To interpret DNA methylation status, the sequencing results should be compared with the original DNA sequence. When unmethylated cytosines (C) are present, they convert to thymine (T) and if a C-peak is observed it indicates the presence of 5-methylcytosine (5mC) in the genome. When both C- and T-peaks appear, it suggests partial methylation or potentially incomplete bisulfite conversion. The proportion of 5mC to C can be determined by analyzing the relative square area of these two peaks.
Using the restriction enzyme-based method is another way to interpret DNA methylation status. These methods rely on specific enzymes called methylation-specific restriction endonucleases (MSREs), such as HpaII, SmaI, MspI or NotI to selectively cut DNA. HpaII is frequently used because it can recognize and cleave the CCGG sequence only when it's not methylated. If a methyl group is added to the CpG dinucleotide (forming CmCGG), HpaII won't cut the DNA at that site. Additionally, a combination of HpaII and MspI, which have different sensitivities to methylation, can be utilized for more detailed analysis.
A third way to interpret DNA methylation status is through affinity enrichment-based methods. These methods involve using antibodies against 5-methylcytidine or methyl-CpG binding domain (MBD) proteins to isolate methylated DNA from the rest of the DNA. For instance, in the Methylated DNA Immunoprecipitation (MeDIP) assay, a specific antibody is utilized to recognize and capture DNA containing methylated cytosines. This method requires single-stranded DNA for antibody binding, and the methylated DNA is then recovered using protein G magnetic beads.