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Background Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored. Methods We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis. Results The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone. Conclusions The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.

Background Recent studies using single molecule, real-time (SMRT) sequencing revealed a substantial population of analyzable long cell-free DNA (cfDNA) in plasma. Potential clinical utilities of such long cfDNA in pregnancy and cancer have been demonstrated. However, the performance of different long-read sequencing platforms for the analysis of long cfDNA remains unknown. Methods Size biases of SMRT sequencing by Pacific Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies (ONT) were evaluated using artificial mixtures of sonicated human and mouse DNA of different sizes. cfDNA from plasma samples of pregnant women at different trimesters, hepatitis B carriers, and patients with hepatocellular carcinoma were sequenced with the 2 platforms. Results Both platforms showed biases to sequence longer (1500 bp vs 200 bp) DNA fragments, with PacBio showing a stronger bias (5-fold overrepresentation of long fragments vs 2-fold in ONT). Percentages of cfDNA fragments 500 bp were around 6-fold higher in PacBio compared with ONT. End motif profiles of cfDNA from PacBio and ONT were similar, yet exhibited platform-dependent patterns. Tissue-of-origin analysis based on single-molecule methylation patterns showed comparable performance on both platforms. Conclusions SMRT sequencing generated data with higher percentages of long cfDNA compared with nanopore sequencing. Yet, a higher number of long cfDNA fragments eligible for the tissue-of-origin analysis could be obtained from nanopore sequencing due to its much higher throughput. When analyzing the size and end motif of cfDNA, one should be aware of the analytical characteristics and possible biases of the sequencing platforms being used.

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 is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5′ 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as “founder” end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.

The relationship between placental pathology and the maternal syndrome of preeclampsia is incompletely characterised. Mismatch between placental nutrient supply and fetal demands induces stress in the syncytiotrophoblast, the layer of placenta in direct contact with maternal blood. Such stress alters the content and increases the release of extracellular vesicles into the maternal circulation. 5’-tRNA fragments (5’-tRF) constitute the majority of short RNA in these vesicles. Here we characterise 5’-tRF profiles in syncytiotrophoblast vesicles in preeclampsia, reporting differential expression of >900 fragments. We detected preeclampsia-dysregulated 5’-tRF in peripheral plasma, where we identified a placentally-derived load. We demonstrated these 5’-tRF induce sterile inflammation in macrophages and monocytes. Our findings suggest syncytiotrophoblast-derived 5’-tRF may contribute to distant vascular inflammation, previously described in preeclampsia. This is the first demonstration of endocrine actions of vesicle-bound 5’-tRF, which may have relevance in other disciplines studying their functions, such as immunology and cancer biology.

Supplemental Tables S1-S5. Characteristics of the 45 normal tissues included in the PCDH10 expression analysis (S1); Characteristics of the 9 normal tissues included in the MALAT1 expression by ISH analysis (S2); Characteristics at primary treatment of 142 endometrial cancer patients from MoMaTEC* trial included in the MALAT1 investigation (S3); PCDH10 RNA expression from 142 prospectively collected endometrial cancer tumors, 19 metastatic lesions and 18 endometrial hyperplasia samples in relation to clinicopathologic data (S4); Oligos used for qRT-PCR, ChIP-PCR, Cloning and Bisulfate genomic sequencing (S5).

Background Human plasma contains RNA transcripts released by multiple cell types within the body. Single-cell transcriptomic analysis allows the cellular origin of circulating RNA molecules to be elucidated at high resolution and has been successfully utilized in the pregnancy context. We explored the application of a similar approach to develop plasma RNA markers for cancer detection. Methods Single-cell RNA sequencing was performed to decipher transcriptomic profiles of single cells from hepatocellular carcinoma (HCC) samples. Cell-type-specific transcripts were identified and used for deducing the cell-type-specific gene signature (CELSIG) scores of plasma RNA from patients with and without HCC. Results Six major cell clusters were identified, including hepatocyte-like, cholangiocyte-like, myofibroblast, endothelial, lymphoid, and myeloid cell clusters based on 4 HCC tumor tissues as well as their paired adjacent nontumoral tissues. The CELSIG score of hepatocyte-like cells was significantly increased in preoperative plasma RNA samples of patients with HCC (n = 14) compared with non-HCC participants (n = 49). The CELSIG score of hepatocyte-like cells declined in plasma RNA samples of patients with HCC within 3 days after tumor resection. Compared with the discriminating power between patients with and without HCC using the abundance of ALB transcript in plasma [area under curve (AUC) 0.72)], an improved performance (AUC: 0.84) was observed using the CELSIG score. The hepatocyte-specific transcript markers in plasma RNA were further validated by ddPCR assays. The CELSIG scores of hepatocyte-like cell and cholangiocyte trended with patients’ survival. Conclusions The combination of single-cell transcriptomic analysis and plasma RNA sequencing represents an approach for the development of new noninvasive cancer markers.

