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The Epigenomics Profiling Core is funded, in part, through the Department of Epigenetics and Molecular Carcinogenesis and is open to researchers across MD Anderson and in the Texas Medical Center. The core is located at the MD Anderson Cancer Center Basic Sciences Research Building. The DNA Methylation Analysis arm of the core is a fully equipped  molecular biology laboratory with dedicated instrumentation including a Qiagen Pyrosequencing machine, an Illlumina MiSeq instrument, and an Oxford Nanopore Technologies GridION sequencer. 

The main obstacle to DNA methylation analysis is that methylated cytosines cannot be detected simply by sequencing. During PCR amplification, methylated cytosines are not differentiated by the DNA polymerase and like unmethylated cytosines, they are paired with guanosine dinucleotides. Thus, capturing methylated cytosines depends on indirect methods. The most commonly used methods are (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. Among these methods, bisulfite conversion is the gold-standard due to its high resolution when combined with sequencing methods. In this way, every single cytosine can be identified as methylated or unmethylated.

After bisulfite-conversion, gene-specific targets are amplified in a PCR reaction with primers that produce amplicons from both the methylated and unmethylated alleles. Selected CpG sites are quantified in a pyrosequencing reaction. The advantages of this method are sensitive and quantitative reading of methylation with medium to high throughput.

In the image to the right, a region near the transcription start site of the gene HAND1 was selected for assay design based on prior microarray experiments. The amplified region covered four CpG sites, which are 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%), but the same gene is unmethylated in normal peripheral blood lymphocytes (6.3%).
 

A combination of enzyme-based and bisulfite-based methods, RRBS is one the most popular genome-wide methods used today due to its ability to accommodate various starting amounts of DNA. DNA is digested with a restriction enzyme and selected for size. Post-adapter ligation ensures enrichment for CpG islands. The DNA is then bisulfite-treated and amplified with universal primers. The resulting RRBS libraries are checked for quality and then transferred to a next-generation sequencing facility for final processing. In general, between 400,000 and 1 million CpG sites are evaluated in a single experiment.

The image shows a genome browser view of RRBS data for the prostate cell lines RWPE-1 and Du145. Each vertical bar shows the methylation density 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.

WGBS is currently the only truly whole-genome approach for DNA methylation analysis of the entire human genome and covers approximately 90% of all CpG sites. In this method, the entire genome is fragmented by sonication, and modified adaptors are ligated to the DNA fragments prior to bisulfite-conversion. Then, as in RRBS, the amplified DNA is subjected to high-throughput sequencing.

The genome browser view on the right shows WGBS data compared to RRBS data for TP73 in the prostate cell lines RWEP-1 and Du145. Multiple differentially methylated regions are visible across the gene body.