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Short Read Sequencing

Sequencing by binding (SBB) chemistry delivers a breakthrough in sequencing accuracy. Compared to sequencing by synthesis (SBS) utilized by traditional short-read sequencers, SBB chemistry gives ~15x lower error rates at any given cycle so that you can achieve near-perfect accuracy in short-read sequencing without having to sequence at unnecessary depths.

What is SBB sequencing?

PacBio SBB sequencing is a Q40+ short-read sequencing technology that works by measuring the light signals from fluorescently labeled nucleotides when they are bound –but not incorporated– by a polymerase on a DNA strand. A computer then matches these light signals with a corresponding nucleotide base call. Unlike other short-read technologies, SBB separates the binding and subsequent extension steps of the sequencing process which eliminates the errors introduced by molecular artifacts.

How does SBB sequencing work?Next-level SBB sequencing chemistry proceeds by four primary steps: Initiation, Interrogation, Activation, and Incorporation.

  • With SBB technology, sequencing is initiated by a polymerase on the DNA strand with a reversible blocker on the 3’ end to prevent additional bases from fully incorporating into this growing strand (step 1).
  • Then, during interrogation fluorescently tagged bases flood the flow cell. Unlike traditional SBS sequencing, these tagged bases do not include a blocker. Once the appropriate tagged base binds to the DNA strand, a fluorescent signal is emitted, and this is measured using powerful optics (step 2).
  • In a crucial difference from SBS sequencing, this tagged nucleotide base is not incorporated into the DNA strand but is washed away after the base signal is captured by the sequencing instrument. Thereafter, the 3’ end of the nucleotide is activated via the removal of the reversible blocker (step 3).
  • This ensures the appropriate base will be incorporated from unlabeled, blocked nucleotides that flood the flow cell which blocks additional incorporation until the appropriate time in the next cycle (step 4).

How is SBB different from traditional SBS?

No molecular scarring 

The key innovation behind the breakthrough accuracy of SBB sequencing lies in the fact that the fluorescently tagged nucleotide, which signals the base call, is not directly integrated into the DNA strand, as is typical in SBS sequencing. Separating these steps allows for separate optimization of the two steps, thereby enhancing the polymerase fidelity, signal-to-noise levels, and eliminating the detrimental effects of molecular scarring that occurs in conventional SBS sequencing when the fluorescent tag is removed from the growing DNA strand. With SBS sequencing, the cleaving needed to remove each fluorescent tag leaves residual linker arms attached to each nucleotide base (referred to as molecular scarring). Over the course of many cycles, these molecular residues cumulatively interfere with the polymerase as it attempts to read the DNA strand, which negatively affects the accuracy of the reads.

Minimal duplication 

In addition to avoiding the accumulation of sequencing errors caused by molecular scarring, PacBio SBB sequencing has a much lower duplication rate than conventional short-read technology. Unlike reads that overlap, duplicated sequences add no additional information value to an analysis and are typically removed via bioinformatics in a process called deduplication. Because SBB sequencing creates fewer of these redundant sequences, less total data and computational resources are needed for the deduplication process, making for an overall more streamlined analysis.

Minimal index hopping 

SBB sequencing causes minimal index hopping in multiplexed samples. Unlike SBB, the amplification methods of the patterned flow cells used by legacy SBS sequencing can cause a portion of unique indexes to cross over between sample pools during sequencing. This results in increased noise in the data caused by reads from one sample being erroneously assigned to the other. PacBio SBB sequencing does not have this problem.

Next-level accuracy

By eliminating molecular scarring, reducing duplication, and minimizing index hopping, SBB sequencing delivers extraordinary read accuracy with an unprecedented error rate of only 1 in 10,000 bases or less (Q40+).

Sequencing By Binding On The Onso System

The Onso system is an integrated hardware and software short-read sequencing platform focused on delivering best-in-class accuracy, resulting in over 90 issued patents and numerous pending applications in the US and abroad. The Onso system utilizes SBB chemistry to free researchers from the limitations of existing short-read sequencing platforms, providing ≥90% of bases at Q40+ accuracy.

A new era of short-read accuracy is here SBB Q40+ accuracy on the Onso system

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