By Monica Heger
Researchers at the University of Chicago have combined a chemical labeling method for 5-hydroxymethylcytosine with sequencing on the Pacific Biosciences RS, allowing for single-base resolution of the epigenetic marker, which is involved in cell development and may play a role in some human disease.
The chemical labeling method is similar to a method published last year in Nature Biotechnology by Chuan He and collaborators at the University of Chicago, which uses beta glucosyltransferase to modify the hydroxymethylated cytosine with glucose. In that paper, the team applied the technique to Illumina sequencing.
Now, in conjunction with PacBio, the team has incorporated the tagging method with sequencing on the PacBio RS for single-molecule hydroxymethylation sequencing. They published their results last month in Nature Methods.
The University of Chicago has also licensed the chemical labeling technology to Active Motif, which has developed a kit called Hydroxymethyl Collector.
In developing the method on the PacBio, the researchers took advantage of the polymerase kinetics in the company's sequencing technology. As the polymerase incorporates nucleotides, if a base is modified, such as with a methyl or a hydroxymethyl group, there is a detectable pause, allowing the modifications to be identified.
To put the method to work, the team first uses beta-glucosyltransferase to add glucose to the hydroxymethylated region, similar to its original protocol. The next step is to add a cleavable biotin-containing capture agent that they developed to be compatible with PacBio sequencing, which uses biotin to immobilize the DNA polymerase. The glucose- and biotin-labeled hydroxymethylated DNA fragments are then captured with streptavidin beads.
The researchers found that their modifications increased the pauses in the polymerase incorporation to a detectable level. "The stop is 10 to 20 times bigger than for a normal base," He said. "That allows you to conclusively say, 'Here's the hydroxymethylated cytosine.'"
The researchers tested the method on synthetic DNA fragments with known 5-hydroxymethyl cytosines and on biological samples from mouse embryonic stem cells.
They found that the method was specific to 5-hydroxymethylated cytosine, as it did not capture any DNA that contained 5-methyl cytosine. Additionally, because the PacBio sequences one strand of DNA at a time, it is strand specific.
The method "takes advantage of the real-time aspect of the PacBio technology, which allows you to observe the DNA polymerase in real time," said Jonas Korlach, a scientific fellow at PacBio.
He's lab at the University of Chicago is applying the method to sequence hydroxymethylation across the entire mouse embryonic stem cell genome. The modification is thought to play a role in development, and hydroxymethylation has been found to increase four- to five-fold as a mouse matures. Additionally, hyrdoxymethylation appears to be enriched in genes involved with neurodegenerative diseases, hypoxia, and angiogenesis, said He.
It's unclear how hydroxymethylation is involved in these diseases, but "there's clearly a link," he said.
While a number of researchers use bisulfite sequencing to detect methylation, a modification that is also involved in disease, much less is known about hydroxymethylation because it has so far been difficult to sequence, said He.
Eventually, hydroxymethylation sequencing and methylation sequencing could be combined so that one run on the PacBio would detect both epigenetic markers, said He, but that will first require some optimization.
PacBio is currently developing a direct methylation sequencing protocol that takes advantage of the pause in polymerase incorporation to detect methyl groups. The company published a proof-of-principle paper on the method last year in Nature Methods (IS 5/11/2010).
In theory, the same method can be used to detect hydroxymethylation, but the pause elicited from a hydroxymethyl group is more subtle, making detection tricky.
Currently, in order to detect methylation or hydroxymethylation using PacBio sequencing, the company recommends 25-fold coverage to detect 4-methylcytosine and 250-fold coverage for 5-methylcytosine and 5-hydroxymethylcytosine. The Hydroxymethyl Collector kit, however, can reduce coverage to five-fold for 5-hydroxymethyl cytosine.
Because PacBio's throughput is around 90 megabases per SMRT cell, the company currently recommends its standard methylation and hydroxymethylation techniques only for small genomes such as bacteria, or isolated DNA regions such as the mitochondrial genome.
Korlach noted, however, that the targeted pulldown chemical labeling technique for hydroxymethylation will make it possible to do larger genomes.
"We're working with customers and internally, and with Active Motif to validate this kit and get some real-world applications and biological samples," he said.
Additionally, as the PacBio's throughput increases, direct methylation sequencing and hydroxymethylation sequencing will be available on whole human genomes, said Korlach, though he did not provide a timeline for when the company expects that to be possible.
In theory, the chemical labeling technique will be applicable to any sequencing platform, said He, adding that he has been working with Illumina on developing a protocol for the company's technology. He expects the protocol will be available next year.
Additionally, He said he is also working to developing a similar labeling technique for methylation sequencing. Applied to PacBio sequencing, it would dramatically increase the polymerase pause, which should reduce the fold coverage needed to detect methylation.
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