Investigation into the Limit of Detection of Massively Parallel Sequencing for Forensic DNA Sequence Profiling

Abstract

Laser microdissection (LMD) is a valuable tool in forensic science, enabling the separation of cell mixtures in order to facilitate DNA analyses of pure populations. As LMD is most commonly applied to particularly difficult or limited samples, it is critical to determine the limit of detection of downstream DNA analyses to ensure optimal use can be made of what sample is available. This thesis investigates the limits of detection of massively parallel sequencing (MPS) looking at the numbers of sperm and epithelial cells needed to produce full profiles when amplified with the ForenSeq™ DNA Signature Prep Kit and sequenced on a MiSeq FGx™. Sampling of sperm and epithelial cells was carried out via LMD with a Leica LMD6000 laser microdissector and bioinformatic analysis undertaken via ForenSeq™ Universal Analysis Software, implementing the default thresholds defined by Illumina®. Sample analysis was carried out in parallel via capillary electrophoresis (CE) for the purpose of comparison. Full ForenSeq™ profiles were obtained from as few as 75 epithelial cells and 150 sperm cells, with the limit of detection determined to lie lower than this. In comparison CE produced full profiles from as few as 50 epithelial cells, however even the largest sperm sample size (200 cells) did not produce any full profiles. Bioinformatic analysis was successful in producing full profiles that were concordant with CE data, however it was noted that several of the default quality thresholds included in the ForenSeq™ Universal Analysis software may require further optimisation to better facilitate interpretation of unknown and potentially mixed samples in the future. Although samples were successfully amplified and analysed following the manufacturer’s recommended protocol this work highlighted several aspects of library preparation and sequencing which could benefit from further optimisation. Specifically, library yield could benefit from additional clean up steps and optimisation of reagent quantities when carried out on samples with a low amount of input DNA, particularly samples obtained via LMD. Although ultimately the limit of detection was not determined the research carried out here was successful in showing the compatibility of MPS with laser microdissected samples, as well as providing a number of insights into how the integration of these processes could be improved upon in future work.