Breaking the double-stranded limitation: single-stranded cfDNA sequencing technology opens a new era of 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 fragment. 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 under such library preparation method. The emergence of single-stranded cfDNA sequencing library preparation methods effectively addresses this limitation, 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 clinical applications of plasma cfDNA, laying the foundation for its broader utilization.