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

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.

Background Although the characterization of cell-free extrachromosomal circular DNA (eccDNA) has gained much research interest, the methylation status of these molecules is yet to be elucidated. We set out to compare the methylation densities of plasma eccDNA of maternal and fetal origins, and between small and large molecules. The clearance of fetal eccDNA from maternal circulation was also investigated. Methods We developed a sequencing protocol for eccDNA methylation analysis using tagmentation and enzymatic conversion approaches. A restriction enzyme-based approach was applied to verify the tagmentation results. The efficiency of cell-free fetal eccDNA clearance was investigated by fetal eccDNA fraction evaluations at various postpartum time points. Results The methylation densities of fetal eccDNA (median: 56.3%; range: 40.5–67.6%) were lower than the maternal eccDNA (median: 66.7%; range: 56.5–75.7%) (P = 0.02, paired t-test). In addition, eccDNA molecules from the smaller peak cluster (180–230 bp) were of lower methylation levels than those from the larger peak cluster (300–450 bp). Both of these findings were confirmed using the restriction enzyme approach. We also observed comparable methylation densities between linear and eccDNA of both maternal and fetal origins. The average half-lives of fetal linear and eccDNA in the maternal blood were 30.2 and 29.7 min, respectively. Conclusions We found that fetal eccDNA in plasma was relatively hypomethylated compared to the maternal eccDNA. The methylation densities of eccDNA were positively correlated with their sizes. In addition, fetal eccDNA was found to be rapidly cleared from the maternal blood after delivery, similar to fetal linear DNA.

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.

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.

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 Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. Plasma EBV DNA is a validated screening tool for NPC. In screening, there are some individuals who do not have NPC but carry EBV DNA in plasma. Currently it is not known from screening if there may be any genotypic differences in EBV isolates from NPC and non-NPC subjects. Also, low concentrations of EBV DNA in plasma could pose challenge to such EBV genotypic analysis through plasma DNA sequencing. Methods In a training dataset comprised of plasma DNA sequencing data of NPC and non-NPC subjects, we studied the difference in the EBV single nucleotide variant (SNV) profiles between the two groups. The most differentiating SNVs across the EBV genome were identified. We proposed an NPC risk score to be derived from the genotypic patterns over these SNV sites. We subsequently analyzed the NPC risk scores in a testing set. Results A total of 661 significant SNVs across the EBV genome were identified from the training set. In the testing set, NPC plasma samples were shown to have high NPC risk scores, which suggested the presence of NPC-associated EBV SNV profiles. Among the non-NPC samples, there was a wide range of NPC risk scores. These results support the presence of diverse SNV profiles of EBV isolates from non-NPC subjects. Conclusion EBV genotypic analysis is feasible through plasma DNA sequencing. The NPC risk score may be used to inform the cancer risk based on the EBV genome-wide SNV profile.

Significance We observed the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. We found that the plasma eccDNA molecules were longer than their linear counterparts. Among such eccDNA molecules, those of fetal origin were shorter than those of maternal origin. Characteristic dual-repeat patterns of eccDNA junctions might shed light on their possible generation mechanisms and provide them with distinctive signatures over linear cell-free DNA. Furthermore, the closed circular structure of eccDNA might allow resistance to exonucleases and thus higher stability of these molecules over their linear counterparts. These features of eccDNA provide opportunities for research and biomarker development. This work represents an example in the nascent field of plasma DNA topologics., We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5′-untranslated regions (5′-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.

