CBI MethylSeq Analysis

Detection of methylated cytosine in DNA requires sequencing of converted DNA, through either bisulfite-treatment or more recently enzymatic treatment. This converts unmethylated cytosines to uracil, which is interpreted as thymine in sequencing. Methylation occurs primarily at CpG contexts, and rarely at non-CpG contexts (CHG or CHH). DNA methylation is often associated with transcriptional repression, and changes in DNA methylation patterns occur during normal developmental processes. In cancer and other developmental diseases, these processes can be disrupted, resulting in altered methylation patterns. The Cancer Bioinformatics (CBI) team has experience in analyzing DNA Methyl-Seq experiments.

Experiment Setup

Enzymatic treatment of DNA for methylation sequencing is highly preferred over older bisulfite-treatment methods, as the latter is damaging to DNA resulting in very low alignment yields. Commercial kits are available for processing samples. For mammalian genomes, typically 200M read pairs are sufficient for median whole genome coverage of at least 10X. Multiple biological replicates (at least two, preferably three) should be sequenced for each condition when doing differential analysis.

Alignment

Methyl-Seq reads must be aligned to the genome using special-purpose aligners due to the C to T conversion. In our experience, the commercial Novocraft Novoalign software consistently provides the best alignment rates relative to other options available. CBI maintains a license for this software.

Analysis

CBI employs a number of software packages for analysis of Methyl-Seq and detection of differentially methylated regions, including but not limited to USeq, bsseq, MethylDackel, and WGBS Tools.

Identified regions of interest may be analyzed in similar manner to ChIPSeq in terms of annotation, etc.

Please contact us for assistance with analyzing your Methyl-Seq experiments.