Assays measuring genome-wide DNA methylation.
The Epigenomics Core offers different assays measuring Genome-wide DNA Methylation. This sequence-based technology provides information on DNA methylation status across the genome at different levels of resolution:
- Whole-genome bisulfite sequencing (WGBS): whole-genome base-pair resolution quantification of DNA methylation following bisulfite conversion of unmodified cytosines (considered the gold standard).
- ERRBS (Enhanced Reduced Representation Bisulfite Sequencing): Quantitative base-pair measurement of cytosine methylation at ~2M CpGs (species dependent) genome-wide located at promoters, CpG islands and shores, and intergenic regulatory regions.
- Illumina MethylationEPIC BeadChip v2: we offer this service in partnership with the UM Advanced Genomics Core. We perform DNA quality control and bisulfite conversion on the samples, then bring them to AGC for hybridization and scanning. We also perform QC on the data generated before transfer back to the investigator.
- meDIP-Seq: Investigators may still request meDIP-seq (Antibody-based method for identification of mC-enriched areas across the genome). The core will purchase the kits required for the project.
- We offer NEB’s EM-Seq: this is a whole-genome base-pair resolution technique that uses an enzyme for cytosine deamination instead of bisulfite conversion. DNA must be of good quality and with no contaminants for best results. Contact Managing Director for more information.
- Twist Biosciences Human Methylome Panel: This is a Capture Panel targeting 3.98M CpG across the human genome. Up to 8 libraries can be pooled before Capture. Contact Managing Director for more information.
Targeted DNA methylation allows for the locus-specific detection of cytosine methylation by bisulfite-sequencing. The Targeted DNA Methylation services at the Epigenomics Core include:
- Primer design consultation
- Bisulfite conversion of DNA and PCR amplification of target sequences (Enzymatic deamination can also be used, depending on the project.)
- Amplicon Bisulfite-Sequencing: amplicons generated for each sample are converted in NGS libraires and sequenced on a MiSeq nano flow cell.
- Pyrosequencing (Note: this technology will be discontinued by the end of 2023.)
- Support with data analysis
5-Hydroxymethylcytosine (5hmC) is part of the DNA demethylation process and mediated by the TET enzymes. DNA demethylation has been associated with activation of gene expression. The Epigenomics Core is actively testing new assays to measure 5hmC. At this time, these are the assays that can be used:
- OxBS + BS: this is the only currently validated for 5hmC. Note that this assay can be performed for both NGS sequencing and for MethylationEPIC arrays. We use Tecan’s OxBS kit for either assays. Measurement of 5hmC with this assay requires 2 reactions that are performed side by side: reaction 1 is the usual bisulfite conversion protocol (measures 5mC + 5 hmC); Reaction 2 is performs oxidation of 5hmC in the presence of potassium perruthenate before bisulfite conversion (5mC only). Comparison of the readout of both reactions is needed to impute 5hmC levels, either after NGS sequencing of the libraries, or EPIC array hybridization.
- hmeDIP-seq: Antibody-based method for the identification of 5-hmC-enriched areas across the genome
- hmeDIP: Antibody-based method for the identification of 5-hmC enrichment at specific loci
Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) provides quantitative base-pair resolution of cytosine methylation status at approximately 3 million CpGs genome-wide, located at promoters, CpG islands and shores, and intergenic regulatory regions. This is accomplished through the use of a restriction enzyme that cuts within CCGG sites, followed by bisulfite conversion. The restriction digestion generates low-molecular-weight fragments amenable to NGS library preparation. During bisulfite conversion, unmethylated cytosines are converted to uracil and later as amplified as “T” in PCR, while 5mC and 5hmC are resistant to conversion and are amplified as “C”.
ERRBS Procedure
Genomic DNA (gDNA) is spiked with unmethylated Lambda phage DNA, which is a control to assess bisulfite conversion efficiency. DNA is first digested with MspI, then fragments are end-repaired, A-tailed, and ligated to a methylated Illumina-compatible adapter. Ligation reactions are purified with magnetic beads. Size-selection is performed on an agarose gel, and DNA libraries are bisulfite-converted. PCR is performed with dual-indexed primers to barcode the sample libraries and increase the amount of templates for sequencing. DNA libraries are purified with magnetic beads and QC’ed with the Qubit quantification system and Agilent TapeStation. Libraries are quantified via qPCR and pooled before submission to the Advanced Genomics Core for sequencing.