Significance We revealed a large population of long cell-free DNA molecules (up to 23,635 bp in length) in maternal plasma and developed an approach which leveraged the abundance of CpG sites on long molecules to deduce the tissue of origin of individual plasma DNA molecules based on single-molecule methylation analysis. We illustrated how such an approach may be utilized to achieve noninvasive prenatal testing of monogenic diseases. We also revealed a reduction in amounts of such long cell-free DNA molecules and a different end motif profile in maternal plasma DNA from pregnancies with preeclampsia. Hence, long cell-free DNA molecules represent a valuable resource of biomarker development for pregnancy-associated disorders., In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g.

Cell-free extrachromosomal circular DNA (eccDNA) as a distinct topological form from linear DNA has recently gained increasing research interest, with possible clinical applications as a class of biomarkers. In this study, we aimed to explore the relationship between nucleases and eccDNA characteristics in plasma. By using knockout mouse models with deficiencies in deoxyribonuclease 1 (DNASE1) or deoxyribonuclease 1 like 3 (DNASE1L3), we found that cell-free eccDNA in Dnase1l3-/- mice exhibited larger size distributions than that in wild-type mice. Such size alterations were not found in tissue eccDNA of either Dnase1-/- or Dnase1l3-/- mice, suggesting that DNASE1L3 could digest eccDNA extracellularly but did not seem to affect intracellular eccDNA. Using a mouse pregnancy model, we observed that in Dnase1l3-/- mice pregnant with Dnase1l3+/- fetuses, the eccDNA in the maternal plasma was shorter compared with that of Dnase1l3-/- mice carrying Dnase1l3-/- fetuses, highlighting the systemic effects of circulating fetal DNASE1L3 degrading the maternal eccDNA extracellularly. Furthermore, plasma eccDNA in patients with DNASE1L3 mutations also exhibited longer size distributions than that in healthy controls. Taken together, this study provided a hitherto missing link between nuclease activity and the biological manifestations of eccDNA in plasma, paving the way for future biomarker development of this special form of DNA molecules.

Single-stranded ends of double-stranded DNA (jagged ends) are more abundant in urinary DNA than in plasma DNA. However, the lengths of jagged ends in urinary DNA remained undetermined, as a previous method used for urinary DNA jagged end sequencing analysis (Jag-seq) relied on unmethylation at CpG sites, limiting the resolution. Here, we performed high-resolution Jag-seq analysis using methylation at non-CpG cytosine sites, allowing determination of exact length of jagged ends. The urinary DNA bore longer jagged ends (~26-nt) than plasma DNA (~17-nt). The jagged end length distribution displayed 10-nt periodicities in urinary DNA, which were much less observable in plasma DNA. Amplitude of the 10-nt periodicities increased in patients with renal cell carcinoma. Heparin treatment of urine diminished the 10-nt periodicities. The urinary DNA jagged ends often extended into nucleosomal cores, suggesting potential interactions with histones. This study has thus advanced our knowledge of jagged ends in urine DNA.

Circular RNA (circRNA) is an important factor for regulating the progression of many cardiovascular diseases, including acute myocardial infarction (AMI). However, the role of circ_0124644 in AMI progression remains unclear. Hypoxia was used to induce cardiomyocytes injury. The expression of circ_0124644, microRNA (miR)-590-3p, and SRY-box transcription factor 4 (SOX4) mRNA was measured by qRT-PCR. Cell counting kit 8 (CCK8) assay and flow cytometry were utilized to detect cell viability, cell cycle progression, and apoptosis. The protein levels of apoptosis markers and SOX4 were determined by western blot (WB) analysis, and the levels of oxidative stress markers were assessed using commercial Assay Kits. Dual-luciferase reporter assay, RIP assay, and RNA pull-down assay were employed to confirm the interaction between miR-590-3p and circ_0124644 or SOX4. Circ_0124644 was upregulated in AMI patients and hypoxia-induced cardiomyocytes. Hypoxia could inhibit cardiomyocytes viability, cell cycle process, and promote apoptosis and oxidative stress, while silencing circ_0124644 could alleviate hypoxia-induced cardiomyocytes injury. In terms of mechanism, circ_0124644 could target miR-590-3p. MiR-590-3p overexpression could relieve hypoxia-induced cardiomyocytes injury. Also, the suppressive effect of circ_0124644 knockdown on hypoxia-induced cardiomyocytes injury could be reversed by miR-590-3p inhibitor. Moreover, SOX4 was found to be a target of miR-590-3p, and its overexpression also could reverse the regulation of miR-590-3p on hypoxia-induced cardiomyocytes injury. Circ_0124644 silencing could alleviate hypoxia-induced cardiomyocytes injury by regulating the miR-590-3p/SOX4 axis, suggesting that it might be a target for alleviating AMI.

