mtGenome Sequencing of Human Hair Shafts Using an MPS Approach

A next generation sequencing (NGS) approach using massively parallel sequencing (MPS) methods is available to forensic laboratories for routine mitochondrial (mt) DNA analysis, including the availability of software for data organization and interpretation. In addition, the use of a deep-coverage MPS (DCMPS) method allows for resolution of minor sequence variants (heteroplasmy) down to a threshold of 2%, augmenting the weight of an mtDNA match in forensic cases and allowing for the frequent differentiation of maternal relatives. While human hair shafts are a common source of evidence for mtDNA analysis in crime laboratories, before an MPS approach will be adopted the system must be operationally viable. For example, it must work on hairs that have historically given results when using conventional Sanger-type sequencing (STS) strategies, it must be cost effective, and it must provide a cost benefit. One of the clear benefits of MPS analysis is the ability expand the range of sequence generated from the control region (CR) to the entire mtGenome. However, little work has been done to evaluate MPS methods for mtGenome sequencing from hairs. Therefore, we propose to perform mtGenome MPS analysis on hairs typically encountered in forensic cases; hundreds of head and pubic hairs from more than one hundred donors with various levels of chemical and environmental exposure. In turn, we propose to create a large dataset of information regarding the expected levels of drift for heteroplasmic variants observed in individuals who have high levels of heteroplasmy in buccal and blood cells (>10%), low levels of heteroplasmy (2-10%), and no detectable heteroplasmy (<1%). Assessments will include the impact of DNA damage on interpretation of resulting MPS data, and the development of models for predicting levels of heteroplasmy in hairs given observed levels in reference samples such as oral swabs. The outcomes of this work will include an increased knowledge of heteroplasmic drift in hairs, the development of models to support the interpretation of heteroplasmy using a DCMPS approach, and an assessment of operational considerations, all of which will help guide and inform the practices being used by the forensic community. Each of the outcomes will address specific interests of the criminal justice system and National Institute of Justice; specifically, the development of discriminating, accurate, and reliable methods for analysis and interpretation of physical evidence. Partners for the project include Mitotyping Technologies, a SoftGenetics Company, and the Ohio BCI DNA Unit. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF