NEW YORK – In a highly anticipated webinar on Thursday, Roche offered more technical details about its so-called Sequencing-by-Expansion (SBX) nanopore sequencing technology.
The platform, which has been in development for over a decade, is scheduled for a 2026 launch, according to the firm.
During the event, Roche made clear that the platform is underpinned by two fundamental technologies: a semiconductor array chip, which Roche obtained through its acquisition of Genia in 2014, and the SBX chemistry that was originally developed by Stratos Genomics, which Roche acquired in 2020.
In an interview with GenomeWeb this week, Mark Kokoris, Roche's head of SBX technology and a cofounder of Stratos, said there has been "a continual stream of innovation" over the past several years. "[T]his is the coming together of those two technologies."
Co-invented by Kokoris and Robert McRuer, another Stratos cofounder, the SBX chemistry works by enzymatically converting a DNA molecule into a larger surrogate, dubbed Xpandomer. Four distinct novel molecules called X-NTPs, or High Signal-to-Noise Reporters — one for each DNA base — are used during the Xpandomer synthesis.
The Xpandomer, which is more than 50 times longer than the original DNA fragment, then passes through a biological nanopore, creating distinct electrical signals for each X-NTP that are translated back into the original nucleic acid sequence.
"Our approach to efficiently sequencing DNA is to not sequence DNA," Kokoris explained.
During the presentation, Kokoris delineated some of the technical innovations to optimize the Xpandomers for the nanopore readout. For instance, he noted that the researchers introduced a so-called translocation control element, enabling a "clean, reproducible, synchronized modulation of the Xpandomer as it moves through the nanopore."
Additionally, since "most polymerases don't like X-NTPs," Kokoris said, the team introduced enhancer chemistries dubbed Symmetrically Synthesized Reporter Tethers (SSRTs) into the Xpandomer structure.
Furthermore, Kokoris said the team deployed rational design as well as high-throughput screening to engineer and select appropriate polymerases that can incorporate X-NTPs with high affinity.
He also offered an overview of the instrumentation for SBX sequencing, an Xpandomer synthesis instrument as well as an SBX sequencer.
The Xpandomer synthesis platform is essentially a liquid handling system that directs reagents onto a so-called XP chip, where Xpandomers are produced through solid-phase synthesis, Kokoris said.
Meanwhile, the SBX sequencer can achieve single-molecule sequencing using a Complementary Metal Oxide Semiconductor (CMOS)-based sensor module, an array that combines microwells, electrodes, and detection circuits and that handles the analog to digital conversion.
According to Kokoris, a lipid bilayer with embedded protein nanopores is created for every sequencing run. Of the 8 million parallel sensors, the company typically uses about 7 million to 7.5 million functioning pores per run. After each run, the sequencing sensor module can be reused.
Kokoris declined to go into the details of Roche's nanopore design other than noting that the company has its "own pore" for SBX sequencing. He also declined to comment on whether there is any overlap between Roche's protein nanopore and those owned by Oxford Nanopore Technologies, which claims a strong grip on the IP in the field.
Kokoris said his team believes having dedicated instruments for Xpandomer synthesis and sequencing "actually maximizes the flexibility of the technology," though it might also be possible to integrate the platforms down the road.
During his presentation, he highlighted two modes of sequencing for SBX: simplex, which Roche promises to be "ultra-high throughput," and duplex, which has increased accuracy.
For SBX duplex sequencing, a hairpin Y adapter is added to the template DNA molecule during library prep, which in turn produces an Xpandomer that contains both parental strands of the original molecule, resulting in increased sequencing accuracy, Kokoris noted.
The duplex library requires 20 ng to 50 ng of unfragmented genomic DNA or 2.5 ng to 10 ng of cell-free DNA, though Kokoris said there is "room to whittle that down and use even less" DNA input. Additionally, the duplex library is produced using linear amplification rather than PCR amplification to boost accuracy for homopolymer indels, he noted.
Using the HG001 cell line as a benchmarking sample, Roche claimed SBX duplex whole-genome sequencing can achieve a quality score of Q39, with F-1 scores of 99.80 percent for single nucleotide variants and 99.56 percent for indels.
In another sequencing experiment using seven Genome in a Bottle (GIAB) samples, the sequencer generated 5.3 billion duplex reads in an hour with each sample having 34X to 38X coverage.
In terms of read length, Roche quoted a 150 bp to 350 bp insert length for duplex sequencing and 200 bp to more than 1,000 bp for simplex sequencing.
Besides speed and throughput, Roche emphasized the flexible scalability of SBX. "The one thing on our mind from the very beginning of SBX was flexible operation," Kokoris said. "We want to be able to sequence up and down the throughput spectrum with impunity."
Raw sequence data from the sensor are base called instantly, according to John Mannion, head of computational sciences at Roche's molecular labs systems. "Real-time analysis is important," he said during the webinar. "We want to make sure that we are able to compress that data as it's streaming off and then allow users to move that to their location of secondary analysis."
In addition, Mannion said duplex sequencing, mapping, alignment, and germline variant calling are "all accelerated through various means."
Regarding the detection of epigenetic signatures, Kokoris said the technology is "compatible with all the conversion chemistries," and the team is still working to decide whether to optimize duplex or simplex sequencing for such assays.
In addition, Kokoris said the SBX workflow is compatible with automation, and users can convert their existing Illumina libraries for the technology.
So far, Roche has deployed the SBX platforms to two early-access collaborators: the Hartwig Medical Foundation in the Netherlands and the Broad Institute.
According to Gustav Karlberg, Roche's life cycle leader of sequencing systems, the company will continue to work with these early-access collaborators throughout 2025 to demonstrate the performance of the SBX technology. In addition, Roche is looking to expand the early-access program "in a selective way" by including additional collaborators with other applications and workflows of interest.
After that, Roche aims to first release the SBX platform as research product in 2026, Karlberg said.
Palani Kumaresan, global head of Roche diagnostics solutions, said that at launch, the company will introduce SBX "as an open, standalone sequencing solution that both academic researchers and translational researchers can use to advance their work."
At the same time, "we will have it as part of an end-to-end sequencing ecosystem that we can bring into a clinical setting, along with our assay kits," he added.
Whenever Roche is ready to launch its SBX sequencer, it will find an NGS landscape vastly different from what it was a decade ago, when it acquired Genia and Stratos.
Besides Oxford Nanopore and Pacific Biosciences, which continue to improve their accuracy, throughput, and cost, legacy players such as Illumina are also racing to enhance their read length.
Additionally, China's BGI Group and Santa Clara, California-based AxBio, which maintains operations in the US and China, are also entering the nanopore sequencing space.
Roche declined to disclose the cost of SBX during the webinar. Kokoris said the firm strives to make the technology efficient and cost-competitive.
"If you're developing a sequencing technology, you have to be relentlessly thinking about the efficiencies of your process," he said. "That is top of mind for us always."