DNA Methylation Assessment
Document Type
Other
Publication Date
2024
Abstract
Thanks to advances in sample preparation and sequencing methods, there is an influx of research examining epigenomics in non-model systems. Broadly, epigenetics can be defined as changes to gene expression that do not arise from changes in the DNA sequence. DNA methylation, or addition of a methyl (CH3) group to a cytosine base adjacent to a guanine (CpG) is one of the more commonly studies epigenetic mechanisms, partly because of its environmental sensitivity and potential role in phenotypic plasticity. For a good review of epigenetic mechanisms including DNA methylation in marine organisms see Eirin-Lopez and Putnam (2019).
The purpose of this tutorial is to provide an example of aligning bisulfite-treated and enzymatically converted DNA sequence data to an existing genome, and discuss needs for potential downstream applications. The principle behind creating bisulfite-treated DNA libraries is that when cytosines are not methylated, then they will be converted to uracil, which ultimately result in thymine nucleotides in the sequence data. Wanamaker et al. (2022) offers a comparison of three methods for quantifying DNA methylation at single base-pair resolution using whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS). When aligning bisulfite-converted data to a reference genome the percent methylation at a given CpG loci is determined by examining the ratio of thymines to cytosines. For example if there is a given cytosine adjacent to a guanine (ie CpG locus) with 30% of the reads containing thymine, one would consider this cytosine loci (CpG) to be 70% methylated. Since bisulfite treatment can damage DNA, newer library preparation and sequencing methods (ex. NEBNext® Enzymatic Methyl-seq Kit; EM-seq are starting to gain popularity. As EM-Seq produces loci-level data, alignment of EM-Seq data is similar to WGBS. Also, technologies like Nanopore and PacBio sequencing can directly detect DNA methylation without bisulfite conversion. See Dimond, Nguyen, and Roberts (2021) as example of using Nanopore sequencing. As DNA passes through a nanopore or is sequenced by single-molecule real-time (SMRT) technology, changes in electrical current or fluorescence patterns can indicate the presence of methylated bases.
Recommended Citation
Venkataraman YR, Greiner-Ferris K, White SJ, Roberts SB (2024). DNA Methylation Assessment. MarineOmics. https://marineomics.github.io/FUN_02_DNA_methylation.html
