Over the last few weeks, any number of articles about Roche's new sequencing technology, called "Sequencing by Expansion" or SBX.
- Fierce Biotech here.
- Genetic Engineering News here.
- Biopharma Trend here.
- Genomeweb here (subscription).
- Roche website here. Investor relations deck here (27pp).
- We describe Sequencing by Expansion (SBX), a single-molecule sequencing technology that overcomes these limitations by using a biochemical conversion process to encode the sequence of a target nucleic acid molecule into an Xpandomer, a highly measurable surrogate polymer. Expanding over 50 times longer than the parent DNA templates, Xpandomers are engineered with high signal-to-noise reporter codes to enable facile, high-accuracy nanopore sequencing.
Roche's New Sequencing Technology: Sequencing by Expansion (SBX)
Roche's Sequencing by Expansion (SBX) is a novel single-molecule sequencing technology that addresses long-standing limitations in nanopore sequencing. Developed initially by Stratos Genomics (acquired by Roche in 2020), SBX introduces an innovative biochemical transformation step that improves the accuracy, signal resolution, and throughput of nanopore sequencing.
How SBX Works: A Technical Breakdown
1. The Fundamental Challenge of Nanopore Sequencing
Traditional nanopore sequencing (such as Oxford Nanopore's technology) works by threading a single DNA strand through a nanopore while measuring changes in ionic current. However, this method struggles with:
- High error rates (>3%)
- Difficulty resolving homopolymer sequences
- Signal-to-noise limitations
- Poor spatial resolution of nucleotides
SBX overcomes these issues by transforming DNA into a different, more easily readable polymer (Xpandomer, Xp) before sequencing.
2. SBX’s Two-Step Workflow: Synthesis & Sequencing
Step 1: Synthesis of Xpandomers (Xp)
Instead of directly threading DNA through a nanopore, SBX first biochemically converts the DNA sequence into an expanded polymer surrogate called an Xpandomer (Xp). This conversion follows a structured biochemical process:
Custom Nucleotide Incorporation (XNTPs)
- SBX uses modified nucleotides (XNTPs) with specially designed reporter tags that expand the DNA strand.
- These XNTPs are incorporated by a specially engineered polymerase (Xp Synthase), which efficiently extends DNA into a high-resolution Xp polymer.
Polymerase Enhancing Moieties (PEMs)
- These molecular cofactors stabilize the Xp polymerization process, allowing long, accurate extension beyond 10 bases.
Acid Cleavage Step (Xp Expansion)
- A controlled acid treatment expands the Xp polymer, stretching it over 50 times longer than the original DNA strand.
- Each Xp unit carries a distinct reporter code (instead of individual nucleotides) that is easily distinguishable during sequencing.
Photocleavage & Release
- The expanded Xp molecule is then released from its solid-phase synthesis platform, making it ready for sequencing.
Step 2: Nanopore Sequencing of Xp
Once synthesized, the Xp polymer is threaded through a nanopore for sequencing. Unlike traditional nanopore sequencing, SBX benefits from:
- Well-defined, high-resolution reporter signals
- The expanded polymer structure prevents overlapping signals, allowing precise base calling.
- Stepwise, controlled translocation
- SBX uses a translocation control element (TCE) to regulate movement through the nanopore, reducing errors.
- Four distinct ion current levels corresponding to each DNA base
- The high signal-to-noise ratio enables accurate single-molecule sequencing.
This unique two-step process—expanding DNA into a surrogate polymer first, then sequencing it through a nanopore—provides higher accuracy, longer reads, and better resolution than existing nanopore sequencing technologies.
Why SBX is Attracting Attention
SBX has generated significant interest in the genomics community due to several key advantages over current sequencing platforms:
1. Superior Signal-to-Noise Ratio
- Traditional nanopore sequencing struggles with poor resolution and high error rates (~3% per read).
- SBX expands DNA, allowing cleaner electrical signals to be read with greater fidelity.
2. Higher Accuracy & Read Length
- By using engineered reporter molecules instead of direct nucleotide measurements, SBX can dramatically improve read accuracy.
- Unlike raw nanopore reads, which often need significant bioinformatic error correction, SBX's stepwise process allows for lower raw read error rates.
3. Cost & Speed Benefits
- SBX allows scalable, high-throughput sequencing using an 8-million sensor nanopore array.
- The expansion process reduces the computational burden of base calling, making sequencing faster and cheaper.
4. Compatibility with Read-Intensive Applications
- Whole genome sequencing (WGS)
- Single-cell RNA sequencing (scRNA-seq)
- Spatial transcriptomics
- Somatic mutation detection
- Long-read sequencing applications
5. A Major Leap for Nanopore Sequencing
SBX is being positioned as a game-changer in the nanopore sequencing market, directly competing with Oxford Nanopore but offering greater accuracy, reduced error rates, and a better signal-to-noise profile.
