Tweet
DNA methylation is an epigenetic modification that occurs most commonly at cytosine nucleotides. A methyl group binds to the fifth carbon of the cytosine ring to form 5-methylcytosine (5mC). Not all genes are active at all times and DNA methylation is an epigenetic mechanism cells use to downregulate the expression of certain genes–either for normal development or as part of a disease-related process. Methylation is also involved in chromatin remodeling, limiting access of transcription machinery to gene transcription start sites. Given the number of cellular processes with which DNA methylation is associated, it is no surprise that perturbations in methylation patterns have been shown to be associated with tumorigenesis, neurodegenerative diseases, and certain neurological disorders.
Analysis of cytosine methylation is carried out using bisulfite sequencing. Treatment of DNA with sodium bisulfite converts unmethylated cytosines to uracil, while methylated cytosines remain unchanged. Sequencing of the resulting DNA strand reveals the level of methylation present within the sample. Among the methods currently available to study DNA methylation, Novogene offers two of the most commonly used–whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS).
WGBS
WGBS is a method that analyses methylation levels of the whole genome, also known as the methylome. It involves library preparation with bisulfite treatment, followed by whole genome sequencing of the samples and it efficiently identifies methylated cytosines on a genome-wide scale to a single nucleotide resolution. Analysis of the methylome allows for identification of differentially methylated sites and regions associated with experimental treatments or sample conditions, and bioinformatic analysis of the data produced can provide insight into the cellular processes impacted by methylation levels and the mechanisms at play in disease states.
RRBS
RRBS enriches for methylated areas of the genome by using restriction enzymes to isolate CpG islands. This reduces the complexity of the genome, requiring fewer reads for the same depth of coverage of methylated regions. In turn, this makes the approach a more cost-effective alternative to WGBS. However, because it is a more targeted approach, RRBS carries the disadvantage that methylation in areas of low GC content will be missed as it will not be included in the enrichment step. This is often inconsequential as the majority of methylation occurs in regions of high GC content.
How to choose
Choosing whether to perform WGBS or RRBS on your samples will often come down to weighing up costs with the required data amount. WGBS is the more costly option as it requires a high number of reads to achieve the required sequencing depth, and it requires high amounts of input DNA. However, it provides a genome wide methylation profile, which RRBS does not, and lends itself to the identification of epigenetic biomarkers and therapeutic targets for disease. RRBS requires lower initial DNA input and due to its targeted nature, requires fewer reads to achieve the necessary sequencing depth. This makes it a much more cost-effective method in comparison to WGBS. As mentioned, RRBS will miss certain sites of methylation if they occur outside the enriched areas, however the majority of methylation occurs withing regions of high GC content and in many cases, the information obtained by sequencing these regions is sufficient to answer the research question at hand.
Overall, the choice between WGBS or RRBS will depend on the desired outcome of the sequencing project and the information required from the analysis. Novogene’s expert sales and technical support teams consider each project on a case-by-case basis. If you have an upcoming project that will require analysis of DNA methylation, please get in touch with us at novogene.com and we will be more than happy to assist you with your decision and sequencing strategy.
Eager to learn more? Check out our previous post for more insights!
Analysis of cytosine methylation is carried out using bisulfite sequencing. Treatment of DNA with sodium bisulfite converts unmethylated cytosines to uracil, while methylated cytosines remain unchanged. Sequencing of the resulting DNA strand reveals the level of methylation present within the sample. Among the methods currently available to study DNA methylation, Novogene offers two of the most commonly used–whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS).
WGBS
WGBS is a method that analyses methylation levels of the whole genome, also known as the methylome. It involves library preparation with bisulfite treatment, followed by whole genome sequencing of the samples and it efficiently identifies methylated cytosines on a genome-wide scale to a single nucleotide resolution. Analysis of the methylome allows for identification of differentially methylated sites and regions associated with experimental treatments or sample conditions, and bioinformatic analysis of the data produced can provide insight into the cellular processes impacted by methylation levels and the mechanisms at play in disease states.
RRBS
RRBS enriches for methylated areas of the genome by using restriction enzymes to isolate CpG islands. This reduces the complexity of the genome, requiring fewer reads for the same depth of coverage of methylated regions. In turn, this makes the approach a more cost-effective alternative to WGBS. However, because it is a more targeted approach, RRBS carries the disadvantage that methylation in areas of low GC content will be missed as it will not be included in the enrichment step. This is often inconsequential as the majority of methylation occurs in regions of high GC content.
How to choose
Choosing whether to perform WGBS or RRBS on your samples will often come down to weighing up costs with the required data amount. WGBS is the more costly option as it requires a high number of reads to achieve the required sequencing depth, and it requires high amounts of input DNA. However, it provides a genome wide methylation profile, which RRBS does not, and lends itself to the identification of epigenetic biomarkers and therapeutic targets for disease. RRBS requires lower initial DNA input and due to its targeted nature, requires fewer reads to achieve the necessary sequencing depth. This makes it a much more cost-effective method in comparison to WGBS. As mentioned, RRBS will miss certain sites of methylation if they occur outside the enriched areas, however the majority of methylation occurs withing regions of high GC content and in many cases, the information obtained by sequencing these regions is sufficient to answer the research question at hand.
Overall, the choice between WGBS or RRBS will depend on the desired outcome of the sequencing project and the information required from the analysis. Novogene’s expert sales and technical support teams consider each project on a case-by-case basis. If you have an upcoming project that will require analysis of DNA methylation, please get in touch with us at novogene.com and we will be more than happy to assist you with your decision and sequencing strategy.
Eager to learn more? Check out our previous post for more insights!
- What you can explore with non-coding RNA data
- Expanding Horizons in Genomic Research with Long-Read Sequencing
- WGS vs WES: Which Genetic Sequencing Method is Right for You?
- A Basic Guide to RNA-sequencing
- Uncovering the Genetic Basis of Rare and Complex Diseases through Whole Genome Sequencing (WGS)
- Long-read Sequencing Technology Explained
- In the Lab: A Closer Look at DNA Methylation Sequencing Techniques
- How to Choose Normalization Methods (TPM/RPKM/FPKM) for mRNA Expression