Alternative Sample Processing Techniques for Rootless Hair Shafts and other Challenging Samples



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Evidence recovered for forensic testing often includes challenges that will impede typical genetic analysis using short tandem repeat markers with capillary electrophoresis platforms. This can include degradation, inhibition, and the lack of sufficient template for successful processing. To increase successful analysis of samples, optimization of testing protocols has been implemented with varying success in forensic crime laboratories. Even with these improvement attempts, it is sometimes necessary to perform alternate analysis on forensic samples. For some cases the sequencing of mitochondrial DNA is performed as a secondary option to traditional workflows. One sample type that is commonly considered challenging and relegated to mitochondrial sequencing is rootless hair shafts. Given these challenges, novel strategies that attempt to improve the recovery of genetic information are often proposed to either circumvent or supplement existing analysis options. These include amplification of markers that are not STRs and analysis of any amplified DNA with alternative instrumentation such as massively parallel sequencing (MPS) platforms. In this research several approaches are implemented to improve the recovery of genetic information from challenging sample types, with a focus on rootless hair samples. The first series of experiments focused on the extraction and amplification of DNA. Several variables were tested during this process including hair pre-treatment, extraction procedure, amplification strategy, and instrumentation. Results from this research showed that while hair decontamination did decrease the occurrence of allele drop-in with rootless hair samples, it also drastically decreased the recovery of DNA. Optimized extraction techniques can increase the recovery of DNA from challenging sample types. Lastly, with challenging samples, it is necessary to concentrate amplification strategies on small amplicon fragments to improve success. To improve results with mitochondrial DNA sequencing, a novel MPS workflow was rigorously assessed for use with forensic samples. Testing parameters included limits of detection, concordance with alternate sequencing techniques, mixture detection, repeatability, and success with forensic type samples. Overall, the workflow tested performed similarly to or better than the comparison methods with a high potential for implementation in the immediate future. Another major concern with many of the challenging forensic sample types is the potential for genetic content to be mixed or contaminated from an external source. To better characterize the occurrence of mixtures in MPS, mixture samples were created using known profiles and amplified for MPS. This data was analyzed to identify potential strengths and weaknesses when compared to capillary electrophoresis. The MPS platform included Y-STR results that would need to be generated using a separate STR amplification on CE. The limit of detection for MPS is also an improvement. The information gathered from isoalleles and additional STR markers can also lead to more discriminatory profiles when looking at some DNA mixtures. However, there are limitations, with a noticeably less reproducible peak height ratios being generated on the MPS platform. The implementation of probabilistic genotyping software with MPS data should be considered to ease mixture interpretation.



Biology, Genetics