High-resolution profiling of human cytomegalovirus cell-free DNA in human plasma highlights its exceptionally fragmented nature

Congenital CMV is one of the most common causes of birth defects yet is not screened for in the United States.

CMV is the leading cause of newborn deafness via congenital infections. Despite this, it is unscreened for in routine prenatal screenings. The current 'gold standard' of congenital CMV diagnostics PCR on amniotic fluid. Amniocentesis is an invasive procedure with a non-zero risk of complications occurring. Here, we attempt to detect CMV/HHV-6 reads using cell-free DNA sequence from a cohort of 2,208 pregnant women.

We aligned cell-free DNA sequence to the CMV and HHV-6 reference genomes and extracted blast-verified CMV reads

After aligning to the CMV Merlin reference genome (NC_006273.2) all aligned reads were extracted, repeatmasked, and run through blast. The scripts containing more information regarding these pipelines are available at reproducibility/CMV_reproducibility.sh and reproducibility/HHV-6_reproducibility.sh

CMV DNA is detectable in cfDNA sequence, and circulates at a smaller fragment size than human chromosomal DNA

We show that approximately 5% of samples sent for NIPT had detectable CMV-specific reads, which routinely outstripped the analytical sensitivity of our CMV qPCR assay when performed on cfDNA. We also discovered that CMV cfDNA exists in circulation at a smaller fragment size than human chromosomally-derived cfDNA. We confirmed the highly fragmented nature of CMV in cfDNA in seven CMV-positive specimens from solid organ transplant patients.