Fig. 2: Detection and mapping of DNA base modifications.

a Schematic representation of the hairpin constructed with seven different base modifications. Each oligonucleotide contained a DNA base modification and the linker was constructed by annealing on a splint template. A detailed protocol how this hairpin is produced is available as Supplementary Fig. 1. b Detection of six out of the seven modifications present on the same molecule by sequentially injecting the antibody corresponding to each base modification. The six different experiments were aligned using the reference oligonucleotide bindings. c By plotting the hybridization time versus binding frequency, it is possible to cluster true positives (in this case, the m⁵C modification with the anti-m⁵C antibody) from the false positives (principally the hm⁵C modification). Each point represents the cumulative binding data for the m⁵C antibody as determined for each modified base on each individual hairpin (note that for many hairpins, there were no false binding events, so fewer points are plotted). By thresholding the time and frequency, we can eliminate the false positives. d Antibody binding positions were mapped to the hairpin molecule within 1 bp resolution for the majority of the molecules. Whisker boxes represent 50% of the points with the average as a line within the box and the median as a cross.