Mitochondrial DNA (mtDNA) is a useful target for analyzing forensic samples when nuclear DNA (nDNA) profiles are difficult to obtain, for example, when a forensic hair sample does not have follicular tissue attached. Obtaining mtDNA does not often depend on the presence of follicular tissue because mtDNA is present in the hair shaft (Andreasson et al., 2006). However, mtDNA analysis presents challenges that nDNA analysis does not. One such challenge is interpreting heteroplasmy, which is a mtDNA mutation phenomenon that causes more than one mtDNA sequence to be present in an individual. Heteroplasmy has the potential to be useful in forensic mtDNA typing because the presence of heteroplasmy at identical sites in both a casework and a reference sample could not only help confirm a match, but also increase the significance of the match (Paneto et al., 2007).
Massively Parallel Sequencing (MPS) can overcome the challenges of forensic mtDNA typing. MPS methods are highly sensitive approaches for clonally amplifying and sequencing many samples at once. MPS methods can resolve complex mixtures (from ≥3 contributors) (Kim et al., 2015) and can also quantify mtDNA mutations. The level of sensitivity achieved with MPS allows for better detection of low-level mutations unobtainable with Sanger sequencing, which is the current forensic mtDNA typing method. The aims of this study were to use an MPS method to better characterize low levels of heteroplasmy across tissues and to compare MPS to Sanger sequencing. In this study, I used the Roche® 454 GS Junior MPS platform to sequence the mtDNA hypervariable regions (HVI/HVII) from human head hair, pubic hair, and buccal samples. I compared the MPS sequencing results to the Sanger sequencing results for different tissues from the same sample sets.
Point heteroplasmy was detected in more samples when MPS was used than when Sanger sequencing was used. The frequency of heteroplasmy was highest in head hairs, followed by pubic hairs and then by buccal swabs. The 454 was able to quantify levels of heteroplasmy, which cannot be achieved with Sanger sequencing. The 454 detected low levels of heteroplasmy better than Sanger sequencing by reporting levels as low as 1.14% (with at least 500X coverage). With Sanger sequencing, only instances of heteroplasmy at about 10% or above could be confidently reported. The 454 also detected more somatic mutations and more heteroplasmy differences within and between different tissue types. Roughly equal numbers of somatic and germ-line heteroplasmy were observed, as well as four heteroplasmic “hot spots” at positions 150, 183, 185, and 189 in HVII. Finally, there was no significant difference in the frequency of heteroplasmy with an increase in an individual’s age.
In conclusion, this study improves our understanding of forensic mtDNA sequencing by better characterizing the mutations inherently present in mtDNA. This study also further argues for the use of MPS platforms for forensic mtDNA sequencing.
(Publisher abstract provided.)