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DNA Methylation Service
Overview
Understanding the complex role and patterns of DNA methylation is a very active field of research around the world. DNA methylation is one of several modifications that normal DNA goes through after each replication. In vertebrates, DNA methylation typically occurs at CpG sites, where a cytosine is directly followed by a guanine in the DNA sequence. This methylation results in the conversion of the cytosine to 5-methylcytosine. CpG sites are often found at higher density near gene promoters where they are collectively referred to as CpG islands. The methylation state of these CpG sites can have a major impact on gene activity/expression. Neoplasia is often characterized by frequent abnormal increases or decreases in DNA methylation. If the genes affected by abnormal methylation happen to be involved in regulating cell proliferation, uncontrolled cell division can occur. In addition, the overall methylation state in a cell might also lead to chromosome instability and increased mutation rates, resulting in cancer.
Introduction
The Genotyping Division of the RPCI Genomics Shared Resource provides DNA methylation services and utilizes the Sequenom MassARRAY Compact for targeted methylation assays and the Illumina Infinium platform for genome-wide high-performance methylation analysis.
Sequenom's MassARRAY EpiTyper platform utilizes mass spectrometry (MS) for the detection and quantitative analysis of DNA methylation using Homogeneous MassCLEAVE (hMC) base-specific cleavage and matrix-assisted laser desorption/ionization time-of-flight MS. The MassARRAY system is ideal for detection of methylation, for the discrimination between methylated and non-methylated samples, and for the identification of differentially methylated sites through quantitative analysis of methylation. Base-specific cleavage of 1ug bisulfite-treated DNA results in clear differences between the mass signal patterns of methylated and non-methylated DNA. Semi-quantitative methylation analysis is performed by comparing signals (the area under the peak) representing methylated and non-methylated DNA. Depending on the sequence content, up to 85% of the CpGs within a target region are represented in the cleavage reaction. The mass spectrometer is configured to accept two, miniaturized 384-pad SpectroCHIPs that can be sequentially analyzed in a single, automated run. Methylation calls are performed by the EpiTyper software v1.0 (Sequenom) and written to an Oracle 8i database to:
All applications are supported by SEQUENOM’s available hardware, software and proprietary SpectroCHIP consumables, which automatically generates analysis data and assigns methylation levels to CpG units. The Genotyping facility employs state of the art equipment including Qiagen, Matrix and Tecan robotic pipetting devices, 4 x 384 well PTC-225 thermalcycling machines, a Samsung chip spotting device, and the MassARRAY compact system for determining the absolute mass of short DNA sequences. This high-tech equipment forms the core of the genotyping facility and enables throughputs in excess of 10,000 reactions per day.
Our service includes primer and assay design through to the detection of methylation spectra. All steps involved are highly automated and are tracked using a laboratory management system. In this method, CpG island PCR followed by IVT and RNase cleavage is performed in a single well. The size of the cleavage products are determined directly by the MassARRAY, yielding relative methylation measurements. Currently, the system permits the methylation status of up to 192 different CpG islands per sample in a single 384 well plate.
Illumina's Human methylation27 BeadChip uses the Infinium assay to interrogate >27,000 CpG sites per sample as single-nucleotide resolution. The 12-sample BeadChip is supplemented with > 1,000 cancer related genes and promoter regions of 110 miRNA genes. After bisulfite conversion of 500ng genomic DNA, each sample is whole-genome amplified (WGA) and enzymatically fragmented. The bisulfite-converted WGA-DNA samples are purified and applied to the BeadChips. During hybridization, the WGA-DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. The two bead types correspond to each CpG locus—one to the methylated (C) and the other to the unmethylated (T) state. Allele-specific primer annealing is followed by single-base extension using DNP- and Biotin-labeled ddNTPs. Both bead types for the same CpG locus will incorporate the same type of labeled nucleotide, determined by the base preceding the interrogated "C" in the CpG locus, and therefore will be detected in the same color channel. After extension, the array is fluorescently stained, scanned, and the intensities of the unmethylated and methylated bead types measured. DNA methylation values, described as beta values, are recorded for each locus in each sample via BeadStudio software. DNA methylation beta values are continuous variables between 0 and 1, representing the ratio of the intensity of the methylated bead type to the combined locus intensity.
Project Design
Because of the unique characteristics of any methylation project and the high quality we aim for, we meet with each investigator who wishes to use our service. Sequenom Primer and assay design will be carried out and a QC methylation run performed. A second meeting will occur after this QC run to discuss results. Depending on those results, we will either proceed with full-scale methylation analysis or consider alternative methods as necessary. Methylation results are provided to the user in an electronic format as an excel document (methylation ratios) and image (epigram).
Methylation Failures for Targeted Methylation - Sequenom
All customers must be aware that not all CpG islands/genes submitted can produce successful targeted assays. At the PCR design stage, there is a drop-out rate of 10-15% of sequences which fail assay design completely. This can be due to the sequence surrounding the CpG site of interest containing repeats or CG rich regions. If it is possible to design an assay, there is then a further chance of the assay not generating good quality methylation data due to amplicon size, quality of bisulfite treated DNA and restrictive PCR conditions. In the past, we have seen up to a 10% further loss of methlaytion assays at this stage.
Our goal is to achieve >85% success rate in methlylation calls for a CpG site, with a minimum cut-off of >80%. When lower success rates are achieved, it is the user’s choice as to whether to have the data.
Methylation Analysis
Illumina - Global Methlylation Analysis using Illumina's Human methylation27 BeadChip requires 12 samples to fill a slide. We require 1ug of high molecular weight DNA (via picogreen quantitation) per sample. Contact us for details and pricing.
Sequenom - Targeted Methlylation Analysis using Sequenom Mass Spectrometry is best suited for assaying 30s - 100s of samples for 10s-100s of CpG islands. Orders consisting of < 20 samples and < 5 CpG islands are more expensive per assay than larger projects because smaller sample sets do not fill all positions of the 384 pad, single use SpectroChip. We require 1ug DNA (dried down in 96-W plates) and 1500-2000 bp of sequence (as indicated). Contact us for details.
The price for Sequenom methylation analysis depends on the number of DNA samples and regions assayed. A rough breakdown is described below:
Plate cost: ($1,100 per 384 samples) – As with all MassARRAY analyses, the SpectoChips hold 384 samples and are single use. We recommend that you maximize the number of methylation reactions to reduce overall costs. Each DNA sample requires a T-reverse reaction, so 380 samples assayed for one CpG island will require one chip. Other combinations to fill one chip: 96 samples x 4 CpG regions, 48 samples x 8 CpG regions, 24 samples x 16 CpG regions, etc
Oligo and set up fee: ($50 per set) – Each CpG region assayed requires primers for PCR amplification. If providing oligos, the primer stocks must be 100uM.
Bisulfite treatment ($4.25 per sample). 1ug of bisulfite treated DNA will yield enough for 50 Sequenom or 4 Illumina methylation assays. You can certainly provide the BIS DNA for the Sequenom analysis, but it must be freshly prepared and stored frozen. Bisulfite treatment for Illumina must be performed by the GSR.
These fees are RPCI CCSG investigator rates, non member and outside users please inquire about external pricing.
To use the Methylation service, we require that you electronically submit DNA samples (96-W plate format) and CpG island sequence (with identifier). After acceptance, we require a completed order form and 1ug DNA. DNA must be eluted in water as TE will interfere with the genotyping assay.
