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Startup Directed Genomics Will Focus on Solving NGS Sample Prep Challenges


NEW YORK (GenomeWeb) – Directed Genomics, a startup that is currently being incubated within New England Biolabs, is aiming to develop technology to tackle problem areas of the next-generation sequencing workflow, with its first focus on target enrichment technology.

Directed Genomics is currently leveraging resources from New England Biolabs, which is providing support both in terms of lab equipment as well as employees. The two companies also recently said that they plan to work together to develop target enrichment technologies.

President and Founder Cynthia Richard said the company is focused on "trying to make novel, robust solutions for next-generation sequencing to increase the efficiency of the platforms."

Before founding Directed Genomics, Richard led validation of new technologies in the genomic services lab at the HudsonAlpha Institute for Biotechnology and before that led the development and production of New England Biolabs' NEBNext DNA and RNA library prep kits.

Richard told In Sequence that target enrichment was "an obvious first choice" because current technologies have too many inherent biases. The firm is aiming to launch its first product, a 50-gene cancer panel, early next year.

Essentially, there are two main methods of target enrichment – those that rely on hybridization and those that rely on PCR. Each has drawbacks, she said. For instance, hybridization techniques are often only 20 percent to 30 percent on target. PCR-based target enrichment tends to have significant amplification bias and an inability to distinguish between the two strands of DNA.

Directed Genomics, meantime, is developing a technique that aims to draw on the strengths of both techniques. The startup's strategy is to first fragment DNA and then do a "quick hybridization," Richardson said. Short single-stranded DNA fragments are hybridized to biotinylated probes and captured on beads, which "defines the 3' end" of the DNA, she explained.

Next, enzymatic reactions remove the 3' sequences that are off target and a sequencing specific adaptor is added to the captured sequences. A similar step is then performed to define the 5' end and a platform-specific 5' adaptor is added. In order to detect unknown translocations, a nonspecific probe is used, which leaves the 5' end undefined. Finally, the 3' adaptor is cleaved, which releases the bead, and then the library is amplified by PCR. The protocol takes a total of six hours.
Richard said that it is the enzymatic step to remove the off-target sequence that helps increase the specificity of the hybridization, and because there are two steps to define the 3' and 5' ends, strand specificity is also conserved. In addition, tethering the regions of interest to beads reduces the amount of material lost, enabling a lower starting input, she said.

The firm described its technology in a poster presentation at the American Society for Human Genetics meeting in San Diego last month, demonstrating a cancer panel comprising 30.3 kb of target sequence from 200 exons on 50 cancer-related genes.

They tested the panel on a colorectal cancer cell line with varying amounts of input DNA — 1 μg, 500 ng, 100 ng, and 50 ng. For each input amount, approximately 97 percent of aligned reads were on target and 100 percent of the targets were covered. Coverage uniformity was 94 percent.

Additionally, the researchers created cell lines with known mutations at frequencies that varied between 2 percent and 30 percent and evaluated the panel's ability to detect 21 variants. In the majority of cases, the detected variant frequency was concordant with known variant frequency with some discrepancies at the lower frequency variants. Only one variant was not detected at all.

Sequencing was performed on Illumina's MiSeq, but Richard said that the technology is platform agnostic. She added that while the firm will target the research and translational market, she thinks the technology will "be a good fit in the clinical market."

The firm is now lining up beta testers for its product and also plans to demonstrate the technology's ability to work with cDNA and hopes to present data from this application in February at the Advances in Genomics and Biology Technology meeting in Marco Island, Fla.