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

gkaf678), Thompson and Byrd examine potential issues in the mapping of DNA G-quadruplexes (G4s) by CUT&Tag and related methods [ 1 ].The authors conclude that detection of G4s in chromatin is significantly confounded by artefacts arising from background Tn5 enzymatic activity independent of the primary G4-targeting probe.We highlight several concerns regarding the authors' meta-analysis of published data, including the problematic use of computational tools developed initially for other experimental modalities, such as A T AC-seq and ChIP-seq, for G4-CUT&Tag data without explicit disclosure of the analytical methods and code.Given that CUT&Tag is widely used and the technology is rapidly evolving, it is timely to promote best practices in the profiling of G4s and other features.A critical point is to clarify the inherent differences between G4-targeted and untargeted CUT&T ag libraries.CUT&T ag methods rely on the selective recognition of a molecular feature on chromatin by a high-affinity probe, followed by the recruitment of Tn5 transposase to insert adapters in the vicinity of the target [ 2 ] to prepare a sequencing library.This is performed under high salt conditions to suppress the otherwise high-affinity transposase binding (utilized in A T AC-seq) and reduce non-targeted tagmentation.Depending on the target, this creates an overall sparse distribution of mapped read fragments across the genome, with local enrichment around the target and minimal random tagmentation, compared to other methods.In the absence of a target, e.g. if the primary probe is omitted, or a non-specific antibody (e.g.serum IgG) is used in place of the primary probe, substantially less DNA is acquired in library preparation (Fig. 1 A).Accurate quantification of those libraries is challenging, and when they are sequenced, this often leads to over-representation of polymerase chain reaction duplicates.This is a crucial point as it influences downstream analysis.The combination of targeted tagmentation with the