What is Sequencing by Expansion, and how do you analyze the data?

SBX: a new sequencing technology bringing NGS to new speeds

Image from the SBX pre-print by Kokoris et al.

Sequencing by Expansion (SBX) is a new nanopore-based sequencing technology that converts DNA into a highly measurable surrogate called an Xpandomer to boost the signal-to-noise ratio when sequencing. This innovative technology achieves sequencing speeds superior to older techniques and could expand NGS applications in healthcare, where fast turnaround times are essential.

According to a recent bioRxiv paper from Roche, single-molecule sequencing by expansion technology overcomes the challenge of sequencing noise faced by traditional nanopore approaches, as the synthetic Xpandomer is up to fifty times longer than original DNA or RNA molecules, meaning it generates well-separated signal peaks for more accurate base calls.

But how does sequencing by expansion work, and what does it mean for your omics data analysis pipeline?

Sequencing by expansion in a nutshell

Sequencing by expansion doesn’t directly sequence DNA (or RNA). It uses DNA as a template to create a surrogate molecule called an Xpandomer that represents the DNA sequence with large reporters (1).

Four expandable NTPs (X-NTPs) form the backbone of these Xpandomers, and each includes a reporter code for detection, enhancers for Xpandomer synthesis, a translocation control element for nanopore transit, and an acid-cleavable bond for expansion after replication.

These X-NPTs act as substrates for template-dependent, polymerase-based replication. The polymerase, called XP synthase, incorporates the large X-NTP monomers in the Xpandomers, which then feed through a complementary metal-oxide-semiconductor-based (CMOS-based) nanopore. These nanopores can scale to array sizes of eight million, indicating the potential for ultra-high throughput uses like single-cell sequencing. You can check the official video from Roche for a visual explanation.

https://medium.com/media/dd3747d5b6236fb119b2236a339b1744/href

Why should you care about sequencing by expansion?

According to Roche, sequencing by expansion combines flexibility and throughput with accuracy, which is a balance that other sequencing approaches sometimes struggle to achieve.

The company reports that the technology is capable of sequencing seven genomes in one hour at over 30x depth. In one hour, it can generate over 5 billion duplex reads.

These reads can be flexible in length, from 50bp to over 1000bp long. While traditional short-read sequencing approaches like those from Illumina can’t yet achieve these lengths, long-read sequencing platforms like PacBio and Oxford Nanopore easily surpass this.

However, while sequencing by expansion technology clearly holds promise, the pre-print only provides proof-of-concept with a 222 base DNA template and lacks direct benchmarking data comparing the accuracy of sequencing by expansion technology to Illumina, PacBio, or Oxford Nanopore. This makes direct comparisons or claims difficult.

In one study, researchers found that Oxford Nanopore technology is limited by raw read average error rates of approximately 3%, but this can vary by the basecaller or version used (2). Roche states that sequencing by expansion ‘demonstrated F1 scores of >99.80% (SNV) and >99.56% (InDel) for HG001 whole genome samples’.

This is impressive accuracy, but as sequencing by expansion technology is in its early stages, its true error rates remain to be seen. Future direct benchmarking comparisons will help.

Meanwhile, the potential of a fast and precise sequencing technology is highly encouraging for both healthcare and precision medicine. Although NGS has been widely used in research — with advanced techniques like spatial transcriptomics — these approaches often overlook the critical requirements of healthcare, where speed and accuracy are vital. With nearly instantaneous and accurate results, SBX could be the key to broadly expanding the use of NGS in diagnostics and treatment decisions.

Sequencing by expansion data analysis: What we know

Very little, unfortunately! The preprint provides limited information on how users would analyze sequencing by expansion data, but given the technology is nanopore based, it is highly likely that raw outputs would be compatible with existing analysis pipelines and standard FASTQ formats.

With this, it would be possible to plug-and-play into existing bioinformatic or AI-based analysis pipelines, and upstream analyses would be largely dependent on the type of experiment performed (whole-genome sequencing, RNA-seq, single-cell RNA-seq, etc.).

The technology is a work in progress and will no doubt hit the market in the next few years.

As the authors state: ‘The data presented here were captured in January of 2020 using an early chemistry that demonstrates all the critical functionality of the SBX technology and is a prelude to newer chemistry now being applied to large CMOS arrays.’

We can expect big things for the future of sequencing by expansion, especially in fields where time is of the essence.

Struggling with your omics data analysis? Schedule a free consultation with Nexco Analytics to learn how we can help.