Untargeted CUT&Tag reads are enriched at accessible chromatin and restrict identification of potential G4-forming sequences in G4-targeted CUT&Tag experiments.

G-quadruplex DNA structures (G4s) form within single-stranded DNA in nucleosome-free chromatin. G4s modulate gene expression and genomic stability, so high-throughput, genome-wide mapping of G4s has generated strong research interest and methodological innovation. Recently, the Cleavage Under Targets and Tagmentation (CUT&Tag) method has been adapted to map G4s using an antibody, a nanobody, and G4-binding small molecules to target Tn5 tagmentation to G4s. These novel methods have generated high-resolution maps of G4s, but we have observed a strong colocalization between untargeted and G4-targeted CUT&Tag signal enrichment, leading us to wonder whether this colocalized signal enrichment would impact G4 mapping using these methods. We observed that the genome-wide signal distribution of untargeted CUT&Tag libraries was highly correlated with that of both cell-line-matched ATAC-seq libraries and cell-line-matched G4-mapping CUT&Tag libraries. When peaks were called from G4-mapping CUT&Tag libraries using the SEACR algorithm with inclusion of the respective matched untargeted CUT&Tag libraries, certain peaks at potential G4-forming sequences were excluded, slightly enhancing precision with which G4s are mapped while limiting recall of potential G4s. Consequently, we recommend that care be exercised when interpreting G4-targeted CUT&Tag experiments unless untargeted tagmentation is taken into account or minimized through protocol optimization.