Eight years ago, 10x Genomics launched the first commercially available single-cell RNA sequencing (scRNA-seq) product for the company’s flagship instrument, the 10x Chromium. (This occurred one year after the launch of GemCode—the Chromium’s earlier iteration.) The instrument was a game changer that offered researchers an efficient approach to single-cell experimentation. After years of impressive growth, the Chromium remains the workhorse of the single-cell biology field. Not only that, but the company’s innovation shows no sign of slowing down. The director of a genomics core facility tells GEN that 10x Genomics’ Chromium Single Cell Gene Expession v4, which launched at the Advances in Genome Biology and Technology meeting last February, is “much better than version 3” and that “the company continues to make iterative improvements all the time.”
But many single-cell researchers share a common gripe: the experiments are expensive to run. Making the transition from bulk RNA sequencing to scRNA-seq can be a steep challenge for some investigators who must sacrifice sample size to be able to afford it. “Democratization” is a buzz word at single-cell meetings.
There are more than 5,000 Chromium instruments placed around the world, and they are a common feature of university core facilities. But many researchers still have to work hard to find one. For example, GEN heard about a researcher in Bilbao, Spain, who has to drive their samples more than 300 miles to use the nearest Chromium in Madrid. It is fairly common for researchers to bike samples across busy cities or to use the subway to reach a Chromium.
An interesting trend has emerged over the past few years: several newer companies have launched kit-based products that offer single-cell researchers an alternative that doesn’t involve additional instrumentation. Here, GEN relates what several of them have to say about their technology, their products, and their views on the future of single-cell technology.
Go big or go home
“It’s a really exciting time in ‘single cell,’” proclaims Alex Rosenberg, PhD, co-founder and CEO of Parse Biosciences in Seattle, WA. He points to two exciting trends. First, many researchers are entering single cell for the first time. Second, current single-cell users are scaling up. The result is that more groups are utilizing single cell to its full potential.
“Every year,” Rosenberg observes, “the size of projects is doubling with respect to the number of cells people are running.” The decreasing cost of sequencing has helped. As more and more researchers gain single-cell experience, they have realized the value of the technology and are expanding into much larger projects.
In addition, the single-cell community is aware of advances happening in generative AI. There is a big push, Rosenberg notes, to create foundational models for single-cell data that can, for example, predict which cells are going to respond to drug treatment or which cancer cells are going to elicit an immune response. But more data are needed. The largest models have been trained on about 50 million cells. “That’s really not that big in the scheme of things,” he says. In comparison, Open AI trained on data in the range of 10 trillion tokens. People realize that, for now, the single-cell field is data-limited.
Parse considers itself part of the trend in scaling up. One researcher can run a million cells in a single Parse experiment. The company has launched kits to perform B-cell receptor and T-cell receptor profiling—an area where scaling up makes sense given the complexity of the immune repertoire. Parse is also working on making single-cell experimentation easier, both on the front end and the back end.
Data analysis has been challenging. It takes a lot of time and work—even for a bioinformatician. Earlier this year, Parse acquired Biomage, the developer of Cellenics, software that allows single-cell data analysis in a browser without requiring the use of Python or R. That will make data analysis easier for the average biologist. Parse is also enabling automation on its workflow through a partnership with Integra Biosciences. The automation instrument is sold as a bundle with Parse’s kits to ease the pipette-heavy workflow.
Long reads in Rosario
In the middle of the COVID-19 pandemic, a group of scientists in Argentina decided to share their thoughts about the future of single-cell technology. Their first prediction, recalls Elizabeth Tapia, PhD, co-founder and CSO of ArgenTAG, was that the scarcity and inability to access single-cell instruments in South America would persist. Their second prediction was that long reads would gain traction in transcriptomics.
In 2020, ArgenTag was founded by Tapia along with Pilar Bulacio, PhD, Joaquin Ezpeleta, Sofía Lavista Llanos, PhD, and Leandro Ciappina. Today, Bulacio, Ezpeleta, Lavista Llanos, and Ciappina serve the company as CTO, Director of Engineering, Director of Process Development, and CEO, respectively. ArgenTAG has an R&D facility in Rosario, Argentina, and an office in the United States. The company is currently in early access mode and expects to launch its first kit in early 2025.
