Johan Strömqvist, PhD, has always been interested in “seeing” molecules, particularly in three dimensions (3D). Strömqvist worked with optical single-molecule imaging for his PhD and then founded a company in 2010 to look at protein-protein interactions to see how different compounds modulated those interactions to improve the attrition rate of drug candidates in clinical testing. Later on, Strömqvist began working with fiberoptics innovator Bengt Sahlgren—who was instrumental in the rise of Hexatronic, the largest Nordic fiberoptic company—to take confocal imaging to the next level.
Around 2014, in the basement of a suburban home in Stockholm, Strömqvist and Sahlgren began working together on an optical technology for proteomics and spatial biology. After ditching a multiplex method for looking at multiple genes using barcodes, like a spatial gene-expression platform, the duo switched their attention to in situ next-generation sequencing (NGS). That’s when they realized they could use their optical system to sequence using a 3D matrix for hugely parallel reactions in three dimensions.
“We realized that the technology scales with a cube, which is a game changer because all NGS technologies, including long-read applications, scale with a square,” Strömqvist told GEN Edge.
“What Illumina did was scale reads with a square, which is why it took over Sanger sequencing—they went from one dimension to two dimensions. Now we’re going to three dimensions.”
Too good to be true?
A few key innovations combine to make Single Technologies’ 3D DNA sequencing a potential game changer in NGS technology.
The first innovation is the development of a high-speed confocal scanner that enables imaging of large areas in three dimensions with single fluorescence molecule sensitivity. This is an approach that has been explored by the likes of Illumina but was abandoned because of speed issues leading to the use of line scanning.
The second innovation concerns ditching the concept of a single, two-surface flow cell patterned with billions of nanowells for a 3D matrix. Strömqvist explained that their matrix works a bit like a stack of Post-it notes containing DNA information. According to Strömqvist, this format enables 3D DNA sequencing in situ.
“If you take all the Post-it notes and put them on the table, you could fill a huge table,” said Strömqvist. “So, if you have a mouse brain, you could either chop it up into small 10-micron slices and distribute it over a huge area, or you can basically keep it pretty much like it is.”
Strömqvist and Sahlgren have been working out the kinks to turn their technology, which they call “3D DNA sequencing,” from a basement DIY project into a commercial solution with their startup, Single Technologies. According to Strömqvist, Single Technologies has generated data over the past two years using a prototype of their fully automated 3D DNA sequencer Theta and is currently developing two commercial systems that should be ready by the start of 2025.
However, not everyone is convinced that the revolution is happening at Single Technologies. In a breakdown of Single Technologies’ patents, sequencing technologist Nava Whiteford, PhD, said that Single Technologies doesn’t describe much innovation in their intellectual property (IP) outside of the optical system. Whiteford said the patterned flow cell—which seems to be (in addition to the imager) the core innovation for Single Technologies—is not described.
Strömqvist also claimed that Single Technologies’ 3D DNA sequencing can be applied to almost any fluorescent-based sequencing chemistry. Whiteford is dubious, noting that the company lacks employees with the backgrounds required to develop a new sequencing chemistry.
According to Whiteford, the best bet for Single Technologies may be to partner with a company with existing sequencing chemistry or to be acquired by an existing sequencing company, a situation in which Single Technologies could provide a throughput advantage with their improved optical system.
Keith Robison, a well-known NGS expert and founder of the “Omics! Omics!” blog, is also skeptical, telling GEN Edge that he hasn’t seen anything “solid” from Single Technologies. Single Technologies has only raised €16 million ($17 million) so far, through a mixture of traditional fundraises and equity financing over the past decade.
Citizen Kain
However, Single Technologies does have its converts, perhaps most notably Bob Kain, who gained fame for his role in the development of Illumina’s Hi-Seq, which brought the sequencing cost of a human genome down to around $1,000. Since leaving Illumina in 2014, Kain has been advising several NGS companies. Kain told GEN Edge that, besides what he’s seen from Single Technologies, he hasn’t been particularly impressed.
“While many companies out there have interesting improvements in sequencing, they all have certain drawbacks. I don’t think they’re compelling enough to get around the barriers to entry that Illumina has put up into switching costs,” said Kain.
“The reality behind sequencing with Hi-Seq and Nova-Seq is really all about how many bases per reagent dollar and bases per day you can get at the appropriate accuracy in the long run. That’s what Illumina wins with.”
But Kain also doesn’t think that Illumina has made any major improvements to NGS or innovated much beyond the Hi-Seq.
“The Hi-Seq was a series of exponential technologies that went into one instrument, and as those technologies individually moved forward, the instrument itself could get higher performance without major reinvention,” said Kain. “Even the Nova-Seq is more of a reinvention based on the user interface, things like that, and how you handle reagents. It’s not a reinvention of the main components.”
When Kain first heard about Single Technologies’ 3D DNA sequencing, he thought it was just an interesting, very early-stage technology that wasn’t far enough to get him to buy in.
“Interesting technologies often mean problems to solve,” said Kain. “But over months, I realized that they have been quietly solving these problems for a while, and the hurdles I might have thought of when I understood their technology were already addressed. I was really impressed by that.”
Searching for a spark
Last December, Kain flew to Stockholm to visit Single Technologies. He was impressed by how well the technology was developing and the state of their instrument. In April 2024, Kain joined Single Technologies as an advisor to support the commercialization of Theta.
“When I saw this technology, I thought this optical architecture had the potential to bring the cost per human genome down to $10 and below,” said Kain. “It’s a new exponential curve. Maybe it’s a little dramatic, but it’s like going from tubes to transistors and transistors to microcircuits…Each time, you get on a new exponential curve.”
But this technology that Kain touts as being transformational to NGS hasn’t materialized. The only live wire coming from Single Technologies pertains to a project called “Regenerar,” which aims to use epigenetic manipulation to reprogram glial cells in the brain into neurons. Single Technologies is not playing a leading role in the project, which has received €3 million ($3.2 million) from the European Commission’s European Innovation Council Pathfinder program, but rather is providing its 3D DNA sequencing technology.
Though Kain and Strömqvist want to capitalize on the purported agnosticism of their platform and stand alone at the top of the NGS pyramid, the solution to summiting may require partnering with a company with a proven sequencing chemistry. The best way for Single Technologies to show the exponential cost-saving and experimental value of “digitizing” samples is to prove it, and to do so they may not need to reinvent the sequencing chemistry wheel.
It is unclear if Single Technologies will be able to survive financially through the four years of the Regenerar project. Perhaps Kain’s endorsement will trigger a chain reaction of excitement and investment, taking Single Technologies from patents and prototypes to the pinnacle of an already crowded NGS market
But without more cash, Single Technologies might struggle to make it out of the basement.
