Abstract
Forensic genetics has undergone numerous developments and innovations, from its beginnings with RFLPs to the current use of STR-based DNA profiling. Research in this field is exploring potential future methods, with DNA methylation studies gaining increasing popularity. Current studies utilise DNA methylation for distinguishing identical twins, identifying body fluids, ethnicity identification, and age estimation, among other applications. Significant progress has been made in age estimation using CpGs, demonstrating low error rates in determining the ages of unknown samples based on the method and model used. These studies highlight the importance of the chosen method and model in accurately determining unknown ages.Technological advancements now allow for the extraction of even more data from genetic samples than the entire genome itself. One of the latest technologies, Oxford Nanopore Technology (ONT), surpasses others in DNA methylation analysis. It offers more accurate and precise analyses by using native DNA directly, eliminating the need for chemical conversions and reducing potential errors and biases. Another key feature is its portability, allowing for field-ready use and minimizing laboratory requirements, thus reducing errors associated with sample packaging and transfer. However, a significant drawback for forensic sciences is the relatively high amount of input DNA required.
In our study, 27 samples from individuals aged between 21 and 77 were sequenced using ONT. Raw data from 26 samples were processed using bioinformatics tools for basecalling, with quality scores determined via the NanoPlot package, and DNA methylation data processed. Further analysis of the data was conducted using third-party DNA methylation analysis packages, including Nanopolish, Deepsignal, Megalodon, and Remora. Visualisation was performed using Methplotlib, Nanomethviz, and Methylartist packages. Based on the evaluation of visualisation packages, further analyses were continued with Methylartist. A total of 2688 graphs were scanned in 1 million bp blocks across all autosomal chromosomes to identify regions showing age-related changes. Genetic coordinates of fragments potentially related to age were extracted for more detailed analysis using another function of the Methylartist package. Although various scripts were used to analyse 3235 regions in detail, statistically significant results were not found. The obtained data were compared with previously acquired CpG data, validating the data in high-coverage regions.
Subsequently, samples were filtered to investigate age-related regions at single base resolution using data from four different methods, considering that low coverage regions might lead to errors. The results indicated that some regions showed high values proportional to age, not only in a single method and sample group but also in similar points across all methods. When investigating the genomic locations of these regions, two regions corresponded to lncRNA regions, while others did not overlap with any known functional genetic regions. These findings suggest that lncRNA regions may play a significant role in ageing, and further detailed analyses of the unknown regions could reveal their functions and potential roles in ageing.
| Date of Award | 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Sponsors | Ministry of National Education |
| Supervisor | Alexander Gray (Supervisor) & Christian Cole (Supervisor) |