NEW YORK – Single-cell genomics startup Factorial Biotechnologies is commercializing an intracellular library preparation technology that the company said can "dramatically simplify" the workflow of single-cell sequencing.
The San Carlos, California-based startup is slated to release its product to early-access customers this year and is hoping to launch it commercially in 2024.
Established in 2020, Factorial Biotechnologies was conceived by Cofounder and CEO John Wells, who came from Lasergen, a company that was later acquired by Agilent Technologies.
"A few years ago, I saw single-cell data scaled for the first time. It got me really excited about the potential for it," said Wells. "I spent really about a year looking around at single-cell technologies and couldn't find anything that I thought would do for the single-cell market what Illumina did for the NGS market, which is to [enable] broad access."
To that end, Wells thought about developing a technology that would get rid of the need for physical cell isolation and DNA extraction of typical single-cell workflows. "People have done PCR inside cells before," he said. "That was the insight I had: If you could do PCR inside of cells, could you do a whole library prep inside of cells?"
Factorial’s other cofounder and CSO, Katie Zobeck, who is also a molecular biologist, helped turn Wells’ idea into an actual workflow.
"For the last two and a half years, we've just been getting the technology to work well enough that we can start to have early-access customers," Wells said. The company's team achieved proof of concept in early 2020, he added, and started raising money the same year. To date, Factorial has "raised a fairly small amount of money" from investors, he said, and is in the process of raising a larger round of Series A funding.
Factorial’s core technology relies on so-called extraction-free library prep, independent of single cells. While the company, which recently came out of stealth mode, has yet to release details about its approach, Wells offered a broad overview of the workflow.
The library prep process starts with fixing and permeabilizing the cells, followed by library prep, which involves enzymatic DNA fragmentation, end repair, A-tailing, and ligation. After that, libraries are PCR-amplified in situ using Illumina indexing primers. Cells are then lysed, and the libraries are purified with SPRI bead cleanup. Notably, Wells said, the company also devised a chemistry that can attach cells to magnetic beads to facilitate buffer exchange during the workflow.
Branching off this core technology, Wells said, the company has also developed a barcoding scheme to achieve intracellular library prep for single cells.
For that, instead of incubating the cells with standard Illumina indexing primers during amplification, custom oligos containing different barcodes are introduced into the reaction and undergo isothermal amplification, turning them into primers for amplifying the precursor libraries. As the precursor libraries are amplified by barcoding oligos, they are labeled with a unique pair of barcodes and are subsequently sequenced and computationally deconvolved to get single-cell data.
Besides this, Wells said, the in-cell library prep product is also compatible with droplet microfluidics or the split pool method to achieve single-cell resolution. "There are a variety of things that our extraction-free library prep [technology] could sit upstream of," he noted.
Generally speaking, Wells believes there are multiple advantages of the company’s extraction-free library prep technology compared with their traditional counterparts. For one, he noted, with a turnaround time from cells to libraries of four to five hours, the technology can speed up the sample prep process. In addition, conventional library prep methods, which typically require reagent transfer between tubes, can be both "cumbersome from a workflow standpoint" and incur a loss of material. In contrast, the company’s method is all done in a single tube, reducing its complexity.
So far, Factorial has demonstrated its method in mammalian and yeast cell suspensions. In one internal experiment, Wells said, the company mixed human and mouse cells and showed "very good separations" between the two sample types. "Greater than 95 percent of the cells were pure," he noted, which "shows we didn't have nucleic acids or barcodes leaking out of one cell and going into another cell."
However, Wells said the company still has many kinks to sort out ahead of a full commercial launch. "We have good proof-of-concept data," he said. "It's just a question of getting the workflow optimized so that we can generate high-quality data."
For instance, the company’s standard workflow is currently optimized to handle 16,000 cells — the number of cells that can generate 100 nanograms of genomic DNA, which is a standard input for library prep. However, Wells said the firm is also further pushing its technology so that it can accommodate both a higher and much lower number of cells.
Additionally, beyond mammalian and yeast cells, Factorial is working to make the method compatible with other sample types such as fresh and formalin-fixed, paraffin-embedded (FFPE) tissues. "That's a very important market to us," Wells said. "I think being able to be compatible with solid tissues and FFPE is absolutely important to us."
Even though the company has only sequenced its libraries with Illumina platforms for now, Wells said it is certainly also interested in long-read sequencing platforms and "[doesn't] see any reason" why its extraction-free library prep technology cannot be compatible with them.
In terms of cost, Wells said the material costs of the company’s method are "significantly lower" than that of microfluidics-based single-cell technologies. Still, the company is "more focused on workflow, content, and [data] quality" at the moment than competing on costs, he said.
To enhance its offerings, Wells said Factorial is currently working with Twist Bioscience to do single-cell hybridization capture. Down the line, the firm also plans to offer multiomics products by enabling RNA and DNA applications.
Near term, Factorial is planning to make its extraction-free product available to early-access customers in the second quarter. "We have customers lined up," Wells said, noting that the company is about to kick off a collaboration with genome engineering company Inscripta but is still looking for additional partners.
The single-cell library prep product is scheduled for early-access in the back half of the year, Wells said. As the company rolls out its projects, people can also expect to see more data comparing Factorial's technology with other library prep and single-cell workflows.
Moving forward, Wells said the company, which currently has 10 employees, will continue to expand its R&D and commercial teams. In addition to its headquarters in San Carlos, it has an office in Houston.
IP-wise, Wells said the company has filed patent applications and provisional patents pertaining to its extraction-free library prep technology and cell barcoding, as well as for the chemistry to conjugate cells onto beads during sample prep. "We have taken [IP] very seriously since the early days," he noted. "We feel as good as we can for a company at our stage."
When it comes to applications, Wells said the company will apply its technology toward CRISPR, cell and gene therapy, immuno-oncology studies, and beyond.
"It’s a really foundational technology," Wells said of extraction-free library prep. "I am excited to see what people can do with it that we maybe haven't even thought of yet."