Next-generation sequencing (NGS) has transformed our understanding of biology from human health and disease to plants, animals, and the world of microbes. Past advances fueled a brilliant era of genomics, but core technology gains have since stagnated. Avidity base chemistry (ABC) is an original sequencing technology that leverages the power of avidites to disrupt establishment models with foundational improvements to cost, quality, and flexibility.
Like sequencing-by-synthesis (SBS), ABC sequencing amplifies and sequences DNA libraries, including those prepared from RNA. Differences in how each technology approaches this function, however, allow NGS applications to evolve beyond SBS.
ABC innovations start with rolling circle amplification (RCA). The flow cell surface captures DNA templates that RCA then replicates and organizes into polonies, where many copies of the template exist as a single concatemer.
RCA improves data quality in two key ways:
- Low-binding surface chemistry creates negligible, nonspecific binding and increases the contrast-to-noise ratio (CNR).
- Copying only the original DNA template removes a common source of error propagation and data decline: PCR.
Two tasks, two steps
Any polymerase-based method of sequencing must accomplish two tasks:
- Detect nucleotide incorporation
- Advance the template to the next position
SBS locks these tasks together as a single step, advancing the register when a blocked, dye-labeled nucleotide is added to the growing complementary strand. Covalent incorporation enables a persistent signal for base detection but requires micromolar reagent concentrations to complete the reaction in a reasonable timeframe.
ABC separates these steps, optimizing enzymes and enzyme conditions for each and consuming mere nanomolar reagent concentrations to create stable complexes for base detection.
Avidites and the power of affinity
Avidity refers to the cumulative strength of multiple affinities of noncovalent binding interactions, which is achieved when multivalent ligands bind to multiple sites in a substrate. Avidites—dye-labeled polymers carrying many identical nucleotides—leverage this strength to label and image polonies with minimal reagent consumption.
Although avidites have similar association rates as the dye-labeled monovalent nucleotides of SBS, ABC maintains the advantage: avidites show no disassociation during the > 1 minute needed for base detection, even at nanomolar concentrations. After base detection, buffers wash away the avidites without the DNA scarring that cleaving often leaves.
Spotlight on homopolymer regions
ABC is not only less expensive than SBS—it is also more accurate. The accuracy gain is particularly prominent in homopolymer regions, which maintain low error rates pre- and post-homopolymer. Element researchers attribute the higher accuracy to RCA, an engineered high-fidelity polymerase, and two additional factors:
- Synergistic binding of multiple nucleotides on a single avidite makes sure that only the correct avidite binds to a polony.
- A binding disadvantage for out-of-phase DNA copies that are inside the polony and lack other out-of-phase neighbors to serve as avidity substrates.
Novel technology with unexplored potential
Avidites enable a modular design that presents clear paths forward for further innovation and improvement, making ABC a highly extensible technology. The polymer core, number and choice of dyes, and length and structure of nucleotide linkers are set up for parallel optimization that increases signal, decreases cycle times, lowers reagent concentrations, and tunes other parameters to neatly align to different applications.
For example, a novel polymerase and optimized reagent formulation have already enabled 2 x 300 sequencing. The results are excellent. Cloudbreak™, which accelerates run times by 20% and enables linear library loading, is another example.
Publication and proof-of-concept
Entwining low cost and high quality, ABC takes a first-principals approach to stretch beyond the limits of SBS and empower more labs to do more science. In a seminal publication, Element researchers describe the kinetics of ABC in detail, including proof-of-concept data from human whole-genome sequencing (WGS) and single-cell sequencing. Datasets provide further visibility into ABC performance.