Fig. 2: Implementation of BitSeq.

a, Design of a prototype for high-throughput fluorogenic fuzzy sequencing. b, Photograph of a sequencing module, which consists of a flowcell connected to a manifold for liquid routing and placed on a thermoelectric Peltier device for temperature control. c, Photograph of a sequencing flowcell, which is made of a glass slide and a FOP, with a flow channel in between with shape defined by laser-cut double-sided adhesive. IPA, isopropanol. d, Scanning electron microscopy image of the microwell array made by selective etching of FOP. These femtolitre microwells are placed inside the flow chamber. e, Size of a microwell. f, Running procedure for one sequencing cycle. First, the microbeads with clonal amplified templates for sequencing are introduced into the microwells and tethered on the inner surface of the microwells. Then, the reaction buffer is primed into the flow chamber and all microwells are sealed by oil flowing into the chamber to separate each microwell from cross-talk. The SBS reaction is triggered by elevation of the temperature. The fluorogenic product is proportional to the bases that are incorporated during each reaction cycle. When the reaction is finished, the flowcell is cooled to take fluorescence images. g, One typical field of view from 171 tiles per cycle and the procedure of fluorescence intensity extraction. The microwells that contain beads generate fluorescence after reaction of each cycle, and higher intensity indicates longer degenerate polymer length (DPL) in that reaction cycle. Each microwell is addressable and indexed to produce an intensity series that can be later deduced into bit sequences. h, Read length distribution of bit sequences. i, Error rate of BitSeq.