Methylation sequencing is the process in which DNA methylation is quantified using sodium bisulfite and subsequent nucleotide base conversion. DNA methylation is an epigenetic modification that alters gene expression, often as a repressor. In eukaryotes, the nucleotide cytosine is commonly methylated to form 5-methylcytosine (5mC), especially when accompanied by guanine (CpG). DNA methylation occurs in both animals and plants, playing significant roles in embryonic development, tissue regeneration, and pathogenesis.
To uncover the methylome, GENEWIZ Multiomics and Synthesis Solutions from Azenta Life Sciences uses wet and dry lab solutions, including Methyl-Seq, for global whole genome and targeted 5-methylcytosine (5mC) detection.
Methylation sequencing is the process in which DNA methylation is quantified using sodium bisulfite and subsequent nucleotide base conversion. Methylation sequencing is used to detect the DNA methylation status of both the starting DNA strand and the DNA methylation patterns of the resulting double-stranded DNA (post-amplification). In analyzing the results of methylation sequencing, a comprehensive overview of DNA methylation patterns across the genome is uncovered, aiding scientists in understanding the epigenetic mechanisms that regulate cellular development, disease, and responses to environmental factors.
Methylation sequencing works by first converting unmethylated cytosines into uracil using sodium bisulfite. Uracil is read as thymine post-PCR amplification and sequencing. Direct PCR sequencing or cloning sequencing is then used to measure the read counts of thymine (the converted bases).
Since methylated cytosine is not converted by sodium bisulfite, methylation sequencing distinguishes methylated cytosine residues from unmethylated cytosine/converted thymine. DNA methylation status can then be determined and quantified from the total read counts of the converted thymine from direct PCR sequencing or cloning sequencing. Methylation sequencing allows you to map and quantify the presence of methyl groups on DNA strands—a critical component of gene regulation and expression.
Submit cells, tissue, or gDNA for enzymatic conversion of unprotected cytosine
Add adapters to ssDNA fragments
Sequence 2x150 bp on Illumina® platform
Identify 5mC sites, calculate methylation ratios, and perform differential methylation analysis
After protecting modified cytosines from downstream deamination, an enzymatic step deaminates cytosine but does not affect 5caC and 5ghmC. The resulting converted sequence can be used to compare treated and untreated samples, creating a snapshot of 5mC methylation patterns across the whole genome or targeted regions of interest.
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