Distinct fragmentation patterns of circulating viral cell-free DNA in 83,552 non-invasive prenatal testing samples

Aim: The fragmentation characteristics of cell-free DNA (cfDNA) are informative biomarkers in liquid biopsies, including non-invasive prenatal testing (NIPT), as they provide insights into the origins of the cfDNA. Viral infections by DNA viruses can contribute to the available cfDNA in these samples. Here, we characterize the fragment size distribution of viral cfDNA fragments obtained from available anonymous NIPT samples. Methods: A viral database of 224 DNA viruses was generated from the NCBI RefSeq viral database. Paired-end cfDNA sequencing reads from 83.522 NIPT samples that did not map to any of the human chromosomes, or mitochondrial DNA of the human reference genome build GRCh38 (excluding alternative and unplaced contigs) were remapped to the generated viral database. Reads mapping to the 14 most abundant DNA viruses were selected, and fragment size distributions were analyzed in detail. Results: Distinct fragmentation patterns were identified for several DNA viruses, most likely due to differences in viral tropism, chromatinization (binding of nucleosomes), and the topology of the viral DNA. In high viral load parvo B19 positive samples, the fragment size distribution differed between samples, potentially reflecting the state of the infection. Conclusion: These findings outline the potential for liquid biopsies to elucidate the dynamics behind the viral infection, which may potentially have various clinical applications. Our data provide preliminary insights on the use of fragmentomics of viral cfDNA to distinguish between reactivation, reinfection, and primary infection and monitoring the state of viral infections.