DNA G-quadruplexes (G4) are guanine-rich structures that are stable in physiological conditions and can affect gene expression and genomic stability. Chambers et al. developed G4-seq for high-resolution, genome-wide G4 mapping. This mapping technique is based on DNA polymerase stalling at stabilized G4 structures, which can be precisely localized by high-throughput sequencing, as stalling results in a drop in sequencing quality. Applying G4-seq in primary lymphocytes revealed >500,000 G4 structures in the human genome. Notably, ∼70% of these were not predicted computationally, because they contain bulges or long loops. Such non-canonical G4 structures were exceptionally prevalent in gene regulatory regions, especially in 5′ UTRs and splicing sites, and also in oncogenes and (other) regions that are amplified in cancers.