DNA Methylation Analysis

Next-generation sequencing is a powerful means to profile genome methylation status at single-nucleotide resolution. Historically, this has been accomplished using sodium bisulfite. Exposure of DNA to sodium bisulfite converts cytosine (C) – but not methylated cytosine (C*) – to uracil (U). Subsequent amplification of the DNA by PCR converts U to T. 
  
More recently, a two-step enzymatic process has been developed that achieves the same endpoint but avoids much of the DNA damage associated with sodium bisulfite conversion. Once such conversion has been accomplished by either method, the original methylation state – C vs C* – can be read out as the presence of a T vs C at a given CpG.

DNA Methylation Options

Biomodal 5hmc and 5mC sequencing: The Duet EvoC is a complete enzymatic workflow that yields sequencing libraries to accurately detect and distinguish 5mC from 5hmC while also providing the native sequence. Combining this native sequence data with the very high accuracy of the Element AVITI platform results in exceptionally accurate methylation profiles.

Enzymatic Methyl-seq (EM-seq): The enzymatic methyl-seq workflow developed at NEB provides an alternative to bisulfite sequencing. The NEBNext Enzymatic Methyl-seq Kit (EM-seq) combines NEBNext Ultra II reagents with two enzymatic steps to construct sequencing libraries that accurately detect 5mC and 5hmC within the genome. EM-seq libraries result in minimal DNA fragmentation and biases compared to WGBS.

Targeted EM-seq: Targeted EM-seq starts with the same two-step enzymatic conversion to generate indexed libraries in which the unmethylated cytosines have been converted to thymines. These converted libraries are then hybridized to a probe panel comprising all iterations of the methylome for your region of interest. This enzymatic conversion, coupled with probe capture, yields high-quality methylome data with relatively low sequencing cost. 

Whole Genome Bisulfite Sequencing (WGBS): WGBS combines sodium bisulfite conversion with high-throughput DNA sequencing to determine DNA methylation states at individual cytosines. Using this technique, unmethylated cytosines are converted into uracils and then into thymines after PCR.

Reduced Representation Bisulfite Sequencing (RRBS): RRBS is a bisulfite-based protocol that reduces the amount of sequencing required by focusing on a subset of the genome where the majority of the DNA methylation occurs. 

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Contact us at [email protected] for project support or questions. If your project has unique requirements, higher complexity, or needs optimization, our Custom Services can do non-routine protocols not offered through our Standard Services.