In the field of DNA methylation, a diverse array of methods, each with its unique strengths and limitations are available. The working principles of each of the sequencing techniques are quickly discussed below.
1 WGBS (Whole Genome Bisulfite Sequencing)
WGBS involves treating DNA with bisulfite, transforming unmethylated cytosines (C) to Uracils (U). Subsequent PCR converts U to adenine (A), preserving only the methylated C. Following amplification, sequencing unveils genomic sites with methylation.
- Coverage: Offers a genome-wide methylation profile, allowing researchers to explore methylation patterns across all genomic regions.
- Applications: Valuable for fundamental research and clinical studies. Identifies epigenetic biomarkers and therapeutic targets for diseases.
- Cost: More expensive due to high sequencing depth requirements.
- Key Consideration: Researchers need to be aware of biases introduced during bisulfite conversion and PCR amplification.
2 RRBS (Reduced Representation Bisulfite Sequencing)
RRBS utilizes the restriction endonuclease Mspl enzyme to selectively cut the genome. After bisulfite treatment, sequencing in the specific regions such as gene promoters and CpG islands.
- Coverage: Does not provide a genome-wide view; selectively captures regions with high CpG density.
- Applications: Useful for targeted studies, such as identifying differentially methylated regions.
- Cost: Generally more cost-effective.
- Key Consideration: Choose RRBS when specific genomic regions are of interest.
3 MethylationEPIC v2.0 BeadChip (935K Chip)
The 935K Chip is a biochip adorned with millions of DNA probes, each matched to a CpG site in the genome. This chip acts like a molecular detective, spotlighting over 935,000 crucial CpG sites in regions like enhancers and promoters, revealing where DNA is methylated.
- Method: Microarray-based.
- Coverage: High-throughput profiling but may miss certain CpG sites.
- Applications: Enables large-scale screening of methylation patterns.
- Considerations: Limited to predefined CpG sites on the chip.
4 Enzymatic Methyl-sequencing (EM-seq)
EM-Seq transforms 5-mC and 5-hmC into 5-caC and 5-gmC using TET 2 and oxidation enhancers. APOBEC deaminase then converts unmethylated cytosines to Uracils (U). By studying these changes, methylated regions can be discerned.
- Method: Targeted approach capturing specific genomic regions with enzymatic precision.
- Applications: Provides focused insights into methylation patterns.
- Advantages: Precise and efficient.
5 Single-molecule real-time sequencing (SMRT-seq) and Nanopore-seq
SMRT-seq captures the essence of DNA sequencing by associating each base with a fluorescently labeled nucleotide during synthesis. This method provides a real-time picture of DNA synthesis, providing a dynamic understanding of the DNA sequence.
Nanopore-seq utilizes nanoscale pores through which DNA passes, generating distinct electrical signals. As DNA molecules pass through the nanopores, different bases generate distinct electrical current signals, and then the sequencer determines the DNA base sequence.
- Method: New technologies providing long-read capabilities.
- Advantages: Long reads but may exhibit higher error rates.
- Considerations: Evaluate trade-offs between read length and accuracy.
A comparative overview of features of the DNA methylation technique is given in Table 1.
Remember that the choice of a DNA methylation sequencing method depends on your specific research objectives, available resources, and the balance between coverage, resolution, and accuracy. Each method has its strengths and limitations, so consider these factors carefully when designing your experiments.
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