Most human hairs found at crime scenes are telogen hairs. However, forensic laboratories do not have a standardized workflow of analysis for this type of sample. Telogen hairs are fully keratinized and they contain very low amounts or no detectable nuclear DNA (nDNA) but high amounts of mitochondrial DNA (mtDNA). This explains why laboratories have different approaches with respect to DNA analysis. While some laboratories perform nDNA analysis on all telogen roots with great chances of failure, other laboratories prefer to save time, costs and sample by performing only mtDNA analysis. Yet, sometimes mtDNA is highly degraded and limited in amount, which causes amplification failure when using traditional PCR-based methods. The aim of this project was to improve the workflow of telogen hair analysis by combining a microscopic screening method and a quantitative assay to predict the success of nDNA analysis, and a novel non-PCR-based approach to sequence the entire mitochondrial genome.
We utilized Modified Harris Hematoxylin, a nuclear stain, to perform a rapid screening under the microscope to determine the presence of nuclei inside the telogen roots or in the surrounding tissue. We compared the microscopic nuclei counting method to a real-time quantitative PCR assay (q-PCR). The qPCR assay results showed no detectable DNA for 96% of the telogen roots with no visible nuclei. The remaining 4% samples had detectable nDNA amounts, although nuclei were not observed. When > 20 nuclei were observed, 80% of telogen roots had enough quantifiable nDNA to expect partial or full STR profiles according to the qPCR assay. The 20% telogen roots with visible nuclei but undetectable nDNA had < 20 nuclei. In conclusion, the nuclei counting method was consistent with qPCR assay yield; however, the latter method proved to be less subjective and more informative.
To improve mtDNA analysis we propose the substitution of the PCR-based amplification method followed by Sanger-type Sequencing (STS) with the novel non-PCR-based Probe Capture Next Generation Sequencing (NGS) System. The novel approach not only has higher throughput and sensitivity, but also allows for deep sequencing of the entire mitochondrial genome. Of a total of 22 cut shaft samples, 41% failed amplification using PCR-based methodology, which is predictive of STS failure. Moreover, 18% exhibited a weak amplification signal, indicating potential STS failure. Probe Capture NGS System successfully captured and sequenced the entire mitochondrial genome of all shaft samples tested. Of 22 samples, 17 had 100% 5X coverage of the entire genome, while the remaining 5 samples had between 82 and 98% 5X coverage of the entire genome. Probe Capture NGS System overcame amplification limitations in mtDNA analysis.
In conclusion, this study shows that we can improve the workflow of telogen hair analysis by adding the nuclear counting method combined with a qPCR assay as screening approaches to predict nDNA analysis success, and the Probe Capture NGS System for mtDNA analysis.
(Publisher abstract provided.)
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