Breaking the double-stranded limitation: single-stranded cfDNA sequencing technology opens a new era in precision medicine.

Cell-free DNA (cfDNA) in human blood or bodily fluids has become a research and clinical focus since its discovery. The broad application of cfDNA relies on accurate and comprehensive characterization of its biological features. Currently, next-generation sequencing (NGS) remains the primary method for detecting and analyzing cfDNA, with the common library preparation strategy targeting double-stranded cfDNA fragments. Based on this strategy, researchers have identified a characteristic peak of 166 bp in cfDNA. However, short DNA, single-stranded DNA, and other irregular DNA structures and sequence information in cfDNA are often lost with such library preparation methods. The emergence of single-stranded cfDNA sequencing library preparation methods effectively addresses these limitations, enabling systematic characterization of cfDNA's structural and sequence features, thereby providing more accurate non-invasive diagnostic materials for clinical applications. This review systematically summarizes single-stranded cfDNA library preparation techniques and the clinical applications of plasma cfDNA, laying the foundation for its broader utilization.