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Startup TwinStrand Biosciences Aims to Commercialize Duplex Sequencing Technology

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NEW YORK (GenomeWeb) – University of Washington spinout TwinStrand Biosciences has raised around $2 million in seed funding and opened a next-generation sequencing laboratory in Seattle. The startup plans to commercialize technology known as duplex sequencing for applications such as oncology and forensics.

TwinStrand Biosciences currently has seven full-time employees, including CEO Michael Kranda, and is looking to at least double in size by the end of year, co-founder Jesse Salk said in an interview.

Researchers from the University of Washington first described the duplex sequencing technology in 2012 in a study published in the Proceedings of the National Academy of Sciences.

The method involves tagging both strands of DNA with random, but complementary, primers. After each strand is tagged, the molecules are PCR amplified. Then after sequencing, the molecules can be grouped by their tags and compared. Errors introduced from either PCR or sequencing can be filtered out by setting a threshold for the number of matches that should be found among the molecules. Next, the tags are matched to the complementary tags, and again compared, with errors filtered out.

Last year, the researchers published another study in PNAS demonstrating that the method could be used to identify low-frequency TP53 mutations in peritoneal fluid samples from women with high-grade serous ovarian cancer, as well as women without cancer. In the study, they were able to identify a single mutation out of 20,000 molecules.

The researchers found that although the women without cancer did in fact have the same TP53 mutations, they were at very low frequency levels.

Salk said that the company plans to develop research-use-only kits that would include the necessary chemistry and software needed for duplex sequencing and make those available to the broader scientific community. In addition, he said, the firm will look to partner with pharmaceutical or other biotech companies to develop duplex sequencing assays for specific applications.

In such cases, Salk said, the assay would be built and developed in TwinStrand's lab but then licensed to the specific company, which would then run the samples in its own lab.

Already, he said, the company has a handful of partnerships, including three with pharmaceutical companies that are developing the technology for noninvasive cancer diagnostics and risk assessment.  Salk declined to identify the partners.

Since the initial PNAS publications, the researchers have been working to further improve and optimize the duplex sequencing method, including making changes to the biochemistry and software, Salk said.

 In addition, the team has been working on modifying methods for different applications. For instance, he said, DNA can be in different forms depending on the application. For oncology liquid biopsy applications, circulating cell-free DNA is highly fragmented. In forensics applications, DNA is often highly degraded. In other cases, DNA may be high quality. "They're all DNA, but they all have different reasons they have problems with accuracy and recovery," Salk said.

Methods for recovering, amplifying, and sequencing small fragments of circulating cell-free DNA are different than for DNA pieces that are tens of kilobases in size and have to be fragmented, he added.

The company has filed several patent applications on its original method, as well as the subsequent improvements it has made. Upcoming publications will highlight improvements the researchers have made to the method's efficiency as well as build on the results of its previous PNAS study that found low-level TP53 mutations in the peritoneal fluid of healthy women.

"When your sensitivity improves enough to get above the technical background, we see mutations that occur in healthy people that look identical to mutations that occur in cancer," Salk said. And, he added the mutations occur at different levels depending on the person's age and environmental exposures. "Just seeing a mutation that looks like a cancer mutation doesn't mean that you have cancer," he said. And, the work highlights the challenges of using liquid biopsy analysis to screen healthy individuals for cancer.  

A number of well-established companies and startups — chief among them Guardant Health and Foundation Medicine — are developing liquid biopsy tests designed to detect increasingly lower-frequency mutations.

At last month's Molecular Medicine Tri-Conference meeting in San Francisco, a number of researchers and startups presented on various methods to detect low-frequency mutations in circulating tumor DNA, including a method dubbed CAPP-seq that has been commercialized by Roche, a technique developed by University of Cambridge researchers and now being commercialized by startup Inivata, and an approach developed by startup AccuraGen, which plans to commercialize ctDNA assays in China.

Nonetheless, Salk said that one way in which TwinStrand Biosciences will set itself apart is by developing applications not only in the increasingly crowded liquid biopsy field, but also in forensics and other non-medical fields that rely on degraded or low-quality DNA.

For instance, Scott Kennedy, who has been heading up the firm's forensics work said that duplex sequencing could help improve on a common problem of analyzing short tandem repeats (STRs) — PCR-induced "stutter." STR regions are used in human identification, but a PCR artifact known as a stutter creates problems when looking at closely related samples. In the analysis, it can be difficult to determine whether there is more than one sample or whether the polymorphism is due to the PCR artifact, Kennedy said.

In his forensics work, he has found that using duplex sequencing to filter out PCR-induced errors can improve the accuracy of sequencing-based STR typing.

"A lot of DNA you get from crime scenes is low quality, which leads to more artifacts," he said. "Duplex sequencing can get rid of the artifacts, which improves the quality of the final data."

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