Sequencing by expansion: A novel approach to next generation sequencing

Next-generation sequencing enables labs to perform rapid sequencing of DNA to evaluate the underlying causes of disease, which is critical for precision medicine and clinical diagnostics.¹ Roche recently introduced a new sequencing technology known as sequencing by expansion (SBX*). As a new approach in the NGS space, SBX technology converts DNA information into a longer, “expanded” molecule, overcoming the spatial challenges of current nanopore technology and enabling higher signal-to-noise for improved accuracy. This expanded molecule, or Xpandomer, is then fed through a nanopore, driving single-molecule sequencing at incredibly high rates of speed and facilitating rapid access to usable sequencing data.²

In a recent webinar, Mark Kokoris, Head of SBX Technology at Roche Diagnostics, and John Mannion, Head of Computational Sciences, Molecular Lab Systems at Roche Diagnostics, showcased the SBX technology, specifically how the technology works and its potential to address the current needs for genomics research and improve upon translational and future clinical applications.

“Our approach to efficiently sequencing DNA was to not sequence DNA,” said Kokoris. “SBX is a biochemical process using modified nucleotides and enzymes to convert DNA into an expanded surrogate molecule.” He added, “We would rescale the signal to noise challenges of direct DNA measurement.”

SBX technology utilizes a proprietary biochemical conversion process to expand and encode the sequence of a DNA template into an Xpandomer molecule. The building blocks of the Xpandomer are expandable nucleotide triphosphates, or X-NTPs. These high signal-to-noise reporters are the result of sophisticated molecular engineering and include an easily differentiated reporter code corresponding to the original base, a translocation control element for highly controlled transit through the pore, enhancers for robust Xpandomer synthesis, and an acid-cleavable bond for post-replication expansion. The Xpandomer molecule is then routed through a biological nanopore in a highly efficient and accurate manner. Movement of the Xpandomer through the pore is guided by voltage pulses that advance the Xpandomer through the pore one reporter code at a time.²

The highly differentiated reporter codes are easily measured during this translocation process via a scalable complementary metal oxide semiconductor (CMOS)-based array, which combines electrodes, detection circuits, and analog-to-digital conversion. Because the CMOS array contains roughly eight million microwells (each containing a nanopore), measurement occurs in a massively parallel, highly controlled manner. Importantly, these sensors are reusable.² “The key part of the technology is to be able to reuse the sequencing sensor module,” said Kokoris.

The CMOS-based sensor module contains an array of millions of microwells. This module performs a similar purpose to a traditional flow cell on other systems, but with the sensor module, the lipid membrane can be formed multiple times. This leads to the reuse of the same sensor module, significantly lowering the cost of operation. Sequencing is performed by threading Xpandomers through the pores at the rate of hundreds of millions of bases per second per sensor module.² According to Kokoris, the setup is flexible and scalable for either low-throughput or very high-throughput sequencing runs.

Different SBX library structures and workflows are possible depending on application needs. This includes SBX-duplex (SBX-D) reads for double-stranded DNA of 200-300 base pairs and SBX-simplex reads for single-stranded DNA, from less than 200 base pairs and up to 1500 base pairs.² “We wanted to have a technology that could support low throughput, very high throughput, everything in between,” commented Kokoris.

Scientists at Hartwig Medical Foundation were given early access to SBX and used the technology to evaluate clinical research sample pairs, specifically single nucleotide variant (SNV) and indel counts. Broad Institute Clinical Labs was also given early access to SBX, which would be critical in accelerating clinical whole genome sequencing. According to Kokoris, the rapid deployment of SBX systems in these medical facilities, which are operational within days, highlights their efficiency and ease of implementation.

Also in the webinar, Dr. Mannion discussed data processing, providing a deeper dive into the read characteristics and local analysis strategy. For SBX-D characteristics, he details how full-length and partial duplex reads are generated and presents data on base accuracy, insert length distribution, and homopolymer performance. Dr. Mannion also talked about the accelerated local data processing approach that aims to perform base calling, mapping, and alignment in real time. “It does not take multiple hours, or maybe a day or even more to generate full, intact reads. Rather, complete reads, fully intact, are generated on the order of seconds,” says Dr. Mannion. 

“You can quickly imagine a great number of useful features that will be made possible by such real-time integrated analysis, in combination with the SBX chemistry and sensor module,” commented Dr. Mannion. “Our strategy is for the instrument’s local compute to be as fast, as flexible, and importantly, as efficient as the SBX technology itself.”

Kokoris and Dr. Mannion also provided an overview of the workflows and data processing for SBX-D and SBX-simplex reads. This includes discussions on the approach’s performance metrics and simplified library prep. Furthermore, this includes the technical details for read properties, including raw read error profile and accuracy, and computational strategy, including access to open-source analysis tools. 

The SBX technology* will not only support a wide range of applications but will also bring multiple benefits to the lab, including improved turnaround time and the flexibility to accommodate different sample sizes and workflows. According to Kokoris, SBX sequencing will be useful for a broad range of clinical research and translational applications, from genetic diseases to somatic oncology, especially for whole genome, exome, and transcriptome sequencing, as well as epigenetics and proteomics.

Want to hear more about SBX? Click here to watch the on-demand webinar now!

*The SBX technology is in development and not commercially available. The content of this material reflects current study results or design goals.