New Service: ATAC-seq
The Epigenomics Profiling Core (EpiCore) now offers ATAC-seq (assay for transposase accessible chromatin with high-throughput sequencing) to aid analyses of chromatin accessibility, transcription factor binding, and nucleosome positioning across the genome. Contact Dr. Abhinav Jain for details. (email@example.com)
New Service: cfMeDIP
The Epigenomics Profiling Core is pleased to announce that it now offers cfMeDIP (circulating cell-free methylated DNA immunoprecipitation) as part of its DNA methylation analysis repertoire. Please contact Dr. Marcos Estecio to learn how this service can help advance your DNA methylation-related research projects. (firstname.lastname@example.org)
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. The core is located at the MD Anderson Cancer Center Basic Sciences Research Building. We are 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.
Our workflow begins with a researcher-initiated request for consultation and service. Depending on whether a project will investigate the methylation state of an individual gene or the genome one of two workflows is applied:
1) Candidate gene assays are designed and optimized as needed. DNA sample integrity and quality are assessed before bisulfite treatment. Treatment is followed by amplification of the specific region(s) of interest and pyrosequencing.
2) 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. For samples with limited material (under 10 ng of genomic DNA), we use cfMeDIP-seq to investigate genome-wide 5-methylcytosine or 5-hydroxymethylcytosine semi-quantitatively. Once sample quality has been assured, it is either enzymatically digested or sonically sheared into appropriately sized pieces. Adaptors are 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.
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%).
In bisulfite sequencing, as in pyrosequencing, gene-specific targets are amplified by PCR using primers that will 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, 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.
In this method, antibodies that specifically recognize 5-methylcytosine (5mC)- or 5-hydroxymethylcytosine (5hmC)-modified bases in DNA are used to selectively capture genomic fragments carrying these modifications 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 5 ng to 100 ng, and potentially even lower amounts, depending on DNA quality. This method is suitable for evaluating circulating-cell free DNA collected from plasma or serum, as well as genomic DNA obtained from formalin-fixed, paraffin-embedded (FFPE) samples.
The images on the left illustrate 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.
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.
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.