Methylation sequencing is a sequencing approach that unravels epigenetic modifications in the DNA. There are two ways to treat the DNA to uncover these epigenetic modifications. But which one to choose? We want to show the differences between the methods and how they affect the outcome.
Before we look at the methods used to prepare the DNA, let’s have a look at the basics. Epigenetics is the study of how behavior, environment, development, or age, but also drugs and pharmaceuticals, can cause changes that affect how our genes work. Unlike genetic changes, epigenetic modifications are reversible. They do not change the DNA sequence but how it is read. One mechanism of epigenetic changes is DNA methylation: a methyl group is added to cytosine molecules of the DNA. These modifications can be investigated in a special NGS approach called methylation sequencing.
As already indicated, two ways exist to treat the DNA to uncover the methylation patterns. The two presented methods are shown in figure 1. Both methods are based on the idea that unmethylated cytosine bases are converted to uracil bases. However, they differ in the way in which this conversion is achieved. A well-known method is to treat the methylated DNA with bisulfite. In this workflow, unmethylated cytosines are converted to uracils by deamination. The methylated 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) do not react with bisulfite and remain unchanged. The second approach is based on enzymatic reactions. This approach is also called enzymatic methyl-sequencing (EM-seq). It comprises two reactions detecting 5mC and 5hmC. In the first reaction, the enzymes tet methylcytosine-dioxygenase 2 (TET2) and T4-phage β-glucosyltransferase (T4-BGT) convert 5mC and 5hmC into 5-(β- glucosyloxymethyl)cytosine (5gmC) that cannot be deaminated by apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A). In the second reaction, APOBEC3A deaminates the unmodified cytosines by converting them to uracils. After this conversion, sequencing libraries are prepared and sequenced. As shown in figure 1, the resulting sequenced DNA of both treatments contains thymine in the complementary position to uracil. For the evaluation and final identification of methylated cytosines, it is necessary to compare the treated sequences with the sequence of an untreated reference of the same sample.
Figure 1 | Comparison of bisulfite and enzymatic treatment for preparing methylation sequencing.
5m(C) = 5-methylcytosine; 5hm(C) = 5-hydroxymethylcytosines; 5gm(C) = 5-(beta-glucosyloxymethyl)cytosine.
The resulting sequenced DNA of both methods does principally not differ. Nevertheless, when choosing a method, the following should be considered: for complete conversion with bisulfite, relatively harsh conditions such as long incubation times, temperature changes, and high chemical concentrations are necessary. These conditions cause DNA damage, such as fragmentation. As a result, the size of the inserts and the library quality suffer. Consequently, amplification and mapping can be negatively affected. The enzymatic method is much gentler and has less influence on the final evaluation.
Although the method is more complex, CeGaT’s Methylation Sequencing product is based on enzymatic methyl-sequencing. This approach results in a higher library quality, less bias, and more trustworthy results. Your DNA sample is worth being treated gently.