA analysis employing a next-generation sequencing (NGS) panel. A total of 290 samples from 30 autopsy cases were analyzed using an NGS panel targeting the entire mitochondrial genome. Among them, 239 samples were from 25 decomposed bodies with total body scores (TBS) ranging from 3 to 24. Nine types of soft tissue, including heart, liver, kidney, lung, brain, pectoralis muscle, iliacus muscle, aorta, and uterus, were examined. Rib cartilage, a representative hard tissue, and blood samples served as reference materials. Over 90% of the mtDNA sequence was confirmed in 49.6% of decomposed samples, increasing to 78.7% in hypervariable regions. As much as 95–100% of the mtDNA sequence could be retrieved from several highly decomposed soft tissues, comparable to rib cartilage. Among soft tissues, the uterus and aorta showed the shortest regions of uncovered mtDNA, highlighting their potential in decomposed bodies. No significant correlation was found between mtDNA sequencing quality and TBS or the nuclear DNA degradation index. The NGS panel successfully obtained most mtDNA sequences from decomposed soft tissues, suggesting that decomposition does not preclude genetic testing. Employing uterine or aortic tissues as alternatives to hard tissues in forensic contexts could streamline procedures, saving both time and resources.