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Cergentis Plans Update, New Kits for Targeted Resequencing Method; Seeks Partners for Outlicensing

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NEW YORK (GenomeWeb) − A year after launching its targeted sequencing method as a service, Dutch startup Cergentis has automated the technology and launched reagent kits for its protocols. In the near future, the firm plans to introduce an updated version of the method, called targeted locus amplification (TLA), and intends to outlicense it to service providers and diagnostic laboratories.

TLA works by crosslinking DNA that is physically close to a gene or region of interest, digesting the DNA with a frequently cutting restriction enzyme, ligating the fragments into large circles, removing the crosslinks, converting the DNA into smaller circles, PCR-amplifying those circles containing the gene of interest, and sequencing the amplification products.

The method is similar in principle to the 4C or circularized chromosome conformation capture strategy but determines the sequence of entire restriction fragments rather than just their ends. Earlier this week, Cergentis and its collaborators, including the Hubrecht Institute and the University Medical Center in Utrecht, published a detailed description of TLA, including examples of its application, in Nature Biotechnology.

Because TLA requires no prior knowledge about the DNA surrounding the region of interest, it can identify several types of genetic changes, such as single nucleotide variants and structural variants, including gene fusions. In addition, it can provide the haplotype of genomic loci. "The TLA technology is therefore of particular interest once (candidate) genes have been identified and complete sequence information of these genes is important," CEO Max van Min told In Sequence.

The complete workflow for generating TLA templates, amplifying them, and preparing a next-gen sequencing library takes about four days, according to the firm's website. The cost per sample depends on "a variety of parameters" and is "in line with the costs of other targeted sequencing approaches," according to the company.

Hanlee Ji, an assistant professor at Stanford University School of Medicine who has developed another targeted sequencing method, called TLA "an innovative integration of commonly used methods of identifying physically proximal chromatin regions with targeted sequencing" that enables targeted enrichment in the context of chromatin structure. It allows for the capture of regions that "can extend far beyond the small fragment targets associated with traditional resequencing methods involving in-solution bait hybridization," Ji told In Sequence.

A major advantage, he said, is the ability to call structural variants in megabase-sized regions. Another potential plus is that large sets of genes can be captured with a small number of target-specific primers.

One important application of TLA so far has been the analysis of transgenes and their integration sites. "This information is very valuable for research institutes and pharmaceutical companies as it helps them ensure that they are using transgenic cells [or] organisms in which the correct genetic changes have occurred," van Min said.

Another significant application is the analysis of clinically important genes in cancer. Cergentis has been working with leukemia researchers, for example, to characterize mutations in genes that are relevant for prognosis and drug response, he said.

Ji said he is most intrigued by the possibility to use the TLA approach to analyze chromatin structural alterations in a target-specific manner. "Thus, this method may prove to be highly informative for considering aspects of chromatin biology that were previously difficult to resolve," he said.

According to Erik Splinter, the company's chief technology officer, the main limitation of the current protocol is the fact that it requires cells as input material. However, the company has developed a modified protocol, which it plans to launch shortly, that works with isolated DNA as starting material and can crosslink it efficiently. The new protocol is currently in beta testing with research institutes and companies, such as plant breeding firms. Cergentis is working on applications in diagnostic gene sequencing and targeted sequencing of animal, plant, and microbial genes, van Min said.

According to Ji, another drawback of the method is the use of chemical crosslinking, which he said can vary in performance. In addition, TLA might be limited by the use of specific restriction enzymes, and the method "will require some 'tuning' depending on the sample and the quality of its nuclear DNA," he said.

Cergentis is now testing TLA for analyzing FFPE samples, developing new protocols in collaboration with cancer research institutes and other firms, according to van Min.

Since launching TLA as a service last year, Cergentis has introduced several TLA kits, which contain all reagents necessary for processing samples and amplifying regions of interest. In collaboration with PerkinElmer, it has also automated the TLA technology on PE's Janus NGS Express Workstation. The plan is to release additional kits to analyze specific genes, and to outlicense the technology to other service providers, as well as diagnostic laboratories.

So far, Cergentis has provided its TLA service to "the majority" of leading pharmaceutical companies and several cancer research institutes, van Min said, and its customer base "continues to grow."

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