Mitochondrial DNA Variation in Human Hair Shafts Influenced by Physical Characteristics: A Massively Parallel Sequencing Analysis

Background/Objectives: Hair shafts are frequently encountered as forensic evidence, but their limited nuclear DNA content often constrains short tandem repeat (STR) profiling. Mitochondrial DNA (mtDNA) analysis provides an alternative approach; however, the extent to which hair-shaft physical characteristics affect MPS-derived mtDNA read counts, degradation state, and point heteroplasmy (PHP) remains insufficiently characterized. This study aimed to evaluate these associations in human hair shaft samples. Methods: mtDNA hypervariable regions were analyzed in 907 hair shaft fragments from 183 donors using the MGIEasy Signature Identification Library Prep Kit and massively parallel sequencing. Samples were grouped by hair-shaft length, longitudinal segment, scalp region, color, diameter, donor sex, and cosmetic treatment. MPS read counts were used as semi-quantitative indicators of relative mtDNA representation, the HVR7/HVR5 read-count ratio was used as an indirect indicator of degradation state, and PHP was called at a minor allele frequency of ≥10% with a sequencing depth of ≥100× at the site. Results: Under the present workflow, hair shafts generated higher mtDNA read counts than paired bloodstains, and PHP was detected more frequently in hair shafts than in bloodstains (15.93% versus 8.85%). In the exploratory subgroup analyses, longer hair shafts tended to show higher total MPS-derived mtDNA read counts, whereas longitudinally segmented hairs showed a tendency toward decreasing read counts and increasing degradation from the proximal to distal end. In a small paired black/white hair dataset, black hairs showed higher read counts than paired white hairs, but this observation should be interpreted cautiously. Scalp region, diameter, donor sex, and cosmetic treatment showed no consistent effects on read counts or degradation state, although cosmetic treatment was associated with descriptive differences in PHP type and regional distribution. Conclusions: These findings suggest that selected physical characteristics of hair shafts may be associated with MPS-derived mtDNA metrics under the present workflow. However, because several subgroup analyses were based on limited sample sizes and clustered samples, these results should be regarded as exploratory and require validation in larger independent datasets.