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UCSD Researchers Develop Assay Combining Methylation Status, Chromosome Conformation Analysis

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NEW YORK – A team of researchers has developed a method to perform whole-genome bisulfite sequencing for methylation status analysis and the Hi-C chromosome conformation capture assay at the same time.

Led by Bing Ren, a professor at the University of California San Diego School of Medicine, the researchers described their assay, dubbed Methyl-HiC, in Nature Methods this week.

In their proof-of-principle study, the researchers showed the assay worked in both bulk and single-cell applications.

"It enables simultaneous characterization of cell-type-specific chromatin organization and epigenome in complex tissues," the authors wrote.

Ren said the ability to do so is especially useful in single-cell studies. "It was a problem we faced, you couldn't study more than one marker at a time because the cell's DNA is destroyed when analyzing them."

Coming up with the idea may have seemed obvious, but implementing it was not, he said. "We spent a lot of time developing technique-optimizing parameters."

"It's a really clever idea to add the methylation status detection to the Hi-C protocol," said Rachel Patton McCord, a researcher at the University of Tennessee, Knoxville who has worked on developing Hi-C. "And it's a very nice method in that it's not disrupting what you actually get from the Hi-C, you're only adding more info."

Bisulfite sequencing chemically treats DNA so that unmethylated cytosines are converted to uracil and read as a "T," rather than a "C," and Hi-C is a proximity ligation-based assay for analyzing the three-dimensional genome structure.

Ren said his lab began working on the Methyl-HiC method more than two years ago. "The simple concept is that you can perform Hi-C first then treat the ligated product with sodium bisulfite and sequence for methylation status," he said. "In practice, it requires a lot of optimization."

The researchers first posted their results in a BioRxiv preprint last November. 

In their publication, they first analyzed bulk DNA to show that their new assay matches data obtained from the same set of samples but profiled separately with each assay. They then analyzed approximately 150 single cells grown in different media, which showed different methylation levels.

McCord noted that the researchers admitted a sparsity of data for the single-cell protocol and said it would be "technically challenging." But with more people adopting Hi-C methods, she said the extra information on methylation, for only a little extra effort, could be attractive. And in general, Hi-C yields less data at the single-cell level than, say, single-cell RNA sequencing. "With DNA, you only get two copies [per cell]," she said.

The ability to cluster those single cells based on methylation status helped to determine important heterogeneity in genome structure, McCord said. "It looks like they're getting something biologically meaningful out of that."

Ren said that aspect was key to his plans for the tool in his lab. "We could take a mixture of brain samples and study methylation in individual cells, then do cluster analysis based on demethylation programs to sort out neurons from other cells types, then study chromatin looping."

"And in tumors, where you have heterogeneity between different cells, you could uncover signatures of epigenetics going on in individual cells and connect that to chromatin conformation," he said, adding that such studies could provide more precise diagnostic tools for the field. 

The method could also "fill a critical need in our understanding of enhancers" and how they drive gene expression, he said. "We know enhancer functions are cell type specific. Each cell type in the body — neurons, blood cells — they utilize a different set of enhancers to ensure the proper set of genes and proteins are expressed."

Ren said he believes there will be broad interest in his assay from other researchers, due to the combination of information it provides. McCord suggested that researchers who were hesitant to do bulk Hi-C might be interested in the extra data.

The bulk assay costs about half as much and takes half the time compared to running the two assays separately, Ren said. "However, for single cells, there is not an alternative that can accomplish this."

When asked if anyone plans to commercialize the Methyl-HiC assay, Ren suggested Arima Genomics, a company he cofounded and which has commercialized a Hi-C assay his team developed. However, Arima CEO Siddarth Selvaraj declined to say whether the firm would pursue commercialization of the Methyl-HiC assay.

Ren noted that the method requires custom software to decipher the DNA methylation patterns from the reads while simultaneously mapping the chromatin interactions, which was developed by his collaborator Yaping Liu, now a researcher at Cincinnati Children's Hospital Medical Center.

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