Concurrent genotyping of mitochondrial DNA and nuclear DNA in rootless hair shafts and blood samples for enhanced analysis

Hair is an important type of biological evidence at crime scenes. However, the highly degraded nature of DNA fragments in hair shafts poses challenges for the detection of nuclear DNA (nuDNA) through capillary electrophoresis-based short tandem repeat (STR) genotyping. In this study, an all-in-one multiplex system named MGIEasy Signature Identification Library Prep Kit (MGI Tech, China) was employed to the simultaneous genotyping of both mitochondrial DNA (mtDNA) and nuDNA in hair shafts. This system is based on massively parallel sequencing (MPS) technology and encompasses Amelogenin, STRs, single nucleotide polymorphisms (SNPs) and mtDNA hypervariable regions (HVRs) in a single reaction. A total of 370 hair shafts, together with 180 blood samples as the references, were examined. The mtDNA analysis of 110 unrelated blood samples unveiled a total of 150 homoplasmic variants and 105 distinct haplotypes, revealing population polymorphisms in the Guangdong Han Chinese. The study also delved into the detection of mtDNA heteroplasmy, revealing 8.18 % and 16.36 % of individuals with point heteroplasmies (PHPs) in blood and hair shaft samples, respectively. Additionally, hair shafts with DNA extracted using the Investigator method yielded higher average depth of coverage (DoC), lower drop-out rate for SNP genotyping, higher nuDNA genotyped rates and success rates, than those using the MinElute method. In the longitudinal research, a gradual decrease in the total DoC of mtDNA fragments was observed along the length of the hair shaft, from the proximal root to the distal end. In contrast, the DoC of nuDNA exhibited a relatively stable pattern along the length of the hair shafts. The study contributes valuable insights into the simultaneous detection of nuDNA and mtDNA in hair shafts, emphasizing the need for optimized DNA extraction and detection methods for these highly degraded samples.