Expanding cfDNA capture beyond nucleosome-sized fragments to enhance liquid biopsy performance.

e15062 Background: Liquid biopsy is a non-invasive diagnostic approach used for early cancer detection, disease monitoring, and minimal residual disease (MRD) assessment. Accurate liquid biopsy results require comprehensive recovery of circulating cell-free DNA (cfDNA) to capture molecular diversity in low-input and low–tumor burden samples. However, conventional cfDNA purification and double-stranded DNA library preparation workflows are optimized for nucleosome-sized fragments and may systematically exclude ultra-short and single-stranded cfDNA species that may be relevant to cancer diagnostics, potentially limiting assay sensitivity and contributing to false-negative results. Methods: In this study, cfDNA was isolated from plasma, serum, and saliva using a magnetic bead-based purification workflow (MAGicBead) and compared with commercially available cfDNA extraction methods under clinically relevant input conditions. Libraries were prepared using a single-stranded DNA–compatible workflow to capture both double- and single-stranded cfDNA. Fragment size distributions and unique molecular characteristics were evaluated by sequencing-based analysis. To assess clinical usability, the workflow was adapted for hybrid-capture targeted enrichment without modification to standard probe designs and evaluated for compatibility with Agilent SureSelect, TWIST, and IDT xGen platforms. Feasibility and enrichment performance were assessed in plasma from lung cancer patients and healthy controls using standard sequencing and bioinformatics pipelines. Results: The optimized workflow demonstrated improved cfDNA recovery relative to conventional methods and consistently captured ultra-short cfDNA fragments (approximately 35–75 bp) that were not detected using standard workflows. Enzymatic digestion suggested that these fragments were predominantly single-stranded. The workflow was compatible with multiple commercial targeted enrichment platforms, enabling simultaneous capture of nucleosome-sized and ultra-short cfDNA within a single assay. Differences in the relative abundance of ultra-short cfDNA fragments were observed between cancer and healthy plasma samples. In saliva, concurrent recovery of host and microbial DNA supported multi-omic profiling from a single library preparation. Conclusions: This clinically adaptable workflow overcomes fragment size bias inherent to standard liquid biopsy assays and has the potential to enhance sensitivity across multiple liquid biopsy applications, including cancer detection and disease monitoring.