Fig. 4: Visualization and identification of DNA barcoded antibodies at single-molecule resolution.

a Schematic illustrations showing a procedure of sequencing of DNA barcoded antibody (Ab-Oligo) on our system. b A fluorescence image of individual CD55 Ab-Oligos visualized by the 1st virtual terminator incorporation. Scale bar represents 3 μm. Molecules identified as single fluorescence spots via image analysis are encircled with colors corresponding to those shown in d. c Extension traces of individual molecules; reads <5 nt were excluded. Each trace represents the extension of each molecule shown in b. d Average number of identified barcode molecules per FOV. Error bars represent SD (N = 16). Note that for analyses shown in b–d, f reference to identify individual reads contained only CD55 barcode sequences. e Comparison of read lengths between different sequencing directions. f Comparison of identification efficiency between 6Q and 12Q. g Estimation of multiplexing ability. False positive identification (incorrect) was calculated by changing the number of barcode sequences in the reference (1–200 types of sequences). Up to 30 types in a reference, false positive identification was negligible. Colored ribbon plots represent SD.