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TAB-Seq Method Enables Direct Measurement at Base Resolution of Hydroxymethyl Cytosine

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While bisulfite sequencing is a proven technique for studying epigenetic changes, it cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine. Now, researchers from the University of Chicago and elsewhere have developed a genome-wide technique for quantifying 5-hmc sites at base resolution.

Chuan He, director of the Institute for Biophysical Dynamics at the University of Chicago and senior author of the study, which was published in Cell last week, told In Sequence that the group plans to commercialize a kit based on the method.

The kit, which will be marketed by a small reagent company Wise Gene, will be available in a couple of weeks, he said.

The method builds on previous work by the same group, which last year developed a single-molecule version of the technique that is compatible with the Pacific Biosciences RS (IS 12/6/2011).

This new method is designed to be compatible with any sequencing platform, said He, and in the current study the team demonstrated a genome-wide approach using Illumina HiSeq as well as targeted approaches with Sanger sequencing and some validation with the Ion Torrent PGM.

Tet-assisted bisulfite sequencing, or TAB-seq as the method is called, first tags hydroxymethylcytosine using beta glucosyltransferase, which protects 5-hmc. Then Tet proteins oxidize 5-mc into 5-carboxyl cytosine. The 5-hmc sites are protected from oxidation by the glucose molecule.

The DNA is then bisulfite-treated and sequenced. During bisulfite treatment, the methylated cytosines, which have been converted to carboxyl cytosines, behave like regular cytosines and are converted to uracil, while the hydroxymethyl cytosines remain protected.

After sequencing, the hydroxymethylated cytosines are read out as cytosines, and the cytosines without epigenetic modifications as well as the methylated cytosines are read out as thymines.

TAB-seq gives not only base resolution, but also the relative abundance of 5-hmc at each position, He said

At each modified position, one or both cytosine copies can be methylated or hydroxymethylated, explained He. This approach can not only tell the difference between the two modifications, but at each base, it can also tell whether only one allele is modified or if both are modified, and which modification exists at each allele.

Combining the TAB-seq approach with traditional bisulfite sequencing will yield an even more comprehensive picture of both 5-hmc and 5mc. Since bisulfite sequencing encompasses both hydroxymethylcytosine and methylcytosine, subtracting the TAB-seq results from bisulfite sequencing will yield methylation.

The TAB-seq approach is similar to the oxBS-seq technique developed by Shankar Balasubramanian's group at the University of Cambridge that was recently published in Science (IS 5/1/2012), except that the oxBS-seq approach directly measures methylation, while TAB-seq directly measures hydroxymethylation, explained He. Both methods enable the other epigenetic modification to be inferred by comparing to bisulfite sequencing.

He's team tested the method on specific loci using Sanger sequencing, and also the whole genomes of both mouse and human embryonic stem cells using the Illumina HiSeq 2000 and generated genome-wide hydroxymethylation maps.

When the researchers applied the approach to stem cells, they found that hydroxymethylation tended to concentrate in regulatory regions.

While the marker is "almost everywhere" in the genome, said He, "if you look at the sites where hydroxymethylation is most abundant, it's mostly associated with functional elements," like enhancers, transcriptional binding sites, and regulatory elements.

This indicates that there's a "dynamic demethylation going on with these functional elements that regulate gene expression," He said.

Ping Jin, a coauthor of the study at Emory University, told IS that his lab is using the method to study hydroxymethylation's role in neuronal development.

Previous studies have shown that 5-hmc is abundant during neuronal maturation, he said. "Now, we can really look at each individual base in terms of the modification as well as the dynamics during development," he said.

While the functional impact of 5-hmc is still not well understood, Jin said that from this Cell study and other studies, it seems to be involved in gene expression, and that it may "prepare the stem cells to be ready to respond to external stimuli."

While the TAB-seq method offers a number of advances, there are still a few limitations. For one, said He, a significant amount of starting material is required. Currently, it requires two to four micrograms of DNA, but ideally, it would require nanograms, or eventually "even a few cells," said He.

He said he is working on methods to reduce the input requirements, including moving away from bisulfite sequencing altogether.

Another option for reducing input requirements could be to combine the method with a low-input whole-bisulfite sequencing protocol, such as the one developed by Jay Shendure's lab at the University of Washington (IS 4/10/2012). His group uses a transposase tagmentation method to create the sequencing library, and recently demonstrated that it can create a sequencing library with just 10 nanograms of DNA.

Andrew Adey, a graduate student in Shendure's lab and coauthor of the Genome Research paper, which describes the transposase tagmentation protocol, told IS that the two methods could potentially be combined for a low input, 5-hmc sequencing method.

Another limitation of TAB-seq, Adey added, is that it simply "requires a lot of sequencing to do on a large scale," which can become expensive.

However, he said, sequencing costs are continuing to come down, and the TAB-seq method provides an important means of distinguishing between 5-hmc and 5mc. "It's another piece of the puzzle, as far as understanding the epigenetic landscape," he said.

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