The Road Ahead
Roche plans to further refine and commercialize SBX by integrating it with high-throughput sequencing workflows, making it accessible for both research and clinical applications. Future publications will likely showcase:
- Performance benchmarks for germline and somatic sequencing
- Validation studies for clinical applications
- Comparative analyses against existing nanopore and short-read technologies
Given these technical breakthroughs and performance improvements, SBX is poised to disrupt the sequencing landscape, providing a cost-effective, accurate alternative to both short-read (Illumina) and direct nanopore sequencing (Oxford Nanopore).
Roche’s Sequencing by Expansion (SBX) has generated significant interest in the genomics field, largely because it addresses long-standing limitations in nanopore sequencing technology. Traditional nanopore sequencing methods, such as those developed by Oxford Nanopore Technologies (ONT), rely on direct measurement of ion current changes as DNA passes through a nanopore. However, this approach has struggled with fundamental signal-to-noise challenges, resulting in relatively high error rates (typically above 3%), difficulty resolving homopolymer sequences, and suboptimal spatial resolution. SBX offers an entirely new approach, overcoming these limitations by first converting DNA into a distinct surrogate polymer—a process that enhances both signal clarity and sequencing accuracy.
The SBX workflow consists of two key steps: synthesis and sequencing. During synthesis, a DNA strand is enzymatically converted into an Xpandomer (Xp), a polymer that expands up to 50 times longer than the original DNA sequence. This transformation is made possible through the use of engineered XNTPs, specialized nucleotide analogs that incorporate reporter codes rather than direct nucleotide information. A highly optimized polymerase, called Xp Synthase, facilitates the controlled synthesis of these Xp molecules. Additional enhancements, such as Polymerase Enhancing Moieties (PEMs), stabilize the polymerization process and enable longer, high-fidelity sequence extensions. Once the Xp molecule is fully synthesized, an acid cleavage step expands the polymer backbone, making it easier to read in the subsequent nanopore sequencing phase.
In the sequencing step, the expanded Xp polymer is passed through a nanopore, but unlike traditional nanopore sequencing, SBX leverages stepwise, controlled translocation to prevent signal degradation. A Translocation Control Element (TCE) ensures that each nucleotide reporter is positioned precisely within the nanopore before measurement, significantly reducing sequencing errors. Because each Xp molecule contains distinct, high-resolution reporter codes, the electrical signals generated during translocation are far more distinguishable than the raw DNA signals used in conventional nanopore methods. This separation between DNA synthesis and sequence measurement is a key advantage, as it allows each process to be independently optimized for accuracy and efficiency.
SBX has attracted attention not only for its technical innovations but also for its potential to reshape sequencing economics and accessibility. One of the biggest limitations in sequencing today is the tradeoff between accuracy, throughput, and cost. Short-read sequencing technologies (such as those from Illumina) are highly accurate but struggle with long-read applications, while existing nanopore sequencing methods offer long reads but require substantial computational correction due to their higher error rates. SBX bridges this gap, offering a nanopore-based approach that delivers long reads with dramatically improved accuracy, reducing the need for post-sequencing error correction. By decoupling DNA sequencing chemistry from the electrical signal measurement process, Roche has created a method that not only enhances raw sequencing accuracy but also reduces computational overhead, improving both speed and cost efficiency.
Another factor contributing to SBX’s excitement is its potential for read-intensive applications. Traditional nanopore sequencing has been limited in its usefulness for applications requiring extremely high accuracy, such as somatic mutation detection, single-cell RNA sequencing (scRNA-seq), and spatial transcriptomics. SBX, by improving signal clarity and sequencing fidelity, may enable more routine use of long-read sequencing in clinical and research settings. Its ability to accurately process complex genomic regions makes it particularly well-suited for whole genome sequencing (WGS), rare variant detection, and structural variant analysis.
Looking ahead, Roche plans to further refine and validate SBX for high-throughput applications, integrating it with an 8-million sensor nanopore array that could dramatically scale sequencing operations. Future publications and performance benchmarks will likely compare SBX with existing nanopore and short-read technologies, providing deeper insights into its capabilities. If Roche successfully commercializes SBX at a competitive price point, it could disrupt the sequencing landscape by offering a cost-effective, high-accuracy alternative to both Illumina’s short-read dominance and Oxford Nanopore’s direct DNA sequencing. Given these advantages, SBX is positioned to become a transformative technology in genomics, expanding the reach of long-read sequencing into mainstream research and clinical applications.
