Sizing Up Cell-Free DNA

Structure and diversity of urinary cell-free DNA informative of host-pathogen interactions in human urinary tract infection

“…We find that nucleosome structures within transcription regulatory 8 elements are preserved in urinary cfDNA, as was previously described for plasma 9 cfDNA 22 . The occupancy of nucleosomes in gene regions flanking the transcription start 10 site is notably reduced for transcribed genes, consistent with known chromatin 11 alterations in gene promoters during transcription, thus providing a measure of gene 12 expression. Furthermore, we observe that the relative proportion of kidney donor 13 specific cfDNA signals graft tissue injury in the setting of viral infection and host immune 14 cell activation in the scope of bacterial infection.…”

“…In the former 6 subject, we observed a high abundance of both BK polyomavirus (3.54 x10 4 RGE) and 7 parvovirus B19 (2.48 x 10 4 RGE), which correlated with positive results of individual 8 viral-specific PCR tests for BK polyomavirus and parvovirus B19 performed in the clinic. 9 For a third patient, we observed a high abundance of human adenovirus B DNA, in 10 samples obtained up to 15 days before (2.52 x 10 3 RGE), and 9 days after (5.08 x10 2 11 RGE) adenovirus infection diagnosis. These data support the utility of urinary cfDNA 12 sequencing for the detection of both common and uncommon viral agents.…”

“…To 7 account for technical variability and sources of environmental contamination during 8 extraction and library preparation, a known-template control sample was included in 9 every sample batch and sequenced (see Methods). 10 11 We analyzed the fragment length profiles of urinary cfDNA at single nucleotide 12 resolution using paired-end read mapping 10 . This analysis confirmed previous 13 observations of the highly fragmented nature of urinary cfDNA compared to plasma 14 cfDNA 25 (Fig.…”

“…For a single sample, urinary 9 cfDNA did not correlate with the bacterial culture. Raoultella ornithinolytica was isolated 10 in culture, but not detected in cfDNA (see Methods for a detailed discussion of this 11 discordant readout). For two clinically positive samples, the primary suspected 12 causative agent was identified to the genus level (Staphylococcus [reported as 13 coagulase-negative Staphylococcus species] and Streptococcus) by conventional 14 culture.…”

“…The proportion of donor specific mitochondrial DNA was 8 quantified using methods previously described 19 . Briefly, a mitochondrial consensus 9 sequence was determined for the donor and recipient from pre-transplant whole blood 10 samples. Comparison of cfDNA sequence data to the mitochondrial consensus was 11 used to quantify donor and recipient specific mitochondrial DNA.…”