Fragmentation-Induced Coverage Biases in Cell-Free DNA Sequencing Affect the Clinical Sensitivity of Liquid Biopsies

Cell-free DNA (cfDNA) circulating in blood is characterized by epigenetic context-dependent fragmentation that distinguishes it from the stable genomic DNA of solid tissues. Extensive fragmentation and degradation of cfDNA can result in uneven coverage and potential loss of genetic information. Yet, its implications for the sensitivity of non-invasive detection of clinically relevant variants remain unclear. We analyzed clinical genomic data comprising 0.4 million variants across 67,129 samples from 99 cohorts spanning all major cancer types. We demonstrate that locus-specific and allele-specific coverage biases in liquid biopsy, driven by cfDNA fragmentation, affect known oncogenic hotspots in clinically actionable genes, including TP53, EGFR, and NRAS. These biases result in uneven sensitivity for detecting somatic variants. Longitudinal analysis reveals that these biases can compromise the reliability of disease tracking, potentially leading to false-negative conclusions about clonal extinction and minimal residual disease. We also urge caution while assessing clonal hematopoiesis from cfDNA sequencing.