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. 2022 Jul 9;13:3989. doi: 10.1038/s41467-022-31702-9

Fig. 2. INDUCE-seq demonstrates improved sensitivity and dynamic range when compared to alternative DSB sequencing technologies.

Fig. 2

a INDUCE-seq detects both highly recurrent induced DSBs and low-level endogenous DSBs simultaneously with high resolution. Genome browser view (IGB) of INDUCE-seq reads mapped to chromosome 19 and a 120 kb section from HEK293T cells following in situ cleavage with the restriction endonuclease HindIII. (a, top panel) untreated HEK293T DSB data on chromosome 19 reveals low level single endogenous breaks present in untreated cells. (Bottom panel) Highly recurrent enzyme-induced breaks represent the vast majority of reads when viewed at low resolution. An expanded panel shows a close-up region of 120 kb, demonstrating the simultaneous detection of HindIII induced breaks shown in black and endogenous breaks shown in red. b HindIII ranked break sites. Total number of breaks were plotted at each of the 782,602 HindIII restriction sites (Left, primary y-axis). The cumulative break frequency at HindIII sites was calculated and plotted as a percentage on the secondary y-axis. c Mapping of INDUCE-seq reads at a HindIII target site demonstrates the precision of single-nucleotide break mapping on both sides of the break. d and e Comparison between INDUCE-seq and DSBCapture in detecting in vitro cleaved restriction sites by the enzymes HindIII and EcoRV. d INDUCE-seq identifies a similar proportion of HindIII restriction sites (92.7%) to that identified by DSBCapture for EcoRV (93.7%). e A greater proportion of sequenced reads were mapped to restriction sites using INDUCE-seq compared to DSBCapture. f The dynamic range of induced DSB detection using INDUCE-seq. In addition to breaks identified at HindIII on-target sequences (AAGCTT), multiple 1 bp and 2 bp mismatching off-target sites were also identified. INDUCE-seq measured cumulative breaks spanning 8 orders of magnitude, from ~146 M breaks identified at HindIII on-target sites, down to the selected cut-off of 5 breaks identified per site at the least frequent off-targets. Source data are provided as a Source Data file.