Reducing the Number of Mismatches between Hairs and Buccal References When Analysing mtDNA Heteroplasmic Variation by Massively Parallel Sequencing

Abstract

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.