Long-read sequencing identifies aberrant fragmentation patterns linked to elevated cell-free DNA levels in cancer

Circulating cell-free DNA (cfDNA) carries fragmentation patterns that serve as biomarkers of cancer, but standard sequencing approaches miss large portions of the fragment length spectrum. In addition to altered fragmentation patterns, cancer patients often have elevated levels of cfDNA, but the underlying mechanisms are not well understood. To address both questions, we analyzed cancer cases with elevated cfDNA levels using Oxford Nanopore (ONT) sequencing. Long-read ONT sequencing captures the full spectrum of cfDNA fragment lengths and enables cell type inference based on DNA methylation markers. One cohort included cases from several cancer types with elevated cfDNA levels, and a second consisted of patients from a single neuroendocrine cancer study. In each cohort, cases with the highest cfDNA levels showed either hypofragmentation (excess fragments of 1-4 kb) or hyperfragmentation (excess fragments 7.5 kb) lacking these hallmarks and consistent with plasma lysis. By contrast, hyperfragmented samples often had elevated levels of both cancer- and blood-derived DNA, indicating an inflammatory or other system process rather than cancer-specific origin. These findings clarify the distinction between biological and artifactual fragmentation, expand our understanding of cfDNA biology, and highlight long-read sequencing as a powerful tool for biomarker discovery.