ArgenTAG introduced its core technology in Scientific Reports in May 2022, in a paper titled, “Robust and scalable barcoding for massively parallel long-read sequencing.” The paper’s authors reported that the technology is designed to enable multiplex long-read sequencing. They also presented evidence that they used the technology to sequence almost 4,000 barcodes simultaneously on Oxford Nanopore Technology’s MinION platform, achieving a high recovery rate and low crosstalk.
“We took the worst-case scenario of long-read accuracy and improved the accuracy until it became comparable to that for an Illumina sequencer,” Ciappina notes. Then they started deploying barcodes in the cells for a single-cell solution.
Why focus on long reads? “When you do short reads, you’re not getting all the information,” Lavista Llanos tells GEN. “You’re just getting a tip of the information.” She adds that short reads provide just a snapshot.
Lavista Llanos has a background in embryogenesis, a discipline in which isoforms are expressed over hours or even minutes. The ability to collect those data to further explore developmental biology is also important in other disciplines. Indeed, according to Lavista Llanos, the folks at ArgenTAG believe that it is the future of single-cell technology. “Our technology makes sense today,” she remarks, “but it will make a lot more sense in five years.”
ArgenTAG’s workflow uses chip-based technology for partitioning and barcoding. Essentially, the chip’s microwells partition the cells and contain beads that carry the barcodes. This technology differs from competing technologies by performing sequencing on an Oxford Nanopore or PacBio instrument instead of an Illumina instrument.
“Our vision is to decentralize single cell today,” Ciappina declares. “10x Genomics has 5,000 instruments installed, but there is a bigger opportunity—100,000 labs that can benefit from single-cell sequencing but cannot today because of the price.”
Génomique unicellulaire
Stuart Edelstein, PhD, a renowned biologist who trained with Nobel laureate Jacques Monod and held posts at Cornell University and the University of Geneva, has been retired for years. But Wilko Duprez, PhD, head of communications at Scipio Bioscience, tells GEN that Edelstein’s mind “never stops thinking about science.” When 10x Genomics released its single-cell, microfluidics-based technology, Edelstein was hiking the volcanoes of Hawaii. Inspired by porous rocks, Edelstein hit upon an idea that would turn out to be an alternative to microfluidics for single cell. “Why can’t we have something that would fit one cell in each little pocket, like the volcanic rock?” Edelstein pondered. The solution was a hydrogel that physically isolates each cell without microfluidics. Edelstein founded Scipio in 2017 and recruited scientists to bring his idea to fruition.
In May 2022, Scipio launched its first kit, Asteria, which can process up to four samples, each with 15,000 cells. The low throughput may appeal to researchers who want to dabble in single cell or have a low number of samples. The only hydrogel kit on the market, Asteria does not manipulate the cells in any way: no fixing, freezing, sorting, vortexing, etc. Duprez says that cells are “happy and cushioned sitting in the hydrogel.”
How does it work? A single-cell suspension is mixed with a bead solution to form cell-bead pairs with one barcoded bead per cell. In a 50 mL tube, the solution is diluted into the hydrogel, which is in a liquid state. A piston-like tool is inserted to ensure there is a thin, uniform layer of hydrogel spread around the walls of the tube. Once put on ice, the hydrogel solidifies with cell-bead pairs isolated into pockets. After the addition of lysis buffer, the mRNA is captured by a bead. The hydrogel is thawed back to liquid using a buffer (the hydrogel doesn’t turn to liquid when it warms up), and the beads are recovered. From there, the standard RNA-seq protocol is followed starting with on-bead reverse transcriptase and PCR. The Asteria kit does not include library prep at the moment, but Scipio plans to include it in the future.
