Fig. 1: Avidity sequencing workflow and scheme.

a, Sequencing by avidity. A reagent containing multivalent avidite substrates and an engineered polymerase are combined with DNA polonies inside a flowcell. The engineered polymerase binds to the free 3′ ends of the primer-template of a polony and selects the correct cognate avidite via base-pairing discrimination. The multivalent avidite interacts with multiple polymerases on one polony to create avidity binding that reduces the effective K_d of the avidite substrates 100-fold compared with a monovalent dye-labeled nucleotide, allowing productive binding of nanomolar concentrations. Multiple polymerase-mediated binding events per avidite ensure a long signal persistence time. Imaging of fluorescent, bound avidites enables base classification. Following detection, avidites are removed from the polonies. Extension by one base using an engineered polymerase incorporates an unlabeled, blocked nucleotide. A terminal 3′ hydroxyl is regenerated on the DNA strand, allowing repetition of the cycle. b, Rendering of a single avidite bound to a DNA polony via polymerase-mediated selection. The initial surface primer used for library hybridization and extension during polony formation is shown in blue. Sequencing primers (red) are shown annealed to the single-strand DNA polony (gray). Each arm of the avidite (black) connects the avidite core containing multiple fluorophores (green) to a nucleotide substrate. The polymerase bound to the sequencing primer selects the correct nucleotide to base pair with the templating base (inset). The result is multiple base-mediated anchor points noncovalently attaching the avidite to the DNA polony. c, Rendering of multiple DNA polonies with template-specific avidites bound during the binding step of the cycle (polymerase not shown for simplicity). Many avidites bind to each DNA polony generating a fluorescent signal during detection. Multiple long, flexible polymer linkers connect the core to the nucleotide substrates.