Methylome-wide analysis

Accurate characterization of RE 5-methylcytosine (5mC) methylation patterns using traditional next-generation sequencing (NGS), such as whole-genome bisulphite sequencing (WGBS), is challenging due to limitations in bioinformatic tools and the inherent complexity of repetitive sequences. The short-read length of NGS limits the ability to precisely locate specific REs within the genome due to their repetitive nature, leading to ambiguous sequence alignment. Moreover, WGBS is dependent on an optimal efficiency of chemical conversion and PCR amplification, which can introduce biases. Third-generation sequencing (e.g. Nanopore sequencing) offers a significant advantage for studying 5mC methylation and other epigenetic modifications, without the need for bisulphite conversion, on long DNA strands, providing greater genomic context and allowing for the precise mapping of RE methylation events.

Given the above features, Nanopore sequencing will be employed in the discovery phase of the MAMELI study, for the unbiased screening of RE methylation in 200 subjects at two time points (T0 and T1). This method will allow for the identification of specific REs that exhibit modifiable methylation patterns in response to environmental stimuli, without compromising genomic stability. The identified modifiable REs will then be the focus of targeted methylation analysis in subsequent stages of the study.