Our Options for the Analysis of c-methylation of DNA
In “MethylSeq” DNA is digested using the methylation-sensitive restriction enzyme HpaII (C’CGG) and a library for NGS is prepared such that the ends of the generated fragments (=MethylSeq-Tags) are sequenced. Differences in the C-methylation result in a distinct population of fragments and hence tags. The tags are counted and annotated to the genome, to generate a genome-wide high-resolution profile of the C-methylation pattern. Thanks to our “TrueQuant” Technique, PCR bias is eliminated.
Another variant of the approach is to digest bisulfite treated and untreated DNA using the restriction enzyme MspI (C’CGG). The bisulfite conversion, which generates Thymin from unmethylated Cytosin, will lead to a distinct fragment population and tags.
The DNA can as well be sequenced entirely (WGS) or after target enrichment before and after Bisulfite conversion. Our TrueQuant method allows to provide more accurate quantification results in this approach.
Methylation sepcific qPCR
For targeted analysis, we also offer methylation specific PCR. Briefly, DNA is digested using a methylation-sensitive or a dependent restriction enzyme. A subsequent PCR with primers whose annealing sites are complementary to the restriction sites, will only lead to an amplification product, if the sites are depending on their methylation status, not digested. A qPCR allows to quantify the methylation.
Binding-sites of DNA binding proteins such as transcription factors are analyzed after chromatin-immunoprecipitation (ChIP) of the proteins by sequencing the bound DNA. Our TrueQuant method eliminates PCR bias for highly accurate results.
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) uses a transprosase to integrate NGS-adapters into the DNA. As this happens preferentially at DNA sites that are nucleosome free and hence more accessible for the transposase, the genereated DNA fragments are preferentially from nucleosome free sites, which are usually also transcriptionally active. Deep sequencing of the PCR-amplified ATAC-Seq libraries reveals hence the portion of nucleosome free DNA.
Zawada AM, Schneider JS, Michel AI, Rogacev KS, Hummel B, Krezdorn N, Müller S, Rotter B, Winter P, Obeid R, Geisel J, Fliser D, Heine GH.DNA methylation profiling reveals differences in the 3 human monocyte subsets and identifies uremia to induce DNA methylation changes during differentiation. Epigenetics. 2016 Apr 2;11(4):259-72