Sub-nucleosomal organization in urine cell-free DNA

Abstract

Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented and whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we perform comprehensive characterization of fragmentation patterns in urine cfDNA. We show modal size and genome-wide distribution of urine cfDNA fragments are consistent with transient protection from degradation by stable intermediates of nucleosome disassembly. Genome-wide nucleosome occupancy and fragment sizes in urine cfDNA are informative of cell of origin and renal epithelial cells are amongst the highest contributors in urine. Compared to a nucleosome occupancy map based on control urine samples, we observe a higher fraction of fragments with aberrant ends in cancer patients, distinguishing cancer samples with an area under the curve of 0.89. Our results demonstrate subnucleosomal organization in urine cfDNA and are proof-of-principle that genome-wide fragmentation analysis of urine cfDNA can enable cancer diagnostics.Circulating cell-free DNA (cfDNA) has emerged as an informative biomarker in prenatal, organ transplant and cancer patients. Recent studies have shown that genome-wide distribution and fragmentation of cfDNA in plasma is not random. Plasma cfDNA fragments have a modal size of 167 bp, are protected from degradation within mono-nucleosomes and their positioning captures nucleosome footprints of contributing tissues 1 . In cancer patients, these observations potentially enable cancer detection 2 , inference of tissue of origin 3 and inference of gene expression 4 . In addition, deviations from expected fragment size and positioning can be leveraged to improve signal-to-noise ratio for somatic genomic alterations in plasma cfDNA 5 .Collection of blood plasma requires venipuncture and plasma volume obtainable at a single time point is limited. In contrast, urine can be collected noninvasively, with minimal assistance and in larger volumes. However, there has been limited success in diagnostic development using urine cfDNA so far. There are multiple reports that cfDNA fragments are more degraded, shorter and variably sized in urine compared to plasma 6,7 , impeding targeted analysis of genomic alterations.Comprehensive characterization of fragment sizes and positioning in urine cfDNA has not been reported and whether any genome-wide organization is preserved is unknown.We characterized fragmentation patterns in urine cfDNA using whole genome sequencing. To our surprise, urine samples from healthy volunteers predominantly showed a modal size of 80-81 bp, suggesting non-random cfDNA fragmentation in urine. Here, we evaluate this hypothesis and investigate fragment size, distribution and nucleosome positioning in urine cfDNA. We describe correlation between cfDNA fragmentation patterns in urine and chromatin accessibility as well as gene expression in contributing cells. In cancer patients, we report a framework to leverage genome-wide differences in urine cfDNA fragmentat...

Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples

“…Critically for urine cfDNA, which is made up primarily of short fragments (majority <100 bp) [8,[15][16][17], our hybridization capture method is not dependent on DNA fragment length and maintains high recovery across fragments from 25-150 bp (Fig 3D). Combined with an ultrashort hairpin qPCR design [28], we can capture and amplify fragments as short as 25 bp with high efficiency (84% recovery).…”

“…While plasma cfDNA has a peak fragment length of approximately 167 bp, reflecting protection of histone-associated DNA within nucleosomes [14], urine cfDNA is more fragmented due to glomerular filtration of the transrenal fraction and fast degradation kinetics of all cfDNA in urine. The distribution of fragment lengths varies across samples, but the majority of urine cfDNA fragments are expected to be <100 bp [8,[15][16][17], with a wider distribution of fragments around the peak fragment length compared to plasma cfDNA [16]. Some forms of urine cfDNA, including fetal [8], tumor [18], mitochondrial [8,15], microbial [15], and viral [15] cfDNA, are even more fragmented than human nuclear cfDNA.…”

Clinical relevance of blood-based ctDNA analysis: mutation detection and beyond

“…The fragment distribution is also different between plasma and urine, with smaller fragments in urine centred around 82 bp. 84,85 The ability to extract and analyse small fragments of cfDNA therefore appears to be a critically important parameter in the detection of ctDNA. Importantly, wide variability in yield and fragment size across different extraction kits has been reported, making the choice of appropriate isolation method an important analytical parameter.…”

Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

“…Urine cfDNA is a challenging target due to the short length and low concentration of TB-specific fragments. While plasma cfDNA has a peak fragment length of 167 bp, urine cfDNA is more fragmented (7, 16, 17). Recently, new sequencing library preparation methods revealed that very short, formerly-undetectable fragments compose a larger fraction of cfDNA than previously realized (18–21).…”

“…Recently, new sequencing library preparation methods revealed that very short, formerly-undetectable fragments compose a larger fraction of cfDNA than previously realized (18–21). The majority of urine cfDNA fragments are <100 bp, with peak fragment length ranging from 30 – 110 bp (16, 17, 21, 22). Although the fragment length of TB cfDNA has not yet been characterized, bacterial cfDNA is expected to be especially fragmented (peak <60 bp) because it is less protected by DNA-associated proteins (21, 23).…”