Evaluating the Performance of Peak Calling Algorithms Available for Intracellular G-Quadruplex Sequencing

DNA G-quadruplexes (G4) are non-canonical DNA structures that play key roles in various biological processes. Antibody-dependent sequencing is an important tool for identifying intracellularly formed DNA G4s, and peak calling is a crucial step in processing the sequencing data. As the applicability of existing peak calling algorithms to intracellular G4 data has not been previously assessed, we systematically compared and evaluated these algorithms to identify those best suited for G4 detection. We selected seven representative candidates from 43 published peak calling algorithms for detailed evaluation. With precision and recall based on benchmark datasets, as well as consistency with known G4 information and epigenetic signals, we identified MACS2, PeakRanger, and GoPeaks as the most effective algorithms for analyzing intracellular G4 sequencing data, and attributed their superior performance partially to the distribution model of sequencing reads/fragments used in the hypothesis testing step of the peak calling procedures. These findings provide guidance and rationale for selecting peak callers appropriate for intracellular G4 data.