Technology

Sequencing by expansion (SBX) technology

Sequencing by expansion (SBX) - a closer look
Overcoming limitations of today’s next-generation sequencing (NGS) technologies

As Roche Sequencing Solutions looks forward to meeting tomorrow’s biggest needs in genomics, it’s clear that today’s sequencing technologies face several limitations that impact growing needs for faster speed, improved accuracy, greater scalability, and increased flexibility.

Some traditional sequencing technologies leverage a cycle-based approach for measuring the bases of the DNA, which delays access to usable data. While on-market single-molecule nanopore technology addresses this challenge, it can be limited by fundamental signal-to-noise limitations—a result of poor spatial resolution and molecular distinction of nucleobases.

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A new approach to NGS

Roche has responded to the demand for improved performance by developing a new category of NGS technology, called sequencing by expansion (SBX). This powerful approach to NGS has been designed for flexibility and performance, with headroom to scale into the future. Specifically, SBX boasts several advantages, including:

  • Flexible operation that is tunable to sample needs
  • High accuracy with demonstrated F1 scores of >99.80% (SNV) and >99.56% (InDel) for HG001 whole genome samples
  • Very high throughput capable of 7 genomes in 1 hour at >30x; >5B duplex reads in 1 hour of sequencing
  • Flexible read lengths spanning 50bp to >1000bp
  • Ultra-fast workflow options for urgent samples, including sample to variant call format (VCF) in <7 hours
  • Cost efficiency enabled by a scalable and reusable sensor module

Fundamentally, 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 Roche’s proprietary nanopore, driving single-molecule sequencing at incredibly high rates of speed and facilitating rapid access to usable sequencing data.

X-NTPs

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, 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. 

X-NTP structure diagram
xpandomer-synthesis chart

Xpandomer Synthesis

Each of the four, easily differentiated X-NTPs (one for each base), act as substrates for template-dependent, polymerase-based replication. The polymerase, XP synthase, has been carefully engineered to incorporate large X-NTP monomers, enabling >99.3% mean raw read accuracy, uniform GC coverage, and longer read lengths. Polymerase enhancing moieties, or PEMs, are also added to the synthesis reaction to assist the polymerase in properly incorporating X-NTPs into the growing polymer. 

By stabilizing the extending molecule, PEMs play an important role in increasing read length beyond traditional short-read sequencing technologies. Following synthesis of the surrogate molecule, acid-cleavable bonds are broken, allowing the newly synthesized Xpandomer to extend 50X longer than the original DNA molecule. 

Single-molecule measurement

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. 

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 without the convolution issues of traditional nanopore sequencing. The result is the cost-effective measurement of hundreds of millions of bases per second, bypassing the traditional approach of cyclical incorporation and measurement of a single base at a time. 

single-molecule measurement

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

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