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Startup X Gen US Reimagines Library Prep Chemistry for Single-Cell Sequencing Methods


NEW YORK – California startup X Gen US has set out to cut sample preparation time for single-cell RNA sequencing to less than two hours.

The company, a recent graduate of Johnson & Johnson's JLabs incubator program, has developed kits that use a new modified transposase that the firm engineered to cleave RNA-DNA hybrids. The effect is that library prep no longer requires a second strand synthesis before tagmentation, according to CEO Kai Lao, cutting down on the number of steps and time needed.

The Fremont, California-based firm already has kits available for single-cell transcriptomics, accessible chromatin profiling by ATAC-seq, cell-free RNA sequencing, and full-length transcriptome sequencing, as well as genomic DNA amplification kits. Additionally, the RNA-seq kits do not require ribosomal RNA or transfer RNA depletion and can work with as little as 5 picograms of total RNA, Lao said.

"I believe we're the first ones to do truly one-step library prep from crude samples to RNA-seq," he said. "We believe that this could become the underlying biochemistry for all single-cell platforms."

So far, the company, which currently has seven employees, has not validated its kits in a peer-reviewed study, but is hoping to do so in the future. According to Lao, he has raised approximately $5 million in private, pre-Series A financing from two individuals and is seeking more funding.

People who know Lao professionally said he's made important contributions to single-cell sequencing before, and he could well do it again.

"Anything that Kai would do, I would definitely take seriously," said James Eberwine, codirector of a program in single-cell biology at the University of Pennsylvania. Eberwine said it has been several years since he last spoke to Lao and has not discussed the new kits with him. But he said that "the fact Kai is developing them, that bodes well for their utility and the technique."

Lao, he noted, published an early study analyzing the whole transcriptome of a single cell in 2009. "It captured people's imagination and probably set the field in motion in terms of trying to do more single-cell sequencing," Eberwine said.

At the time of the publication, Lao was working on sequencing systems for Applied Biosystems, where he was employed from the days of the Human Genome Project until 2017, when he retired. That summer, he founded X Gen. Sunney Xie, another single-cell sequencing pioneer, who has labs at Harvard University and China's Peking University, as well as several companies, is also involved with X Gen, as technology leader.

At first, X Gen was pursuing assays for 3D structural genomics, Lao said. The firm developed a so-called tag-enabled direct RNA/DNA detection (ted) technology and created a single-cell whole-genome amplification assay that gave higher resolution on chromosome conformation capture. That assay, now available from X Gen as ted-scDNA, is a simplified version of the Dip-C method developed by Xie and published in Science in August 2018.

But the market wasn't quite ready for 3D genomics, Lao said, and he decided to jump back into single-cell analysis when he saw Science name it 2018's "Breakthrough of the Year." In most protocols for RNA-seq on Illumina's platform, RNA is analyzed by creating a complementary DNA (cDNA)-RNA hybrid, then synthesizing a second strand of cDNA, before a transposase designed to cut DNA-DNA duplexes tags and fragments the molecules to create sequencing libraries.

X Gen saw an opportunity to cut out the second strand synthesis by engineering a new transposase. "We looked carefully at one domain called a DDE domain. It's really highly conserved, but it's very similar to HIV integrase," which can cleave RNA, Lao said.

By engineering the amino acids at several locations, X Gen created a new enzyme that could perform tagmentation on RNA-DNA hybrids.

Lao noted that the firm has filed three patent applications for methods to convert DNA-RNA hybrids into next-generation sequencing libraries; however, he said they did not disclose the specific modifications they made to the enzyme complexes.

For now, the kits are only available for plate-based single-cell sequencing methods, which are limited in throughput, compared to droplet- or nano-well based methods. But Lao said X Gen is working on developing a bead technology that could bring the company's chemistry to higher-throughput methods.

X Gen's kits run from $350 to $500 for an eight-well format and $3,360 to $4,820 for the 96-well format. They do not include PCR or Illumina indexing reagents. There's also a kit with an unlisted price, the ted-scDNA/RNA, which, according to its product description, "can obtain both RNA and [the] whole genome from a single cell."

"I'm not so sure where the market is for that," Lao admitted. "That's why I didn't put a price on it." Most single-cell researchers, he said, only care about the accessible genome regions. But developing an assay to do the whole transcriptome plus the whole genome was Lao's white whale. He and a colleague tried to do it a decade ago but were unsuccessful. Now it's there for anyone else who's curious about it. 

Lao mentioned only one customer — an unnamed pharmaceutical company who used the ted-CapAll kit to look at RNAs in exosomes and serum from Alzheimer's patient samples.

Though the kits are available on X Gen's website, Lao said he's looking to collaborate with companies already offering single-cell sequencing platforms. "I believe I need to partner with some bigger company to truly drive single-cell genomics," he said. "I have the technology, but I need platforms."

Lao added that he thought X Gen's kits could be useful for clinical samples, where there's not enough material to perform RNA depletions, or where there is degraded RNA. The simpler method would also help in that regard, he added.

"If you can reduce a complex technology down to one step, you can directly apply it to clinical samples," he said.