The Genomics Shared Resource provides DNA methylation services for both genome-wide as well as targeted studies. For genome-wide methylation analysis the GSR utilizes Infinium MethylationEPIC BeadChip as well as NGS based TruSeq DNA Methylation- whole genome bisulfite sequencing, and TruSeq Methyl Capture sequencing. The GSR utilizes the Agena Bioscinces’s* EpiTYPER platform for targeted methylation assays.
Illumina –Genome-wide methylation analysis using Illumina's Infinium MethylationEPIC BeadChip requires samples to be run in batches of 16. We require 1ug of high molecular weight DNA (via picogreen quantitation) per sample.
Illumina's MethylationEpic BeadChip contains >850K of highly informative CpG sites covering 99% of RefSeq genes, and 95% of all known CpG islands, along with enhancer sits and other content categories. The array encompasses CpG sites outside of CpG islands, Non-CpG methylated sites, differentially methylated sites identified in tumor versus normal, FANTOM 5 enhancers, ENCODE open chromatin and enhancers, miRNA promoters and DNase hypersensitive sites. The MethylationEpic array contains over 90% of the CpGs on the Illumina Methylatio450 plus an additional 350,000 CpGs in enhancer regions.
Whole Genome Bisulfite Sequencing- The TruSeq DNA Methylation Kit converts bisulfite-treated, ssDNA into an Illumina sequencing library. Only 50ng of DNA is required to create a TruSeq DNA Methylation Kit library. All ssDNA fragments are captured during the procedure, elminiating sample loss associated with other methods. WGBS-Seq covers all genes and > 38M CpGs in humans.
TruSeq Methyl Capture- The TruSeq Methyl Capture EPIC Library prep kit, which spans the full human methylome and contains over 3 million CpG sites, prepares targeted Methyl-Seq libraries for NGS. Targeted Methyl-Seq is a cost-effective method for large scale screening studies and can support both screening and biomarker discovery study objectives. As well as covering traditional epigenetic regions, TruSeq Methyl Capture EPIC offers higher coverage of emerging epigenetic regions of interest as well as siginicant coverage of CHG (H stands for A, T or C nucleotides) and CHH sites.
Agena Biosciences* (formerly Sequenom) -Targeted Methylation Analysis using Agena MassARRAY EpiTyper is best suited for assaying 10s - 100s of samples for 10s-100s of CpG islands. Orders consisting of < 10 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. Our service includes methylation primer and assay design through to the detection of methylation spectra.
- Plate: – As with all MassARRAY analyses, the SpectroChips 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: Each CpG region assayed requires primers for PCR amplification. If providing oligos, the primer stocks must be 100uM.
- Bisulfite treatment:1ug of bisulfite treated DNA will yield enough for 50 Agena methylation assays. We require 1ug DNA (dried down in 96-W plates) and a completed sample_plateMAP file using this template.
- Methylation Sequence -Simply submit a target methylation sequence(s) file and we will design and manufacture a methylation assay for you. It is recommended that you provide at least 1,500 bp of sequence and that the region of interest be indicated in the sequence file. Agena EpiTyper requires amplicons from 200-600bp, so some CpG regions may have to be assayed using several PCR primers.
- Project Design: All customers must be aware that not all CpG islands/genes submitted can produce successful targeted EpiTyper 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 methylation assays at this stage. Our goal is to achieve >85% success rate in methylation calls for a CpG site, with a minimum cut-off of >80%.
Core Grant Citation
*Sequenom was recently acquired by Agena Biosciences. This includes the MassArray system, which we use to run SNP genotyping (iplex) and DNA methylation assays. Agena Biosciences has stated that it plans to continue to support the MassArray System while working on new and exciting advances with this technology.