Even if you don’t have a new flat screen HD TV hanging in your living room, most of us have at least seen the beauty that is HD when strolling through the TV section in the local Wal-Mart or Best Buy. The picture is so crystal-clear that watching a game in HD is just like being on the court or on the field with the players, providing a rare opportunity to look giants like LeBron James in the eye.
Now, a team of researchers has developed a new “HD” method for profiling CpG methylation called Bisulfite Capture. The high-resolution, genome-wide approach provides DNA methylation fans an opportunity to feel like a polymerase right there in the DNA strands, counting every methylation site in a genome. With a few tweaks, Bisulfite Capture could zero in on the methylation patterns in rare stem cell populations and track epigenetic reprogramming during differentiation.
Until this work, DNA methylation profiling was more of an “either-or” scenario where researchers had to choose whether to look at small regions in high-res or at whole genomes with less detail. (For background info, check out “Method Spotlight: DNA Methylation Analysis”.) High-res strategies will often combine bisulfite conversion with methods such as mass spectrometry, restriction analysis, or base-specific cleavage. But these combos are limited in scale and are costly—like paying a week’s salary to see the bumps on the basketball, but not being able to see any of the players in a game. Some researchers try to get around that by using bisulfite sequencing, but cost is still an issue, and it’s really only practical for smaller, non-mammalian genomes. So, maybe you can see the game clearly, but only the first quarter.
With genome-wide techniques, scientists usually separate out methylated DNA with methylation-responsive enzymes or with antibodies or protein complexes that specifically bind this type of DNA. The enriched material can then be arrayed or sequenced easily, but these upstream methods don’t always provide the precise mapping needed for some types of studies. Here, it’s like seeing the whole game on a full screen, but the picture’s blurry.
A Genome of Mammalian CpGs Comes into Focus
For many researchers, these technical concessions make the overpriced beer and nachos at a game look trivial. To see the whole game (or genome) clearly, researchers at the Cold Spring Harbor Laboratory, the University of Southern California, Albert Einstein College of Medicine, and Agilent teamed up and developed an effective methylation profiling trifecta starring bisulfite conversion, target enrichment, and deep sequencing.The researchers first bisulfite converted their samples, and then used Agilent’s new SureSelect™ DNA Capture Array to hook their regions of interest. The array-based SureSelect™ DNA Capture Arrays can be custom designed using Agilent’s eArray interface, enabling researchers to design capture probes to any of the four bisulfite converted strands that are created from the methylated and non-methylated fractions. This provides researchers an option to either increase confidence by capturing multiple strands or even investigating mechanisms like asymmetrical methylation. After a few more rounds of PCR amplification, the eluted DNA fragments were ready for deep sequencing. The upshot? You guessed it—coverage of CpGs in larger mammalian genomes with single-nucleotide resolution.
James Hicks, a corresponding author on the paper, says, “I think the big advantages are in assaying ALL bisulfite sensitive C’s independent of prior knowledge of sequence or methylation status (not requiring a known CpG sequence, for example) and the flexibility of chip design for assaying different genomic regions at will.” He adds that, with bisulfite capture, they could assay just as many if not more regions than other methods out there.
When the researchers used the method on a primary skin cell line and a breast cancer cell line, they found complex methylation profiles that would have been missed otherwise. For example, partial methylation patterns and methylation occurring in small, contiguous blocks were observed in some CpG islands. The blocks of methylation, or “punctuation marks”, may have biological functions, say the scientists. In addition, methylated CpG sites and H3K36me3 (found in active genes) were both enriched in exons.
Also, if you want to learn more about how to use arrays for applications like Bisulfite Capture, be sure to check out the SureSelect™ DNA Capture Array over at Agilent.