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NuGen Publishes Details of 'Fundamental' Single-Primer Target Enrichment Method

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NEW YORK (GenomeWeb) – NuGen Technologies plans to develop new applications for its proprietary target enrichment technology for next-gen sequencing, including the analysis of copy number variants, viral integration sites, and methylation sites.

Over the past year, the company has commercialized its method, called single-primer enrichment technology (SPET), for a number of applications to analyze both DNA and RNA, including single nucleotide variant and indel detection and gene fusion identification.

Compared to other target enrichment strategies that are based on hybridization capture or entirely on PCR amplification, SPET promises fast workflows, low nucleic acid input requirements, and flexible designs that can cover a broad range of target sizes, from a single gene to tens of megabases of DNA, according to the company.

"The technology is kind of a fundamental platform for us, and it can be applied to many different types of genomic analyses," Alan Dance, NuGen's vice president of marketing, told GenomeWeb.

He said the firm is developing a number of new products, including one to be launched in the near future that will integrate targeted enrichment panels and copy number analysis into a single assay. This could be particularly useful for cancer research, he said, where sample amounts are often limited.

In addition, the company is "actively working" on applying the technology to study viral integration sites, and to understand the methylation status of targeted regions.

In a paper published in PLOS One today, company researchers described SPET, which is patented in Singapore and patent-pending elsewhere, using it to identify gene fusions involving 401 known cancer genes from fresh-frozen and formalin-fixed paraffin-embedded samples. According to Dance, the paper is the first peer-reviewed publication showcasing SPET, and demonstrated that it is more sensitive than standard RNA-seq for detecting fusions with the targeted genes.

SPET starts with fragmented genomic DNA or cDNA, usually between 10 and 100 nanograms of material. Indexed forward adapters are ligated to the ends of the fragments, followed by a hybridization step with target-specific probes that are designed to bind upstream of the target. Each target has at least two probes — one for each strand — and each probe carries a reverse adapter. The total number of probes can range from just a few to more than 200,000, though the upper limit has not yet been determined, he said. Next, a DNA polymerase extends the probe through the target region and the forward adapter. Using the two adapter sequences as primer sites, the targets can then be PCR-amplified and sequenced.

The entire workflow can be performed in as little as eight hours, but many labs prefer to do the hybridization step overnight, he said.

One advantage of the gene fusion assay over fluorescent in situ hybridization, for example, is that it only requires probes to one fusion partner, so the other gene involved in the fusion can be unknown.

NuGen is not the only firm offering NGS-based assays for the detection of gene fusions. For example, ArcherDX — formerly part of Enzymatics — offers a number of gene fusion assays based on its proprietary anchored multiplex PCR (AMP) technology; and Fusion Genomics sells an assay that uses hybridization-based capture to identify gene fusions in childhood cancers.

However, Dance said NuGen is currently the only one offering a broad screening panel covering hundreds of gene fusions — the company's Ovation Fusion Panel Target Enrichment System includes 500 genes known to be involved in fusion events. In addition, the firm provides customized gene fusion panels that have a turnaround time of three weeks, he said.

Bastiaan Tops of the Radboud University Medical Center in Nijmegen, the Netherlands, is one of the users of the gene fusion technology. According to NuGen, his group has used a custom gene fusion panel to analyze gene rearrangements in sarcomas and has discovered two previously unknown fusions. The group plans to validate the fusions in additional samples and to develop a molecular diagnostic fusion detection test for sarcomas, Dance said.

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