The main obstacle to DNA methylation analysis is methylated cytosines are not differentiated by DNA polymerase during PCR amplification. The detection methylated cytosines depends on indirect methods, most commonly (1) restriction enzyme-based approaches, which take advantage of methylation-sensitive enzymes; (2) affinity-based approaches, in which either antibodies against 5-methylcytosine or against methyl-binding domain proteins are used to collect the methylated fraction of the genome, and (3) bisulfite conversion of non-methylated cytosines to thymidine through a hydrolytic deamination reaction, which takes advantage of the non-reactivity of methylated cytosines to free hydroxyl groups. Each one of these methods has an important application in studying the epigenome. Our EpiCore is equipped to apply each of these methods to your research.
DNA Methylation Analysis Workflow
Our workflow begins with a researcher-initiated request for consultation and service. Depending upon whether a project will investigate the methylation state of a particular gene or the genome, one of two workflows is applied:
(a) Candidate gene assays are designed and optimized as needed. DNA sample integrity and quality are assessed before undergoing bisulfite treatment. Treatment is followed by amplification of the specific regions of interest and pyrosequencing.
(b) For genome-wide studies, reduced representation bisulfite sequencing (RRBS) is applied to questions regarding promoters and CpG islands, whereas whole genome bisulfite sequencing (WGBS) is used to answer research question requiring mapping of all of the approximately 28 million CpG sites in the human genome.
(c) For samples while limited material, in general below 10ng of genomic DNA, we apply cfMeDIP-seq to investigate 5-methylcytosine or 5-hydroxymethylcytosine genome-wide in semi-quantitative fashion.
Workflows (b) and (c) are followed by next-generation sequencing facility to produce dozen to hundreds of millions reads, generally using an Illumina sequencer instrument. Researchers with projects that involve next-generation sequencing services should always consult a bioinformatician before planning the detailed project. For assistance with bioinformatics analyses, MD Anderson users should consult with their institutionally assigned, departmental bioinformatician, or seek advice on potential bioinformatics collaborators from the Epicore directors.
DNA Methylation Analysis Services
Genomic DNA is subject to bisulfite-conversion, and gene-specific targets are amplified in a PCR reaction with primers that produce amplicons from both the methylated and unmethylated alleles at the same time. Selected CpG sites are quantified in a pyrosequencing reaction. The advantages of the method are sensitive and quantitative analysis of methylated cytosines with medium to high throughput.
Figure 1 shows an example of PMA results. The region near the transcription start site of the gene HAND1 was amplified by bisulfite PCR and four CpG sites are included in the sequenced region (highlighted in yellow). In the example, the colon cancer cell line RKO showed high levels of DNA methylation at all examined CpG sites (average = 97%), and normal peripheral blood lymphocytes are unmethylated (6.3%).
Reduced Representation Bisulfite Sequencing (RRBS)
A combination of enzyme-based and bisulfite-based methods, RRBS is one the most popular genome-wide methods due to its ability to accommodate various starting amounts of genomic DNA. DNA is digested with a restriction enzyme and selected for size, promoting enrichment of CG-rich fragments and CpG islands. The DNA is then bisulfite-treated and amplified using barcoded adapters. The resulting RRBS libraries are checked for quality and then transferred to a next-generation sequencing facility for final processing. In general, over one million CpG sites are evaluated in a single experiment.
Figure 2 shows a genome browser view of RRBS data for the prostate cell lines RWPE-1 and Du145. Each vertical bar represents the methylation percent value of one CpG site, ranging from 0% to 100%. A differentially methylated region (DMR) is shown overlapping the CpG island present at the alternative transcriptional start site of the TP73 gene.
Circulating cell-free methylated DNA immunoprecipitation (cfMeDIP)
In this method, antibodies that specifically recognize 5mC or 5hmC modified bases in DNA are used to selectively capture genomic fragments carrying such modification and deplete non-modified fragments. The use of barcoding in library preparations allows for multiplexed sequencing of samples for next-generation sequencing. The methods are compatible with DNA amounts ranging from 5ng to 100ng, and possibly lower amounts can be used depending on the quality of the DNA. The method is suitable for the evaluation of circulating-cell free DNA collected from plasma or serum, and genomic DNA obtained from FFPE.
Figure 3 illustrates the specificity of anti-5hmC antibodies. When applied to sequences with unmodified cytosines and modified cytosines, only 5hmC-marked DNA is capture with over a thousand-fold enrichment. The Genome Browser view and correlation graphic highlight the reproducibility of cfMeDIP-seq to detected 5hmC when using different starting amounts of DNA (5ng and 100ng) and compared to publicly available data for the same cell line.
Whole Genome Bisulfite Sequencing (WGBS)
WGBS is applied to evaluate DNA methylation of the entire genome and covers approximately above 90% of all CpG sites in the human genome. In this method, genomic DNA IS fragmented by sonication to sequencing-compatible size, subjected to bisulfite-conversion, and amplified for library generation after adaptors ligation. The resulting WGBS libraries are then subjected to high-throughput sequencing.