DNA Methylation Analysis Services
The DNA Methylation Analysis Facility is funded by the Center for Cancer Epigenetics and the Department of Epigenetics and Molecular Carcinogenesis. It provides services related to the analysis of DNA methylation for researchers across all MD Anderson campuses and in the Texas Medical Center. Services include: consultation with investigators to choose the best method of analysis; sample preparation, assay design, and sample processing for the most commonly used gene-specific and genome-wide (
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 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 and has been individually or combinatorially applied to individual genes and large-scale analyses. 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.
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 as a whole, one of two workflows is applied.
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.
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 more global questions. Once sample quality has been assured, it is either enzymatically digested or sonically sheared into appropriately sized pieces. Adaptors and then ligated onto the DNA ends before treatment with bisulfite. Following treatment, sequencing libraries are prepared and checked for quality before sending them to a next-generation sequencing facility.
The choice of an appropriate DNA methylation analysis method depends on whether the investigation is still in the discovery stage or whether a set of targets has already been decided. Further, special considerations regarding assay design and detection methods must be addressed when dealing with minimal amounts of DNA or when applying DNA methylation as a molecular marker. The DNA Methylation Analysis Facility will work with investigators to design assays and conduct experiments. To arrange a consultation, please contact Marcos Estécio, Ph.D., at email@example.com.
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 may consult with their institutionally assigned, departmental bioinformatician, seek advice on potential bioinformatics collaborators from the Epicore directors, or contact Arif Harmanci, Ph.D., of UT Health by email.
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 the 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%).
Similar to pyrosequencing, in bisulfite sequencing, gene-specific targets are amplified in a PCR reaction with primers that produce amplicons from both methylated and unmethylated alleles. Sequencing of individually cloned products by Sanger sequencing can reveal the methylation status of dozens of sites and identify allelic-specific methylation.
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, 400,000 to 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.
During whole genome bisulfite sequencing (WGBS) 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. 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.
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.
High-Throughput ChIP-Seq Services
Our workflow begins with a researcher-initiated request for consultation and service. Please always check with us for advice on preparing and submitting your samples. We are able to process both fixed, frozen cell pellets and flash-frozen tissues.
The success of large-scale profiling projects is critically dependent not only on carefully designed
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 may consult with their institutionally assigned, departmental bioinformatician or contact Arif Harmanci, Ph.D., (Arif.O.Harmanci@uth.tmc.edu) of UT Health for consultation (http://harmancilab.org/index.html).
Successful ChIP experiments require high-quality chromatin. As part of our services, we will prepare high-quality chromatin from frozen pellets of fixed cells or from flash-frozen tissues. Please contact us for sample preparation guidelines. Chromatin integrity is assured through quality control measures performed prior to proceeding to ChIP. Once the chromatin is prepared, we will perform ChIP using either our core-supplied, ChIP-validated antibodies, as described below, or investigator-supplied antibodies*.
• H3K4me3 and H3K27me3 (promoters)
• H3K4me1 and H3K27ac (enhancers)
• H3K36me3 and H3K79me2 (gene bodies/active transcription)
• H3K9me3 (heterochromatin)
* Investigator preferred antibodies must be provided by the customer and should have been previously validated for ChIP.