There is much global research interest surrounding the use of cell-free DNA (cfDNA) for liquid biopsies. cfDNA-based non-invasive prenatal testing for foetal chromosomal aneuploidies was the first successful application of cfDNA technology that transformed clinical practice - it has since been rapidly adopted in dozens of countries and is used by millions of pregnant women every year. Prompted by such developments, efforts to use cfDNA in other fields, especially for cancer detection and monitoring have been actively pursued in recent years. Cancer-associated aberrations including single nucleotide mutations, copy number aberrations, aberrations in methylation and alterations in DNA fragmentation patterns have been detected in the cfDNA of patients suffering from a wide variety of cancers. In addition, the analysis of methylation and fragmentomic patterns has enabled the tissue origin of cfDNA to be determined. In this review, different approaches for detecting circulating liver-derived nucleic acids and cancer-associated aberrations, as well as their potential clinical applications for the detection, monitoring and management of hepatocellular carcinoma, will be discussed.

Sesquizygotic multiple pregnancy is an exceptional intermediate between monozygotic and dizygotic twinning. We report a monochorionic twin pregnancy with fetal sex discordance. Genotyping of amniotic fluid from each sac showed that the twins were maternally identical but chimerically shared 78% of their paternal genome, which makes them genetically in between monozygotic and dizygotic; they are sesquizygotic. We observed no evidence of sesquizygosis in 968 dizygotic twin pairs whom we screened by means of pangenome single-nucleotide polymorphism genotyping. Data from published repositories also show that sesquizygosis is a rare event. Detailed genotyping implicates chimerism arising at the juncture of zygotic division, termed heterogonesis, as the likely initial step in the causation of sesquizygosis.

Objective Cell‐free DNA (cfDNA) fragments in maternal plasma contain DNA damage and may negatively impact the sensitivity of noninvasive prenatal testing (NIPT). However, some of these DNA damages are potentially reparable. We aimed to recover these damaged cfDNA molecules using PreCR DNA repair mix. Methods cfDNA was extracted from 20 maternal plasma samples and was repaired and sequenced by the Illumina platform. Size profiles and fetal DNA fraction changes of repaired samples were characterized. Targeted sequencing of chromosome Y sequences was used to enrich fetal cfDNA molecules following repair. Single‐molecule real‐time (SMRT) sequencing platform was employed to characterize long (>250 bp) cfDNA molecules. NIPT of five trisomy 21 samples was performed. Results Size profiles of repaired libraries were altered, with significantly increased long (>250 bp) cfDNA molecules. Single nucleotide polymorphism (SNP)‐based analyses showed that both fetal‐ and maternal‐derived cfDNA molecules were enriched by the repair. Fetal DNA fractions in maternal plasma showed a small but consistent (4.8%) increase, which were contributed by a higher increment of long fetal cfDNA molecules. z‐score values were improved in NIPT of all trisomy 21 samples. Conclusion Plasma DNA repair recovers and enriches long cfDNA molecules of both fetal and maternal origins in maternal plasma., What is already known about this topic? Most of the cell‐free DNA (cfDNA) fragments in maternal plasma have sizes less than 200 bp, with fetal molecules being shorter than maternal ones.DNA damages exist in cfDNA, particularly single‐strand nicks.Occasional no call for noninvasive prenatal testing (NIPT) can be caused by insufficient fetal DNA fraction. What does this study add? Repair of cfDNA by PreCR repair mix can recover a subset of long (>250 bp) cfDNA molecules.Both fetal and maternal long cfDNA are enriched by PreCR repair treatment.Mild but consistent increments in fetal DNA fractions after PreCR repair, which are contributed by higher enrichment of long fetal cfDNA molecules.PreCR repair treatment improves NIPT of trisomy 21 by elevating z scores resulting in better discrimination of aneuploid from euploid samples.

Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We explored the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in the fragmentation of ucfDNA using mouse models. The deletion of Dnase1 in mice (Dnase1-/-) caused aberrations in ucfDNA fragmentation, including a 24-fold increase in DNA concentration, and a 3-fold enrichment of long DNA molecules, with a relative decrease of fragments with thymine ends and reduction of jaggedness (i.e., the presence of single-stranded protruding ends). In contrast, such changes were not observed in mice with Dnase1l3 deletion (Dnase1l3-/-). These results suggested that DNASE1 was an important nuclease contributing to the ucfDNA fragmentation. Western blot analysis revealed that the concentration of DNASE1 protein was higher in urine than DNASE1L3. The native-polyacrylamide gel electrophoresis zymogram showed that DNASE1 activity in urine was higher than that in plasma. Furthermore, the proportion of ucfDNA fragment ends within DNase I hypersensitive sites (DHSs) was significantly increased in Dnase1-deficient mice. In humans, patients with bladder cancer had lower proportions of ucfDNA fragment ends within the DHSs when compared with participants without bladder cancer. The area under the curve (AUC) for differentiating patients with and without bladder cancer was 0.83, suggesting the analysis of ucfDNA fragmentation in the DHSs may have potential for bladder cancer detection. This work revealed the intrinsic links between the nucleases in urine and ucfDNA fragmentomics.

We developed genetic-epigenetic tissue mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung transplant recipients, we showed that, at 72 hr after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles, and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring, and cancer screening.

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.

We developed Genetic-Epigenetic Tissue Mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung-transplant recipients, we showed that, at 72 hours after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring and cancer screening.

Circular RNAs (circRNAs) are a group of RNAs featured by a covalently closed continuous loop structure. This study aimed to uncover the function and mechanism of circ-ubiquitin specific peptidase 36 (USP36) in endothelial cells treated with oxidized low-density lipoprotein (ox-LDL). The levels of circ-USP36, microRNA-98-5p (miR-98-5p) and vascular cell adhesion molecule 1 (VCAM1) were examined by a quantitative real-time polymerase chain reaction (qRT-PCR). The viability, apoptosis and inflammation were detected by (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Western blot assay was performed to detect the expression of apoptosis and proliferation-related markers and VCAM1 protein level. The targets of circ-USP36 and miR-98-5p were searched using starBase website, and dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to validate the above predictions. Ox-LDL exposure induced the upregulation of circ-USP36 in HUVEC cells. Circ-USP36 accelerated ox-LDL-induced apoptosis, inflammatory and viability inhibition of HUVEC cells. MiR-98-5p was a direct downstream gene of circ-USP36. Circ-USP36 promoted the injury of ox-LDL-induced HUVEC cells through targeting miR-98-5p. VCAM1 could bind to miR-98-5p, and the protective effects of miR-98-5p accumulation on ox-LDL-induced HUVEC cells were reversed by the transfection of VCAM1. VCAM1 was regulated by circ-USP36/miR-98-5p signaling in HUVEC cells. Ox-LDL promoted the apoptosis and inflammation but suppressed the viability of HUVEC cells through upregulating circ-USP36, thus elevating the expression of VCAM1 via miR-98-5p.

Objectives Due to the maternally‐inherited nature of mitochondrial DNA (mtDNA), there is a lack of information regarding fetal mtDNA in the plasma of pregnant women. We aim to explore the presence and topologic forms of circulating fetal and maternal mtDNA molecules in surrogate pregnancies. Methods Genotypic differences between fetal and surrogate maternal mtDNA were used to identify the fetal and maternal mtDNA molecules in plasma. Plasma samples were obtained from the surrogate pregnant mothers. Using cleavage‐end signatures of BfaI restriction enzyme, linear and circular mtDNA molecules in maternal plasma could be differentiated. Results Fetal‐derived mtDNA molecules were mainly linear (median: 88%; range: 80%–96%), whereas approximately half of the maternal‐derived mtDNA molecules were circular (median: 51%; range: 42%–60%). The fetal DNA fraction of linear mtDNA was lower (median absolute difference: 9.8%; range: 1.1%–27%) than that of nuclear DNA (median: 20%; range: 9.7%–35%). The fetal‐derived linear mtDNA molecules were shorter than the maternal‐derived ones. Conclusion Fetal mtDNA is present in maternal plasma, and consists mainly of linear molecules. Surrogate pregnancies represent a valuable clinical scenario for exploring the biology and potential clinical applications of circulating mtDNA, for example, for pregnancies conceived following mitochondrial replacement therapy.

