Orientation-aware plasma cell-free DNA fragmentation analysis in open chromatin regions informs tissue of origin

Abstract

Cell-free DNA (cfDNA) in human plasma is a class of biomarkers with many current and potential future diagnostic applications. Recent studies have shown that cfDNA molecules are not randomly fragmented and possess information related to their tissues of origin. Pathologies causing death of cells from particular tissues result in perturbations in the relative distribution of DNA from the affected tissues. Such tissue-of-origin analysis is particularly useful in the development of liquid biopsies for cancer. It is therefore of value to accurately determine the relative contributions of the tissues to the plasma DNA pool in a simultaneous manner. In this work, we report that in open chromatin regions, cfDNA molecules show characteristic fragmentation patterns reflected by sequencing coverage imbalance and differentially phased fragment end signals. The latter refers to differences in the read densities of sequences corresponding to the orientation of the upstream and downstream ends of cfDNA molecules in relation to the reference genome. Such cfDNA fragmentation patterns preferentially occur in tissue-specific open chromatin regions where the corresponding tissues contributed DNA into the plasma. Quantitative analyses of such signals allow measurement of the relative contributions of various tissues toward the plasma DNA pool. These findings were validated by plasma DNA sequencing data obtained from pregnant women, organ transplantation recipients, and cancer patients. Orientation-aware plasma DNA fragmentation analysis therefore has potential diagnostic applications in noninvasive prenatal testing, organ transplantation monitoring, and cancer liquid biopsy.

Sub-nucleosomal organization in urine cell-free DNA

“…We considered non-overlapping windows of 500 kb across the genome annotated using Given a non-random distribution of urine cfDNA fragments across the genome, we hypothesized that genome-wide fragmentation patterns and positioning in urine may be informative of tissue of origin. Based on differences observed in urine cfDNA fragment size in open and closed chromatin regions, we developed a computational approach for correlating fragment size differences between active and inactive genomic regions to DNase I hypersensitive sites (DHS) across 116 cell types and tissues (using a published dataset) [17][18][19] . For each individual sample, we calculated median fragment size within non-overlapping 500 kb windows for all autosomes and normalized all median values to z-scores.…”

Fluid-based assays and precision medicine of cardiovascular diseases: the ‘hope’ for Pandora’s box?

“…Remarkably, a recent work by Sun et al has demonstrated that another epigenetic biomarker, such as cfDNA fragmentation pattern, could also be informative for CV diagnosis besides the above-mentioned DNA methylation. 114 Owing to their reversibility, most attention should be given to circulating epigenetic-sensitive changes, which may provide innovative drug targets. In particular, individual DNA methylation profiles may be useful to identify a personalised dietary interventions consisting of consuming foods containing methyl donors to attenuate cardiac phenotypes.…”

In case you missed it: The Prenatal Diagnosis editors bring you the most significant advances of 2019

“…In 2019, pioneering researchers at the Li Ka Shing Institute of Health Sciences consolidated these understandings of fetal cfDNA in a publication that opens further opportunities for prenatal diagnosis, oncology, and transplant medicine. In this proof‐of‐principle study, the authors proposed a new method for nucleosome positioning profiling and quantitative determination of the relative contributions of various tissues in plasma DNA by fragmentation pattern analyses . In this elegant paper, the investigators capitalise on the knowledge that cfDNA fragmentation is not random but occurs in tissue‐specific patterns that reflect open chromatin regions and nucleosome position and is deducible by sequencing coverage patterns.…”

“…The approach used in this report was to first screen the pregnant mothers' germline DNA, and the cfDNA in maternal plasma is reflex tested in mothers subsequently identified as carriers to determine the fetal genotype. Much of the data reported in this paper comes from modelling experiments using spiked‐in genomic DNA, which may be a useful starting point for assay development but, as discussed above, we know that fetal cfDNA is different from maternal cfDNA or postnatal DNA . There appears to be a high inconclusive rate in this study, although the FF was reported at greater than 10% in most cases.…”