Single-base-pair resolution detection of nicks in genomic DNA. (A) Schematic of an E. coli genome used to test DENT-seq, in which red arrows represent the activity of Cas9 nickase paired with one guide RNA and the purple arrow represents the activity of Cas9 nickase paired with a different guide RNA. Circularized plot data (all colors) show normalized sequence coverage. Green signals represent the locations of called MACS2 peaks with quality score greater than 0.4. Red signals represent locations of such peaks wherein a nick is identified with the exact location of that nick represented by a black dot. (B) Cumulative number of nicks contained within MACS2 peaks (green) and cumulative number of MACS2 peaks containing no nick (red) as a function of P-value threshold. The left gray dashed line represents the P-value where the final nick-containing peak is identified, and the right gray dashed line represents the P-value where the first peak not containing a nick is identified. This panel uses only the coverage signal and does not incorporate mutational data. (C) Normalized sequence coverage (top) and base transition rate (bottom) for four MACS2 peaks that contain nicks resulting from Cas9 nickase in conjunction with one of the guide RNAs used (corresponding to the red arrows in A). The second set of plots from the left represent a single MACS2 peak, which encompassed two nicks owing to their close proximity. (D) Normalized sequence coverage (top) and base transition rate (bottom) for the MACS2 peak that contains the nick resulting from Cas9 nickase in conjunction with the other guide RNA used (corresponding to the purple arrow in A). Coverage is lower for this guide, but there are still nucleotide positions proximal to the Cas9 nickase target site with high mutational signal that enable nick identification.