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.

Liquid biopsies that analyze cell-free DNA in blood plasma are used for noninvasive prenatal testing, oncology, and monitoring of organ transplant recipients. DNA molecules are released into the plasma from various bodily tissues. Physical and molecular features of cell-free DNA fragments and their distribution over the genome bear information about their tissues of origin. Moreover, patterns of DNA methylation of these molecules reflect those of their tissue sources. The nucleosomal organization and nuclease content of the tissue of origin affect the fragmentation profile of plasma DNA molecules, such as fragment size and end motifs. Besides double-stranded linear fragments, other topological forms of cell-free DNA also exist-namely circular and single-stranded molecules. Enhanced by these features, liquid biopsies hold promise for the noninvasive detection of tissue-specific pathologies with a range of clinical applications.

Interaction between eosinophils and dermal fibroblasts is essential for provoking allergic inflammation in atopic dermatitis (AD). In vitro co-culture of human eosinophils and dermal fibroblasts upon AD-related IL-31 and IL-33 stimulation, and in vivo MC903-induced AD murine model were employed to investigate the anti-inflammatory mechanism of IL-1 family cytokine IL-37 in AD. Results showed that IL-37b could inhibit the in vitro induction of AD-related pro-inflammatory cytokines IL-6 and TNF-α, and chemokines CXCL8, CCL2 and CCL5, increase autophagosome biogenesis-related LC3B, and decrease autophagy-associated ubiquitinated protein p62 by regulating intracellular AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling pathway. In CRISPR/Cas9 human IL-37b knock-in mice, IL-37b could significantly alleviate MC903-stimulated ear tissue swelling, itching sensation and the level of circulating cytokine IL-6 and ear in situ expression of AD-related TNF-α, CCL5 and transforming growth factor-β. Moreover, IL-37b could significantly upregulate Foxp3+ regulatory T cells (Treg) in spleen and ear together with significantly increased serum Treg cytokine IL-10, and decrease eosinophil infiltration in ear lesion. IL-37b knock-in mice showed a distinct intestinal microbiota metabolic pattern upon MC903 stimulation. Furthermore, IL-37b restored the disordered gut microbiota diversity, through regulating the in vivo autophagy mechanism mediated by intestinal metabolite 3-methyladenine, adenosine monophosphate, 2-hydroxyglutarate, purine and melatonin. In summary, IL-37b could significantly ameliorate eosinophils-mediated allergic inflammation via the regulation of autophagy mechanism, intestinal bacterial diversity and their metabolites in AD. Results therefore suggest that IL-37 is a potential anti-inflammatory cytokine for AD treatment.

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.

BACKGROUND There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. METHODS Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. RESULTS Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by β-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. CONCLUSIONS Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.

Direct detection of F8 and F9 sequence variants in maternal plasma of hemophilia carriers has been demonstrated by microfluidics digital PCR. Noninvasive prenatal assessment of the most clinically relevant group of sequence variants among patients with hemophilia, namely, those involving int22h-related inversions disrupting the F8 gene, poses additional challenges because of its molecular complexity. We investigated the use of droplet digital PCR (ddPCR) and targeted massively parallel sequencing (MPS) for maternal plasma DNA analysis to noninvasively determine fetal mutational status in pregnancies at risk for hemophilia. We designed family-specific ddPCR assays to detect causative sequence variants scattered across the F8 and F9 genes. A haplotype-based approach coupled with targeted MPS was applied to deduce fetal genotype by capturing a 7.6-Mb region spanning the F8 gene in carriers with int22h-related inversions. The ddPCR analysis correctly determined fetal hemophilia status in 15 at-risk pregnancies in samples obtained from 8 to 42 weeks of gestation. There were 3 unclassified samples, but no misclassification. Detailed fetal haplotype maps of the F8 gene region involving int22h-related inversions obtained through targeted MPS enabled correct diagnoses of fetal mutational status in 3 hemophilia families. Our data suggest it is feasible to apply targeted MPS to interrogate maternally inherited F8 int22h-related inversions, whereas ddPCR represents an affordable approach for the identification of F8 and F9 sequence variants in maternal plasma. These advancements may bring benefits for the pregnancy management for carriers of hemophilia sequence variants; in particular, the common F8 int22h-related inversions, associated with the most severe clinical phenotype.

