Table 1. Advantages and limitations of techniques to map and detect G-quadruplexes (G4s).

Methodology	Technique	Uses and advantages	Limitations
Mapping by chain-extension stalling	G4-seq RNA G4-seq (rG4-seq)	Identifies in vitro nucleic acid sequences with the potential to form G4s in the genome (G4-seq) or transcriptome (rG4-seq)	Performed on extracted DNA or RNA. Thus, the influence of the cellular environment, for example proteins or chromatin structure, on the G4 landscape is not considered
Chemical mapping	Potassium permanganate–S1 nuclease footprinting	Maps genome-wide multiple types of non-B DNA structures in the chromatin context	Relies on motif-annotation algorithms to map the non-B-DNA structures (including G4 formation) Cannot accurately discern individual non-B DNA structures at sites containing large clusters of non-B DNA Readout is an averaging of structural states Can shift the dynamic equilibrium of structural states and hence may not reflect true intracellular structures. Readout is not specific to G4 structures
	Selective 2’-OH acylation analysed by primer extension (SHAPE)	Provides quantitative, single-nucleotide-resolution RNA structural information	Structural information is lost at both the 5´ and 3´ ends of an RNA because the technology depends on primer extension Readout is not specific to G4 structures
	In vivo dimethyl-sulphate (DMS) footprinting	Determines nucleic acid (DNA and RNA) secondary and tertiary structures at single-nucleotide resolution DMS easily and rapidly penetrates cells and all cellular compartments	Readout is an averaging of structural states High cellular toxicity Can shift the dynamic equilibrium of structural states and, hence, does not reflect true cellular structures DMS reactivity depends on solvent accessibility and local electrostatic environment
Antibody-based mapping	G4 chromatin immunopre-cipitation sequencing (G4 ChIP-seq)	Genome-wide mapping of DNA G4s in the chromatin context	Possible biases introduced by sample fixation and fragmentation Relies on antibody specificity, target accessibility and cell-population averaging Cannot determine on which DNA strand G4s are located Antibodies against G4-binding proteins provide indirect evidence of DNA G4s, which relies on the specificity of the protein for G4s
	ChIP-seq of G4-binding proteins	Genome-wide mapping of G4 DNA binding proteins in the chromatin context
	Individual-nucleotide resolution ultraviolet crosslinking and immuno-precipitation (iCLIP)	Identifies all RNA sequences bound to the RNA binding protein (RBP) of interest	Relies on the specificity of the RBP to bind RNA G4 Cannot account for protein binding to unfolded G4 sequence motifs Relies on cell-population averaging
Imaging	Immunofluorescence	Single-cell resolution of G4 abundance Possible to detect DNA and RNA G4s simultaneously	Requires cellular fixation and permeabilization Relies on the specificity of the antibody Does not provide sequence context Undetermined resolution: do detected G4 foci represent one or several G4s?
	Fluorescent G4-stabilizing ligands	Allow the study of dynamic formation of G4s in fixed and live cells	Fluorescence is sensitive to cellular changes in pH, polarity (hydrophilic versus hydrophobic compartments) and viscosity, making discrimination of G4-specific from non-specific binding events difficult The dynamic equilibrium of G4 formation may be shifted by the experiment and thus will not reflect the true cellular state Lack of sequence context Relies on ligand specificity and half-life

G4-seq, genome-wide DNA polymerase-stop assay followed by high-throughput sequencing; rG4-seq, transcriptome-wide reverse transcriptase stalling assay followed by high-throughput sequencing; S1 nuclease, a single-strand nuclease.