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New Bisulfite Sequencing Method Uses Next-Gen to Lower Cost, Increase Throughput

As the National Institutes of Health requests its first applications for grants under the new NIH Roadmap Epigenomics program (see Short Reads in this issue), researchers are developing new methods for analyzing epigenomic modifications, such as DNA methylation, on a genome-wide scale.
One of them is Alex Meissner, a postdoctoral fellow at the Whitehead Institute for Biomedical Research. Working with the Broad Institute, he and his colleagues have developed a new method for analyzing DNA methylation that involves next-generation sequencing.
The method, which has not yet been published but has been presented at conferences, promises to reduce the cost and increase the throughput of bisulfite sequencing while delivering the method’s high resolution.
“We are trying to scale up the classic bisulfite sequencing, which is still considered to be the gold standard for methylation analysis,” Meissner told In Sequence last week.
Sodium bisulfite converts unmethylated cytosine to uracil while leaving methylated cytosines unchanged. By comparing sequence data from treated and untreated DNA, researchers can analyze which C’s were originally methylated.
However, traditional bisulfite sequencing, which uses Sanger sequencing, is also the most expensive methylation analysis method, “basically preventing it from being used at a genome-wide scale,” Meissner said.
Most of that cost is related to sequencing, he said. “Some of the new high-throughput sequencing technologies, such as Solexa sequencing, now have reduced these costs at least 10- to 100-fold and increased the throughput a lot so you can easily just scale up the sequencing with that,” Meissner said.
Epigenetics researchers would like to study DNA methylation on a genome scale because of its role in developmental biology and in cancer. Other methods to analyze methylation on a genome-wide scale exist, such as chromatin immunoprecipitation coupled with microarrays, also called ChIP-chip. But unlike bisulfite sequencing, they do not provide nucleotide resolution, Meissner explained.
However, it is not clear if that kind of resolution is always required. “For some parts of the genome, you may not need nucleotide resolution,” he said. “But it can’t hurt. The question is if it’s cost-effective enough, and that part, we still have to see.”
‘Too Complicated, Too Big, Too Expensive’
To reduce the cost of bisulfite sequencing, Meissner and his colleagues used  Illumina Genome Analyzers at the Broad to analyze bisulfite-treated DNA taken from mouse embryonic stem cells. They chose Illumina’s instrument because it provided the highest throughput of the platforms available, Meissner said. He added that the method could work with other platforms as well. “Whichever one is more cost-effective will be ultimately the one [we use],” he said.

“Next-gen sequencing is having a powerful impact on the development of new techniques for DNA methylation analysis.”

While the ultimate goal is to sequence the entire genome in this manner, “we can’t do [that] at the moment,” Meissner said. “It’s too complicated, it’s too big, and too expensive.”
Instead, the scientists used a restriction-enzyme-based method to reduce the representation of the genome, an approach they published in 2005 in Nucleic Acids Research.
In that published study, they analyzed DNA fragments ranging in length from 500 to 600 base pairs, which comprised approximately 0.5 percent of the genome.
Other researchers have started using other next-generation sequencing platforms for large-scale DNA methylation studies. John Edwards, an associate research scientist at the Columbia University Genome Center, for example, has used Applied Biosystems’ SOLiD sequencer to analyze methylated and unmethylated fractions of the genome, generated by methylation-sensitive and methylated-dependent restriction enzymes (see In Sequence 10/16/2007).
Also, researchers at the University of Missouri-Columbia have used 454 sequencing to analyze more than 100 bisulfite PCR products in a single sequencing run. They published their method in a recent issue of Cancer Research.
“Next-gen sequencing is having a powerful impact on the development of new techniques for DNA methylation analysis,” Edwards told In Sequence by e-mail this week. “That said, I don't think anyone will have a ‘perfect’ method in the near future. Any technique will still have significant trade-offs in terms of resolution, the ability to analyze repetitive elements, throughput, cost, and sensitivity, to name just a few of the relevant factors.”
Meissner agreed that it is not clear yet which methylation assays will be best suited for large-scale projects, like the Roadmap Epigenomics program. “The jury is still out on which is the best method in terms of how you generate the reference epigenome, and what’s the most cost-effective way of getting one or two, and then … getting many,” he said.

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