Inference of forensic body fluids/tissues based on mitochondrial DNA copy number: a preliminary study

The inference of body fluids and tissues is critical in reconstructing crime scenes and inferring criminal behaviors. Nevertheless, present methods are incompatible with conventional DNA genotyping, and additional testing might result in excessive consumption of forensic scene materials. This study aims to investigate the feasibility of distinguishing common body fluids/tissues through the difference in mitochondrial DNA copy number (mtDNAcn). Four types of body fluids/tissues were analyzed in this study - hair, saliva, semen, and skeletal muscle. MtDNAcn was estimated by dividing the read counts of mitochondrial DNA to that of nuclear DNA (RR_mt/nu). Results indicated that there were significant differences in RR_mt/nu between different body fluids/tissues. Specifically, hair samples exhibited the highest RR_mt/nu (log₁₀RR_mt/nu: 4.3 ± 0.28), while semen samples showed the lowest RR_mt/nu (log₁₀RR_mt/nu: -0.1 ± 0.28). RR_mt/nu values for DNA samples without extraction were notably higher (approximately 2.9 times) than those obtained after extraction. However, no significant difference in RR_mt/nu was observed between various age and gender groups. Hierarchical clustering and Kmeans clustering analyses showed that body fluids/tissues of the same type clustered closely to each other and could be inferred with high accuracy. In conclusion, this study demonstrated that the simultaneous detection of nuclear and mitochondrial DNA made it possible to perform conventional DNA analyses and body fluid/tissue inference at the same time, thus killing two birds with one stone. Furthermore, mtDNAcn has the potential to serve as a novel and promising biomarker for the identification of body fluids/tissues.