Sequencing By Xpansion (SBX) is a DNA sequencing method that uses a simple biochemical reaction to encode the sequence of a DNA molecule into a highly measurable surrogate called an Xpandomer. This single molecule approach produces enough Xpandomer in a single drop reaction to sequence an entire human genome 1000X over. To achieve this, an Xpandomer replaces each DNA sequence with a sequence of large, high signal reporter molecules using the SBX molecular expansion technology. The DNA sequence is then read out as the Xpandomer reporters pass sequentially through a nanopore detector. SBX is a molecular engineering platform that benefits from core design principles that separate the multiple molecular functions. This systems approach enables efficient development and incorporation of improvements to SBX and is key to reconfiguring and optimizing Xpandomer measurement for different detection platforms.
SBX SEQUENCING DEMONSTRATION
Stratos Genomics demonstrated 36-base DNA sequencing in July 2012 and rapidly improved to 210 bases by Sept 2013. This proof-of-concept included conversion of target DNA to Xpandomer and sequence read-out of the Xpandomer with a wild-type alpha-hemolysin nanopore. Data presented is averaged with a 1 ms filter and the overlaid red lines are autocalled state levels. Repeated bases (homopolymers) are resolved by alternately encoding between the normal base state and an additional homopolymer reporter state (Hp). The base type associated with an Hp state is determined by the previous state it transitioned from. After demonstrating 210 base reads, efforts were refocused on the X-NTP development.
The probe-based building blocks used to synthesize Xpandomers are called Xprobes. For most process development and the proof-of-concept demonstration, a library of hexamer Xprobes were used, each of which encoded for its 6-base probe sequence. To encode all sequences, a full library of 4096 hexamer Xprobes is required. To simplify the commercial product, a new single-base expandable nucleotide called an X-NTP is in development that will reduce the library requirement to 4, one for each base. X-NTPs have additional advantages of polymerase incorporation, faster kinetics and very low-cost synthesis. At the end of 2013, a major milestone was achieved with the synthesis of the full X-NTP library (A,C,G,T).