Previous studies found that cell-free DNA (cfDNA) generated from tumors was shorter than that from healthy cells, and selecting short cfDNA could enrich for tumor cfDNA and improve its usage in early cancer diagnosis and treatment monitoring; however, the underlying mechanism of shortened tumor cfDNA was still unknown, which potentially limits its further clinical application.

Using targeted sequencing of cfDNA in a large cohort of solid tumor patient, sequencing reads harboring tumor-specific somatic mutations were isolated to examine the exact size distribution of tumor cfDNA. For the majority of studied cases, 166 bp remained as the peak size of tumor cfDNA, with tumor cfDNA showing an increased proportion of short fragments (100-150 bp). Less than 1% of cfDNA samples were found to be peaked at 134/144 bp and independent of tumor cfDNA purity. Using whole-genome sequencing of cfDNA, we discovered a positive correlation between cfDNA shortening and the magnitude of chromatin inaccessibility, as measured by transcription, DNase I hypersensitivity, and histone modifications. Tumor cfDNA shortening occurred simultaneously at both 5′ and 3′ ends of the DNA wrapped around nucleosomes.

Tumor cfDNA shortening exhibited two distinctive modes. Tumor cfDNA purity and chromatin inaccessibility were contributing factors but insufficient to trigger a global transition from 166 bp dominant to 134/144 bp dominant phenotype.