The State of the Union Address and the drama/commentaries that accompany it have come come and gone, but a fresh Nature Review produced by USC’s Peter Laird now provides the epigenetics community with a State of Methylome Analysis. For the epigenetics researcher, it is every bit as insightful but won’t take as much time to digest, plus the only BS you’ll find in this review will be an abbreviation for Bisulfite Sequencing. Laird discusses all of the current methods for genome-wide DNA methylation analysis, bioinformatics, benefits and future directions. So if you’re new to the field and want to get a handle on the available options for analysis, or a seasoned vet that’s been plowing away with a certain method for years, you’ll want to take a look at this comprehensive look at the technology landscape and make sure you haven’t missed any developments.
At EpiGenie, we couldn’t help but notice a couple of emerging areas that could be key in the coming years for researchers looking at DNAm. A couple of points that caught our attention as important emerging trends in the DNAm space: The need for low amounts of input sample DNA, and coverage of low density CpG regions.
Low Sample Input
OK this one might be obvious as almost every researcher wants to use less sample, but working with minute samples isn’t just a convenience issue in epigenetics, it just might be one of the biggest obstacles in understanding epigenetics. Larger sample requirements force researchers to analyze mixtures of cell types that can mask the really interesting action in any given cell’s epigenome. As evidenced by the NIH Epigenomics Roadmap Initiative, developing sequencing-based methylation analysis methods with the ability to analyze small amounts of DNA is high on researchers’ wish lists.The ability to work with minute samples will provide researchers with the muscle to obtain a truer methylation profile within each population. Some methods highlighted in the review that do more with less include:
- Reduced Representation Bisulfite Sequencing (RRBS)– By removing unmethylated sequences, RRBS gets the job done with less sample. Just watch out for the classic bisulfite-based problems of incomplete conversion and PCR bias.
- Enzyme-Seq – Pretreatment with enzymes mean that you sequence only fragments with methylation, but keep in mind that fragment size bias could derail your analysis.
- Whole Genome Shotgun Bisulfite Sequencing (WGSBS) – This whole genome approach leads to very broad coverage, as long as your bisulfite reaction is dialed in.
Coverage of Low CpG Density Regions
Recent work as shown that DNAm is a player in regions other than just CpG Islands. Last year we highlighted several studies that point to Gene Bodies and CpG Shores as hot spots for interesting methylation activity. These areas can have lower CpG densities than the CG dense promoters, so choosing an approach that can deal effectively will be key. The review calls out a couple of techniques that let you look at low density CpG regions.
- WGSBS – Coverage of the entire genome means that you can look at any region you like for methylation. Just remember to keep your bisulfite reaction and PCR amplification steps in check to minimize headaches.
- Enzyme-Seq – This technique can be adapted to sequence low CpG density regions. However, you’ll need to choose your enzymes and fragment size carefully to avoid losing the material you’re looking to study.
Dr. Laird’s review also lays out several factors to keep in mind when setting up your own genome wide methylation study including: Genome coverage/resolution, Accuracy, Sensitivity, Allele Specificity and Bioinformatics, among others. So take a look at the full review in Nature Reviews Genetics, February 2010… Unlike Justice Alito, we think you’ll agree that it’s an informative and useful tool for anyone thinking about undertaking a genome wide methylation study.