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With Focus on Methylation Sequencing, Belgium's NXT-Dx Caters to Epigenetics Researchers

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Taking advantage of the recent boom in epigenetics research, Belgium-based NXT-Dx is banking on offering services catering to this field, in particular DNA methylation sequencing.

The company, founded a year ago based on methylation sequencing technology developed at the University of Ghent, offers genome-wide methylated DNA binding domain, or MBD, sequencing, as well as targeted bisulfite sequencing to researchers worldwide.

Two months ago, the company, which has a handful of employees and uses sequencing instrumentation at the University of Ghent, also added RNA sequencing and real-time qPCR-based microRNA and long non-coding RNA expression profiling services.

According to Maarten Braspenning, director of commerce and operations and a co-founder of NXT-Dx, the interest in epigenetics, initially largely focused on cancer research, has recently grown in areas such as toxicology and immunology. Many researchers from those fields have no experience with methylation sequencing, he said, providing an opportunity for service companies.

The company's name stands for "Next-Generation Diagnostics" because its research services can help others develop diagnostic assays, Braspenning said. However, the firm has no immediate plans to provide diagnostics services. So far, it has been solely funded by its founders and provides its services out of university labs.

The majority of NXT-Dx's customers come from universities and academic research institutions in the US and Europe, but they also include several pharmaceutical and biotechnology companies. The company mainly analyzes human, mouse, and rat DNA samples, although it can work with any type of mammalian cells in principle.

NXT-Dx has an exclusive worldwide agreement with the University of Ghent to commercialize MBD sequencing technology developed by university researchers. While those scientists did not invent MBD sequencing as such, they improved it in several ways, Braspenning said, including optimizing the general process, choosing the best type of protein and salt concentration for methylation capture, and developing a bioinformatics pipeline for cleaning up and mapping the sequencing data.

NXT-Dx has compared its version of MBD sequencing to commercially available kits, he said, and the results of that study are currently under review for publication.

MBD sequencing service requires about 500 nanograms of genomic DNA from the customer, which the company checks for quality prior to fragmentation. After size selection and another round of quality control, those fragments containing methylation are captured by an MBD. The captured fragments are then used to prepare libraries for sequencing on the Illumina HiSeq. Also included in the service are data cleanup and mapping to the reference genome. Besides receiving the raw data, customers obtain access to the company's proprietary H2G2 Genome Browser software for two months to visualize and analyze their data.

According to the firm's website, the turnaround time for the service is about four weeks, including one week of presequencing work, one week of sequencing, and two weeks for bioinformatics analysis.

Unlike bisulfite sequencing, MBD sequencing does not offer single-base resolution, but it is a genome-wide methylation enrichment technique, similar to methylated DNA immunoprecipitation sequencing, or MeDIP-seq, which uses antibodies instead of an MBD.

But according to NXT-Dx, MeDIP picks up many biologically irrelevant methylated regions because it binds to methylated cytosine independent of the CpG context. The company's approach, on the other hand, uses the methylation binding domain of the NERD complex, which has a role in chromatin remodeling and binds to biologically relevant sites.

MBD sequencing also focuses on CpG-dense regions, while MeDIP-seq covers more individual methylated cytosines, which the company says are less relevant, and it is 100 times more sensitive than MeDIP. Finally, its MBD-seq approach discovers 95 percent of the information that bisulfite sequencing provides, the company claims.

Besides genome-wide MBD sequencing, NXT-Dx offers deep bisulfite methylation sequencing of specific DNA areas of interest. Until now, it has been using the Roche 454 sequencing platform for that but is considering moving the application to the Illumina MiSeq.

The recent addition of RNA sequencing as well as microRNA and lncRNA expression profiling services resulted in part from customer requests. For example, many researchers want to link methylation to gene expression, so RNA sequencing is "a nice addition" to the other services, Braspenning said.

NXT-Dx says it differs from many providers of DNA methylation sequencing services in that it offers customers data visualization and analysis software, also developed at the University of Ghent. "There are a lot of labs that can do the methylation pull-down and have their samples sequenced. But doing the analysis afterwards is the biggest problem," Braspenning said.

The company is also offering additional bioinformatics analyses that are not included in the standard service, for example generating lists of differentially methylated regions, and it is currently looking into commercializing its analysis software.

NXT-Dx is not the only firm catering to epigenetics researchers. "More and more providers offer epigenetics/methylation sequencing," said Michael Heltzen, CEO of BlueSEQ, an online platform that connects sequencing service providers with customers. However, he added, there is room for companies like NXT-Dx because "parts of the market are asking for more specialized sequencing service providers."

NXT-Dx says its main competitor is Zymo Research, based in Irvine, Calif. According to Zymo's website, it offers genome-wide methylation analysis using reduced-representation bisulfite sequencing – an approach that employs restriction enzymes to produce fragments rich in CpG sites – as well as genome-wide hydroxymethylation sequencing that combines capture enrichment of hydroxymethylated DNA with next-gen sequencing.

NXT-Dx is currently looking to expand its service portfolio further. One area the company is particularly interested in is hydroxymethylation sequencing, which would require the use of hydroxymethylation-specific antibodies for pull-down, Braspenning said. It is also looking into ChIP-seq, although he cautioned that this might be difficult to offer as a service because it requires many different antibodies for the different DNA-binding proteins.

For the foreseeable future, the company is not planning to employ new sequencing technologies, like those from Pacific Biosciences or Oxford Nanopore Technologies, that promise direct methylation sequencing. "I do think it would take a few more years before that's actually up and running well," Braspenning said. "But once that's there, it is obviously of interest to us to look into."

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