Holistic determination of ends of cfDNA molecules.

Cell-free DNA (cfDNA) end motifs serve as fragmentomics biomarkers for cancer. Prior studies primarily focused on 5' ends, whereas 3' ends were overlooked due to artifactual modification in existing sequencing protocols. We utilized single-stranded library preparation ("2-end sequencing") to assess the native 5' and 3' end motifs (EM5 and EM3, respectively). Additionally, we demonstrated diagnostic power from the nucleotide motifs located immediately upstream and downstream of 5' and 3' ends, named pre-end motifs (PREMs) and post-end motifs (POEMs). These fragmentomics markers collectively achieved an area under the curve (AUC) of 0.95 for hepatocellular carcinoma (HCC) detection. Fragmentomics-based methylation analysis of 3' ends (3' FRAGMA) improved detection of HCC (AUC: 0.97). We further developed "4-end sequencing" to interrogate both ends of both strands of a double-stranded cfDNA molecule, enhancing fragmentomics-based cancer detection. Holistic end profiling adds to the armamentarium of liquid biopsy and sheds light on the biology of cfDNA fragmentation.