Urinary cell-free (cf) DNA holds great potential as a completely noninvasive form of liquid biopsy. Knowledge of the composition of cfDNA by tissue of origin is useful for guiding its clinical uses. We conducted a global survey of urinary cfDNA composition using genomewide bisulfite sequencing. While previous studies focused on detecting cfDNA from a single source at a time, genomewide tissue specific methylation signatures allow us to simultaneously deduce the proportional contribution from each contributing tissue. The proportional contributions derived from methylation deconvolution are highly correlated with those calculated using allograft-derived donor-specific genetic markers in the urine of hematopoetic stem cell and renal transplant recipients. We found a large variation of proportional contributions from different tissues. We then assessed if cfDNA undergoes time-dependent fragmentation in urine by conducting in vitro incubation experiments. In vitro incubation at 37°C showed that urinary cfDNA concentration decreased under first order kinetics with a half-life of 2.6 to 5.1h. This is reflected in parallel by a decrease in the proportion of long fragments and increase in amplitude of 10bp periodicity seen in the cfDNA size profile. This global survey of urinary cfDNA has deepened our understanding of the composition, degradation and variation of cfDNA in the urinary tract and has laid a foundation for the use of genomewide urinary cfDNA sequencing as a molecular diagnostics tool.

BACKGROUND Recent studies have suggested that single-stranded DNA (ssDNA) library preparation can enrich short DNA species from the plasma of healthy individuals, cancer patients, and transplant recipients. Based on previous observations that fetal DNA molecules in the maternal plasma are shorter than maternal DNA molecules, ssDNA library preparation may potentially enrich fetal DNA and provide substantial improvement in noninvasive prenatal testing. METHODS We tested this hypothesis by comparing the maternal plasma DNA sequencing results using 2 types of ssDNA library preparation methods and a standard double-stranded DNA (dsDNA) library method using samples from first- and third-trimester pregnancies. We also evaluated the performance of ssDNA and dsDNA library methods in the noninvasive prenatal detection of trisomy 21 from maternal plasma. RESULTS Short DNA species were significantly enriched in ssDNA libraries. However, contrary to previous speculation, no significant enrichment was observed in the overall fetal fraction in maternal plasma collected in the first trimester. Our use of an ssDNA library did not reduce the variation in chromosomal representation when compared with a standard dsDNA library in the first-trimester plasma samples. ssDNA libraries also showed inferior performance in the noninvasive prenatal detection of trisomy 21 from maternal plasma. Detailed fetal fraction analysis using size-fractionated Y chromosome sequences and fetal-specific single-nucleotide polymorphisms (SNPs) revealed an unexpected finding that short maternal DNA was preferentially enriched over short fetal DNA in an ssDNA library irrespective of GC content. CONCLUSIONS Our findings have shown that ssDNA library preparation preferentially enriches short maternally derived DNA in maternal plasma.

Objective The aim of this study is to develop an approach for analyzing plasma DNA sequencing data for noninvasive fetal chromosomal aneuploidy testing that does not require the comparison with control samples or a series of selected genomic regions. Results We developed the control-free noninvasive fetal chromosomal examination (COFFEE) algorithm by utilizing the size differences between the fetally derived and maternally derived DNA molecules in maternal plasma. We applied COFFEE on three datasets generated in different experimental settings. COFFEE showed 100% accuracy in trisomy 21 testing on these datasets. In contrast, samples analyzed using an existing control-based z-score method would introduce a false-positive result because of batch-to-batch variation, when the tested samples were analyzed using control samples from other batches. We believe that COFFEE is useful for enhancing the cost-effectiveness of noninvasive fetal chromosomal aneuploidy testing particularly in laboratories with small caseloads. Source code and testing datasets for COFFEE are available for download at http://www.cuhk.edu.hk/med/cpy/Research/COFFEE/. Conclusion Control-free noninvasive fetal chromosomal examination is demonstrated to be a versatile data analysis approach and could enhance the application of noninvasive fetal chromosomal aneuploidy detection. © 2017 John Wiley & Sons, Ltd.

