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Background Jagged ends of plasma DNA are a recently recognized class of fragmentomic markers for cell-free DNA, reflecting the activity of nucleases. A number of recent studies have also highlighted the importance of jagged ends in the context of pregnancy and oncology. However, knowledge regarding the generation of jagged ends is incomplete. Methods Jaggedness of plasma DNA was analyzed based on Jag-seq, which utilized the differential methylation signals introduced by the DNA end-repair process. We investigated the jagged ends in plasma DNA using mouse models by deleting the deoxyribonuclease 1 (Dnase1), DNA fragmentation factor subunit beta (Dffb), or deoxyribonuclease 1 like 3 (Dnase1l3) gene. Results Aberrations in the profile of plasma DNA jagged ends correlated with the type of nuclease that had been genetically deleted, depending on nucleosomal structures. The deletion of Dnase1l3 led to a significant reduction of jaggedness for those plasma DNA molecules involving more than 1 nucleosome (e.g., size ranges 240-290 bp, 330-380 bp, and 420-470 bp). However, less significant effects of Dnase1 and Dffb deletions were observed regarding different sizes of DNA fragments. Interestingly, the aberration in plasma DNA jagged ends related to multinucleosomes was observed in human subjects with familial systemic lupus erythematosus with Dnase1l3 deficiency and human subjects with sporadic systemic lupus erythematosus. Conclusions Detailed understanding of the relationship between nuclease and plasma DNA jaggedness has opened up avenues for biomarker development.

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5′ ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson’s absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.

BackgroundDouble-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends.MethodsJagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing.ResultsThe jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83.ConclusionsJagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.

Plasma DNA fragmentomics is an emerging area of research covering plasma DNA sizes, end points, and nucleosome footprints. In the present study, we found a significant increase in the diversity of plasma DNA end motifs in patients with hepatocellular carcinoma (HCC). Compared with patients without HCC, patients with HCC showed a preferential pattern of 4-mer end motifs. In particular, the abundance of plasma DNA motif CCCA was much lower in patients with HCC than in subjects without HCC. The aberrant end motifs were also observed in patients with other cancer types, including colorectal cancer, lung cancer, nasopharyngeal carcinoma, and head and neck squamous cell carcinoma. We further observed that the profile of plasma DNA end motifs originating from the same organ, such as the liver, placenta, and hematopoietic cells, generally clustered together. The profile of end motifs may therefore serve as a class of biomarkers for liquid biopsy in oncology, noninvasive prenatal testing, and transplantation monitoring. Significance: Plasma DNA molecules originating from the liver, HCC and other cancers, placenta, and hematopoietic cells each harbor a set of characteristic plasma DNA end motifs. Such markers carry tissue-of-origin information and represent a new class of biomarkers in the nascent field of fragmentomics. This article is highlighted in the In This Issue feature, p. 627

Background Analysis of circulating tumor DNA has become increasingly important as a tool for cancer care. However, the focus of previous studies has been on short fragments of DNA. Also, bisulfite sequencing, a conventional approach for methylation analysis, causes DNA degradation, which is not ideal for the assessment of long DNA properties and methylation patterns. This study attempted to overcome such obstacles by single-molecule sequencing. Methods Single-molecule real-time (SMRT) sequencing was used to sequence plasma DNA. We performed fragment size and direct methylation analysis for each molecule. A methylation score concerning single-molecule methylation patterns was used for cancer detection. Results A substantial proportion of plasma DNA was longer than 1 kb with a median of 16% in hepatocellular carcinoma (HCC) patients, hepatitis B virus carriers, and healthy individuals. The longest plasma DNA molecule in the HCC patients was 39.8 kb. Tumoral cell-free DNA (cfDNA) was generally shorter than nontumoral cfDNA. The longest tumoral cfDNA was 13.6 kb. Tumoral cfDNA had lower methylation levels compared with nontumoral cfDNA (median: 59.3% vs 76.9%). We developed and analyzed a metric reflecting single-molecule methylation patterns associated with cancer, named the HCC methylation score. HCC patients displayed significantly higher HCC methylation scores than those without HCC. Interestingly, compared to using short cfDNA (area under the receiver operating characteristic [ROC] curve, AUC: 0.75), the use of long cfDNA molecules greatly enhanced the discriminatory power (AUC: 0.91). Conclusions A previously unidentified long cfDNA population was revealed in cancer patients. The presence and direct methylation analysis of these molecules open new possibilities for cancer liquid biopsy.

Significance Single molecule real-time (SMRT) sequencing theoretically offers the opportunity to directly assess certain base modifications of native DNA molecules without any prior chemical/enzymatic conversions and PCR amplification, using kinetic signals of a DNA polymerase. However, the kinetic signal changes caused by 5mC modification are extremely subtle. Hence, the robust genome-wide measurement of 5mC modification has not been achieved. We enhanced 5mC detection using SMRT sequencing by holistically analyzing kinetic signals of a DNA polymerase and sequence context for every base within a measurement window. We employed a convolutional neural network to train a methylation classification model, leading to genome-wide 5mC detection. The sensitivity and specificity reached 90% and 94%, with a 99% correlation of overall methylation level with bisulfite sequencing., 5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human–mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.

International audience; Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a ''CC'' end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.

Epstein-Barr virus (EBV) is associated with a number of diseases, including malignancies. Currently, it is not known whether patients with different EBV-associated diseases have different methylation profiles of circulating EBV DNA. Through whole-genome methylation analysis of plasma samples from patients with nasopharyngeal carcinoma (NPC), EBV-associated lymphoma and infectious mononucleosis, we demonstrate that EBV DNA methylation profiles exhibit a disease-associated pattern. This observation implies a significant potential for the development of methylation analysis of plasma EBV DNA for NPC diagnostics. We further analyse the plasma EBV DNA methylome of NPC and non-NPC subjects from a prospective screening cohort. Plasma EBV DNA fragments demonstrate differential methylation patterns between NPC and non-NPC subjects. Combining such differential methylation patterns with the fractional concentration (count) and size of plasma EBV DNA, population screening of NPC is performed with an improved positive predictive value of 35.1%, compared to a count- and size-based only protocol., Epstein-Barr virus (EBV) is associated with different malignant diseases and circulating EBV DNA is a biomarker for nasopharyngeal carcinoma (NPC). Here, the authors report that plasma EBV DNA methylation profiles show disease-associated patterns and can help to distinguish NPC and non-NPC subjects.

BACKGROUND Cellular mitochondrial DNA (mtDNA) is organized as circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma. METHODS We elucidated the topology of plasma mtDNA using restriction enzyme BfaI cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying BfaI cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme MspI digestion was used for confirming the conclusions based on BfaI cleavage analysis. We analyzed the presence of BfaI cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation. RESULTS Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%–97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%–93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool (r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular. CONCLUSIONS Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins.

Significance Circulating DNA in plasma has many diagnostic applications, including noninvasive prenatal testing and cancer liquid biopsy. Plasma DNA consists of short fragments of DNA. However, there is little information about mechanisms that are involved in the fragmentation of plasma DNA. We showed that mice in which Dnase1l3 had been deleted showed aberrations in the fragmentation of plasma DNA. We also observed a change in the ranked frequencies of end motifs of plasma DNA caused by the Dnase1l3 deletion. In Dnase1l3−/− mice pregnant with Dnase1l3+/− fetuses, we observed a partial reversal of the plasma DNA aberrations. This study has thus linked the fields of nuclease biology and circulating nucleic acids and has opened up avenues for future research., Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3−/− mice carrying Dnase1l3+/− fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3. Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity.