The development of enhanced experimental strategies for the DNA analysis of low-template or compromised forensic sample types

摘要

Single-cell DNA analysis is not routinely carried out in a forensic setting as it is considered unreliable due to challenges associated with DNA amplification, contamination and profile interpretation. In light of the development of increasingly sensitive techniques, the question of the reliability of single-cell DNA analysis in terms of both processing and interpretation is addressed in the first part of this thesis. Optimising all stages of the DNA analysis process has provided a sensitive method which facilitates the successful outcome of a useable profile from single-cells. Although no consensus profile can be generated for this sample type, interpretation guidelines have been set to enable the robust analysis of single cells. It has been concluded that single-cells can be reliably amplified and profiled for forensic purposes.ududBoth DNA and textile fibres have a proven track record in forensic casework yet their analysis is rarely combined. As an application of the aforementioned single-cell DNA analysis, this project explores the possibility that when fibres are transferred from one surface to another, they could also be acting as a vector for the wearer’s own DNA, through cells that have adhered to the fibre surfaces. Fluorescent staining and microscopy is used to detect the cells in situ on the fibre surface, which are then recovered and processed for DNA using the previously optimised single-cell analysis methods, along with a newly developed DNA assay designed for the amplification of low DNA template samples. The results of this study have demonstrated that cells can be visualised in situ on the fibre surface and that there is potential for cell transfer to occur. It has been concluded however, that from a casework point of view, targeting transferred fibres for cells may not be the best approach as it is time consuming and has not been shown to be effective in this study.ududThe final part of this thesis is focused on the efficacy of massively parallel sequencing (MPS) technology for samples that are expected to be severely degraded due to age or exposure to a hostile environment. The ability of both the recently launched Illumina ForenSeq™ DNA Signature Prep Kit for nuclear DNA markers and an in-house method for the sequencing of degraded mitochondrial DNA, have been tested to determine if MPS offers a more comprehensive evaluation of degraded material than the traditional PCR-CE methods. The results of the ForenSeq kit have demonstrated the effectiveness of its low molecular weight STR and SNP markers for amplifying low template, degraded DNA samples, with alleles amplified using less than 20 pg total DNA input. This kit has also therefore shown application in the field of bioarchaeology, as it can provide the biological sex of the sample, biogeographic ancestry information and also aids detection of sample/control contamination. The in-house mitochondrial DNA assay resulted in the successful amplification and sequencing of samples for which no nuclear DNA was amplified. The high depth of read coverage in these samples, average of 18,000, allowed for the identification of even low level variants.