Objectives The objectives of this study were to compare the concentrations, size profiles and major tissue contributors of cell-free DNA (cfDNA) in plasma and in serum. Design and methods Thirteen pregnant women in the third trimester were recruited for this study. We collected EDTA-plasma and serum samples using various collection tubes. We determined their cfDNA concentrations and fetal cfDNA fractions using a zinc-finger X (ZFX)/zinc-finger Y (ZFY) droplet digital polymerase chain reaction (ZFX/ZFY ddPCR) assay. We used paired-end massively parallel sequencing (MPS) to measure plasma and serum cfDNA sizes at single-base resolution. We deconvoluted the genome-wide bisulfite sequencing data with reference to the methylation profiles of different tissues. Results The concentrations of cfDNA collected in Sarstedt Serum Z tubes were found to be significantly higher than those in Greiner Bio-One Vacuette® Z Serum Separator Clot Activator tubes or Vacuette® Z Serum Clot Activator tubes. The concentrations of fetal cfDNA were significantly reduced in samples collected in the Vacuette® serum collection tubes. Fetal cfDNA fractions were significantly reduced in all sera compared to plasma. MPS of serum cfDNA revealed a right shift of the size distributions compared to plasma. Methylation-based tissue mapping of serum cfDNA revealed an increase of cfDNA from neutrophils and B cells but not T cells. Conclusions The use of different serum collection tubes has a significant impact on serum cfDNA concentrations. This effect is likely mediated through the combined effect of genomic DNA release from white blood cells and DNA degradation or removal.

To the Editor: There has been much recent interest in the biology of plasma DNA because of the emergence of noninvasive prenatal testing and liquid biopsy for cancer. Circulating cell-free (cf) DNA displays distinct fragmentation patterns in terms of fragment sizes, genomic distribution, and fragment endpoints (1, 2). Although the mechanism of cfDNA fragmentation remains largely unknown, fragment characteristics can be used to infer epigenetic architecture (3) and gene expression (4) at the site of DNA origin. DNase1 is a major secretory nuclease. We used a DNase1 knockout mouse model [DNase1tm1.1(KOMP)Vlcg] obtained from the University of California at Davis Knockout Mouse Project (KOMP) Repository to investigate the effects of removing DNase1 on the characteristics of circulating cfDNA. Plasma was obtained via cardiac puncture at 10–12 weeks of age from homozygous wildtype ( DNase1 +/+) and knockout ( DNase1 −/−) mice. The blood was put into EDTA-containing collection tubes that were centrifuged at 16000 g at 4 °C for 10 min. The supernatant was then subjected to a second centrifugation at 16 000 g at 4 °C for 10 min to obtain the cell-free plasma. Western blot analysis of total cell lysates from the kidney confirmed the absence of DNase1 in the knockout mice. We used an in vitro DNase activity assay on the basis of the measurement of DNA degradation activity, as previously described (5) with minor modifications. In …

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.

The discovery of circulating cell-free fetal DNA has profoundly transformed the landscape of noninvasive prenatal testing (NIPT) and rapidly found its way into global clinical applications. The fractional concentration of cell-free fetal DNA in plasma DNA of a pregnant woman is an important parameter for understanding and interpreting analytical results of NIPT. Thus, the accurate quantification of fetal DNA fraction is indispensable when NIPT is involved. In this protocol, we describe the bioinformatics workflow to calculate fetal DNA fraction using two programs developed by our group, which provide accurate estimation.

The discovery of circulating cell-free fetal DNA has profoundly transformed the landscape of noninvasive prenatal testing (NIPT) and rapidly found its way into global clinical applications. The fractional concentration of cell-free fetal DNA in plasma DNA of a pregnant woman is an important parameter for understanding and interpreting analytical results of NIPT. Thus, the accurate quantification of fetal DNA fraction is indispensable when NIPT is involved. In this protocol, we describe the bioinformatics workflow to calculate fetal DNA fraction using two programs developed by our group, which provide accurate estimation.

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.