Scipio is located at the Cochin Hospital in Paris, where it has access to clinical samples. The hydrogel is well suited for multisite clinical studies because it is very stable for a long time (at least a month at −80°C). Some clinical studies are multisite and include hospitals or clinics in different locations. Because transcriptomics is time-sensitive, if there are multiple centers collecting samples for single-cell analysis using a kit is easier than having an instrument in five different locations. Hydrogels could be shipped to the same place so that sequencing can be done together. In addition, the Asteria kit can now accommodate CITE-seq data by incorporating tagged antibodies measuring the presence of surface proteins at the single-cell level, therefore achieving multiomics data within a single experiment
Moving into the clinic
The core technology at Fluent BioSciences—pre-templated instant partition sequencing (PIPseq)—originated in the laboratory of Adam Abate, PhD, professor of bioengineering and therapeutic sciences at the University of California, San Francisco. More than a decade ago, Abate and Sepehr Kiani, PhD, chief business development officer at Fluent, started Gnubio (later sold to Bio-Rad Laboratories in 2014), which had developed a technology to inject femtoliter volumes into droplets. In 2018, Abate told Kiani about a new tool he had been working on. Kiani tells GEN that the tool was designed to “do everything RainDance Technologies [another company acquired by Bio-Rad] could do but without any instrumentation.” Once Abate’s laboratory established proof of concept, PIPseq technology attracted investment and led to the founding of Fluent Biosciences in 2020.
The company launched its first kit in July 2022. After five iterations of PIPseq technology, the PIPseq V launched in May 2024. About 30% of Fluent’s customers are newcomers to the field. “We’re really about enabling single cell,” Kiani says. According to Kiani, colleagues of his have met many scientists who are interested in single cell but have shied away because it is too expensive, too complicated, or demands instrumentation they lack.
In the PIPseq kit, a tube has hundreds of thousands (or even millions) of template particles—organic polymers that define the partition and carry a barcode. Fluent sells kits that can manage two to eight reactions and collections of cells that range from a few thousand to hundreds of thousands (with one million cells in early access).
One advantage of Fluent’s technology is that researchers can use it to interrogate the transcriptome multiple times. Researchers can employ the standard method (which sequences the three-prime end), reanalyze the cells for targeted transcriptomics on the same cells, and then go back to perform long reads. Researchers can also process many cells in one sample, which speaks to Fluent’s vision of moving into clinical applications in the future. This large-scale per sample workflow is aligned with clinical utility, Kiani notes. “Fluent is going to own the clinic,” he predicts. “It’s the only thing with the elegance to go into the clinic.”
Other companies introducing single-cell technology in kits include Scale Biosciences, which is located in San Diego, CA, and Singleron Biotechnologies, which has offices in Michigan and Connecticut, as well as in Germany, Singapore, and China. Scale was co-founded by Jay Shendure, PhD, professor of genome sciences at the University of Washington, Cole Trapnell, PhD, associate professor of genome sciences at the University of Washington, and Garry Nolan, PhD, professor of pathology at Stanford University. The company offers multiple kits: a single-cell RNA kit, a single-cell ATAC kit, and a single-cell methylation kit. (One customer told GEN that the methylation kit, in particular, is exciting and sets Scale apart.)
Singleron was founded by Nan Fang, PhD, formerly of QIAGEN and Novogene, and Jing Zhou, PhD, formerly of Dilworth IP and IsoPlexis. Singleron uses a SCOPE-chip microwell microfluidics system that uses gravity to guide cells into wells and is designed for gentle single-cell partitioning.
Regardless of the technology, the company, or the kit, there are an increasing number of options for researchers interested in obtaining gene expression data of single cells. The consensus is that the field is just beginning. Current technologies can reach about 20% of the roughly 250,000 mRNA transcripts in a mammalian cell. The issue is not the abundance of molecular probes. There are enough barcodes to label every mRNA. So, what is the limitation? Hydridization, reverse transcription, mRNA secondary structure, or library complexity? Opinions vary. But given the momentum in the single-cell community, it won’t be long before limitations are overcome.