ERRBS Sample Guidelines
Because of the use of a restriction enzyme, this assay requires gDNA of high molecular weight. It also works best when starting with 50 ng of genomic DNA. Lower input is possible, but we have seen a higher rate of sample failure at lower starting inputs. If you have lower amounts, please contact the Epigenomics Core Managing Director. Typically, ERRBS libraries are sequenced with paired-end 50 cycle sequencing on the NovaSeq 6000, and we aim to obtain 60M to 80M reads per library.
Whole Genome Bisulfite Sequencing (WGBS) is a method for genome-wide profiling of cytosine methylation at single base-pair resolution. During bisulfite conversion, unmethylated cytosines are converted to uracil and later amplified as “T” in PCR, while 5-mC and 5-hmC are resistant to conversion and are amplified as “C”. An advantage of WGBS is that all cytosines in the genome are profiled, not only those in a CpG context. However, higher sequencing read depth is required to obtain equivalent coverage per methylation site, compared to ERRBS.
WGBS Procedure
Genomic DNA is spiked with unmethylated Lambda phage DNA, which is a control to assess bisulfite conversion efficiency. The DNA is then sheared into smaller fragments (~400 bp). Sheared DNA is end-repaired and A-tailed before ligation of methylated Illumina-compatible adapters. Ligation reactions are purified with magnetic beads, and DNA libraries are bisulfite-converted. PCR is performed with dual-indexed primers to barcode the sample libraries and increase the amount of templates for sequencing. DNA libraries are purified with magnetic beads and QC’ed with the Qubit quantification system and Agilent TapeStation. Libraries are quantified via qPCR and pooled before submission to the Advanced Genomics Core for sequencing.
WGBS Sample Guidelines
A minimum of 400 ng of genomic DNA is required for submission. Please indicate the concentration and quantification method on the sample submission sheet. Please contact the core if you have less DNA for submission. Typically, WGBS libraries are sequenced paired-end 100 on the NovaSeq 6000.
Methylated and hydroxymethylated DNA immunoprecipitation followed by massively parallel DNA sequencing (MeDIP-seq/hMeDIP-seq) are antibody based methods for mapping the location of 5mC or 5hmC marks across the genome. This technique can be used for qualitative analysis of DNA methylation profiles, with a resolution of 100-500 bp. It can detect 5mC or 5hmC coverage in both low density and highly repetitive regions with high specificity and minimal bias. An advantage over WGBS (whole genome bisulfite sequencing) is that it is cheaper and easier to analyze, as only small DNA regions pulled down in the IP require sequencing.
MeDIP-seq and hMeDIP-seq Procedure
Purified genomic DNA is sheared, end-repaired, and A-tailed prior to ligation of Illumina compatible adapters. Next the DNA is denatured and immunoprecipitated with specific antibodies against 5mC or 5hmC. Spike-in DNA controls corresponding to 5mC, 5hmC, and unmethylated cytosine are included with sample DNA. Antibody-DNA complexes are recovered with Protein G magnetic beads followed by proteinase K digestion to release the DNA. Samples undergo a qPCR test for enrichment at target loci prior to completion of library preparation. A PCR amplification step incorporates indexed primers to barcode the samples and increases the amount of template available for sequencing. Libraries are QC’d and quantified via qPCR prior to pooling and submission to the Advanced Genomics Core for next generation sequencing.
MeDIP-seq and hMeDIP-seq Sample Guidelines
The customer should provide at least 1 µg of genomic DNA per sample.
At least two biological replicates per experimental group are advised.
Sequencing is performed on the NextSeq 550 (single-end 75) or NovaSeq 6000 (paired-end 50) for MeDIP-seq and hMeDIP-seq libraries. We recommend a minimum of 50M reads per sample.
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