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Understanding DNA Methylation Sequencing: Methods, Applications, and Targeted Approaches

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DNA methylation is an epigenetic modification that involves the addition of a methyl group (-CH3) to DNA molecules. This process usually occurs throughout specific regions of the DNA known as CpG sites, where cytosine (C) is followed by a guanine (G) in the DNA sequence. CpG sites are often clustered in regions called CpG islands, which are found in the regulatory regions of many genes.

DNA methylation plays a crucial role in gene expression regulation and is involved in various cellular processes, including development, differentiation, and diseases. Methylation of DNA can influence gene activity by affecting how genes are transcribed and interpreted by the cellular machinery. When a gene is heavily methylated, it typically leads to the silencing of that gene, as the methyl groups can prevent the binding of transcription factors and other proteins required for gene activation. On the other hand, when a gene is unmethylated or has low levels of methylation, it is more likely to be actively transcribed and expressed. DNA methylation is essential for normal cellular function and development. Dysregulation of this process is linked to various diseases, such as cancer, metabolic disorders, autoimmune diseases and neurological disorders. Researchers continue to study DNA methylation to better understand its role in pathogenesis and to explore potential therapeutic applications.

CpG island & DNA methylation landscape

Figure 1. CpG island & DNA methylation landscape.

When it comes to methylation researches, bisulfite sequencing is the most widely used molecular biology technique that allows researchers to study DNA methylation patterns at single-base resolution. The technique is based on the chemical conversion of unmethylated cytosines (C) to uracils (U), while methylated cytosines remain unchanged(CT conversion). However, bisulfite sequencing has a drawback of causing significant DNA loss during the chemical treatment process. Recently, an alternative method using enzymes(TET2 and APOBEC) instead of bisulfite has been introduced, which also converts unmethylated cytosines to uracils. This enzymatic method offers the advantage of minimizing the loss of DNA as the reaction conditions are less aggressive. Additionally, it allows the same approach as bisulfite sequencing for data analysis.

Overview of methylation sequencing methods

Figure 2. Overview of methylation sequencing methods.




  Targeted sequencing can be a useful approach in methylation sequencing, as it allows researchers to concentrate their efforts and resources on specific genomic regions of interest. Targeted sequencing requires much fewer sequencing reads compared to whole-genome sequencing, making it a cost-effective option while still providing high-resolution methylation data for the regions of interest. Also, by allocating more sequencing reads to a specific set of target regions, targeted sequencing can achieve a higher coverage depth compared to whole genome sequencing (WGS), increasing confidence in the methylation status determination for those regions.



Celemics targeted methylation panel design

Figure 3. Celemics targeted methylation panel design.



 For targeted methylation sequencing, due to the sequence changes caused by bisulfite or enzyme treatment, it requires a unique panel design approach compared to conventional capture panel. If the panel is designed only based on the original reference genome sequence, there will be mismatches at every unmethylated cytosine positions, significantly reducing the panel’s capturing efficiency or causing distortion in the methylation ratio. To address this issue, Celemics has developed a novel panel design approach that incorporates sequence changes arising from methylation into the probes, thereby improving performance. The graph below illustrates the application of Celemics methylation panel on actual samples, demonstrating its ability to distinguish methylation patterns based on the tissue of origin of each sample.Example of targeted methyl-seq data analysis results. This targeted methyl-seq approach can be applied to various sample types, such as fresh tissue, FFPE, cfDNA, etc.


CELEMICS_Example of targeted methyl-seq data anlaysis results_img1730x1818

Figure 4. Example of targeted methyl-seq data anlaysis results.


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