Sequencing by expansion (SBX) chemistry video

DNA bases are separated by only 3.4 angstroms. The width of a few atoms. DNA sequencing methods cannot easily resolve this dimension. Sequencing by expansion technology or SBX, overcomes this limitation by creating a surrogate molecule called an Xpandomer. This molecule is more than 50 times longer than its target DNA, and encodes the DNA sequence information in large, high signal to noise reporters. The building blocks for the Xpandomer are novel nucleotides called X-NTPs.

A modified DNA replication process synthesizes the Xpandomers by sequentially linking the X-NTPs along a target DNA template using a proprietary enzymatic process. This forms a double stranded helix. Each of the four X-NTP types depicted as A, C, G, and T has a tether that is linked between the base and the alpha phosphate. The tether supports a high signal to noise reporter that mirrors the base sequence identity. Located in the center of each reporter is the Translocation Control Element, or TCE, that modulates Xpandomer movement during sequencing.

As the extension proceeds, only an X-NTP that correctly complements the next base on the DNA template will be incorporated. The sequence information of the DNA is now expressed in the reporter sequence of the X-NTP. This process of linking X-NTPs is continued sequentially and extends along the entire length of the DNA template. After synthesis, a reagent is added which degrades the DNA template, and the cleavable bond between each of the X-NTP bases is broken, allowing the backbone to expand. This product is called the Xpandomer.

The Xpandomer is then threaded electrically through a biological nanopore in a deterministic manner. The translocation control element holds the reporter in the nanopore, altering the electrical resistance of the pore to provide one of four signals that uniquely identifies the corresponding base in the original DNA sequence. After measurement, a short high voltage pulse is applied and the Xpandomer is reliably advanced to the next reporter. This process is performed in a highly parallel manner across millions of wells on a single CMOS based sensor module, and can provide for remarkable real time sequencing rates of hundreds of millions of bases per second. Roche's innovative SBX technology can flexibly scale to satisfy a broad range of applications, setting a new paradigm for next generation sequencing technology.