scholarly journals Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease

2018 ◽  
Author(s):  
Joshua Moss ◽  
Judith Magenheim ◽  
Daniel Neiman ◽  
Hai Zemmour ◽  
Netanel Loyfer ◽  
...  
Keyword(s):  
Vascular Endothelial Cells ◽  
White Blood Cells ◽  
Cell Types ◽  
Clinical Findings ◽  
The Body ◽  
Unknown Primary ◽  
Cell Free Dna ◽  
Free Dna ◽  
Circulating Cell Free Dna

AbstractMethylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated usingin silicosimulations as well asin vitromixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.

scholarly journals Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA

2017 ◽  
Vol 114 (36) ◽  
pp. 9623-9628 ◽  
Author(s):  
Mark Kowarsky ◽  
Joan Camunas-Soler ◽  
Michael Kertesz ◽  
Iwijn De Vlaminck ◽  
Winston Koh ◽  
...  
Keyword(s):  
Human Body ◽  
Sequence Data ◽  
Human Microbiome ◽  
Pcr Amplification ◽  
The Body ◽  
Direct Pcr ◽  
Cell Free Dna ◽  
Free Dna ◽  
Novel Taxa ◽  
Circulating Cell Free Dna

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

scholarly journals Detection of somatic mutations in circulating cell-free DNA from patients with esophageal squamous cell carcinoma

2020 ◽  
Author(s):  
Zuyang Yuan ◽  
Xinfeng Wang ◽  
Xiao Geng ◽  
Yin Li ◽  
Juwei Mu ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Solid Tumor ◽  
Somatic Mutations ◽  
White Blood Cells ◽  
Cell Free Dna ◽  
Free Dna ◽  
Circulating Cell Free Dna

Abstract Background: The aim of this study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in circulating cell-free DNA (cfDNA) from patients with esophageal squamous cell carcinoma (ESCC). Methods: Paired multi-regional tumor tissues, cfDNA and white blood cells (WBCs) collected from five ESCC patients before treatment from a prospective study (NCT02395705). Of them, samples from Cohort 1 (E102 and E110) were sequenced by whole-exome sequencing (WES) and those from Cohort 2 (E104, E111 and E121) were sequenced by targeted captured sequencing with a panel of 560 cancer-related genes respectively. To call somatic single nucleotide variations (SNVs) by comparing the solid tumor or cfDNA with matched WBCs, the minimal variant allele frequency (VAFmin) as 0.1% and P value <0.05 were allowed. Results: Genomic DNA (gDNA) and plasma-derived cfDNA from 26 samples were successfully sequenced. In Cohort 1, 596 (596/712, 83%) and 562 (562/796, 71%) were heterogeneous SNVs in E102 and E110 respectively. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R2 = 0.78, P <0.0001). In Cohort 2, 296 (296/323, 92%), 384 (384/423, 91%) and 331 (331/357, 93%) were heterogeneous SNVs in E104, E111 and E121respectively. cfDNA could recover an average of 60.7% (31/51; range, 35.7%-76.2%) of somatic mutations present in matched solid tumors. The correlation of VAFs between cfDNA and matched solid tumor was significantly positive (r2 =0.92, P <0.0001).Conclusions: Both sequencing approaches revealed the highly intratumoral heterogeneity in ESCC and enabled the detection of both ubiquitous and heterogeneous mutations in cfDNA. Further validation in cfDNA is required to define its potential utility for ESCC in clinical practice. Trial registrationAll patients selected in this study were from the registered clinical trial from ClinicalTrials.gov (NCT02395705). Date of registration: March 24, 2015.

Circulating cell‐free DNA release in vitro: kinetics, size profiling, and cancer‐related gene methylation

2019 ◽  
Vol 234 (8) ◽  
pp. 14079-14089 ◽  
Author(s):  
Maria Panagopoulou ◽  
Makrina Karaglani ◽  
Ioanna Balgkouranidou ◽  
Chrisoula Pantazi ◽  
George Kolios ◽  
...  
Keyword(s):  
Related Gene ◽  
Gene Methylation ◽  
Cell Free Dna ◽  
Free Dna ◽  
Cancer Related Gene ◽  
Dna Release ◽  
Release In Vitro ◽  
In Vitro Kinetics ◽  
Circulating Cell Free Dna

scholarly journals Remote immune processes revealed by immune-derived circulating cell-free DNA

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ilana Fox-Fisher ◽  
Sheina Piyanzin ◽  
Bracha Lea Ochana ◽  
Agnes Klochendler ◽  
Judith Magenheim ◽  
...  
Keyword(s):  
Blood Cell ◽  
B Cell ◽  
Immune Cell ◽  
B Cell Lymphoma ◽  
Cell Types ◽  
Cell Free Dna ◽  
Cell Counts ◽  
Free Dna ◽  
Blood Cell Counts ◽  
Circulating Cell Free Dna

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N=242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N=92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with Eosinophilic Esophagitis (N=21) and B-cell lymphoma (N=27) showed selective elevation of eosinophil and B-cell cfDNA respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.

scholarly journals Spatial co-fragmentation pattern of cell-free DNA recapitulatesin vivochromatin organization and identifies tissues-of-origin

2019 ◽  
Author(s):  
Yaping Liu ◽  
Tzu-Yu Liu ◽  
David E. Weinberg ◽  
Brandon W. White ◽  
Chris J. De La Torre ◽  
...  
Keyword(s):  
White Blood Cells ◽  
Three Dimensional ◽  
Cell Types ◽  
Chromatin Organization ◽  
Fragmentation Pattern ◽  
Cell Free Dna ◽  
Sequence Composition ◽  
Free Dna ◽  
Complete Representations

ABSTRACTThree-dimensional chromatin organization varies across cell types and is essential for gene regulation. However, current technologies are unable to assessin vivogenome-wide chromatin organization non-invasively. Here we show that distant correlations in the fragment length of cell-free DNA (cfDNA) recapitulate three-dimensional chromatin organization. The inferred organization is highly concordant with that measured by Hi-C in white blood cells from healthy donors, and is not explained by technical bias or sequence composition. Furthermore, the inferred organization reflects different genomic organization in the various cell types contributing to cfDNA, allowing identification and quantification of tissues of origin. This approach is concordant with previous methods, but with more complete representations of cfDNA. Our results, demonstrated in cfDNA from healthy individuals and cancer patients, may enable noninvasive monitoring ofin vivogenome organization and accurate quantification of cell death in different clinical conditions.

scholarly journals Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

Tumor Biology ◽  
2019 ◽  
Vol 41 (8) ◽  
pp. 101042831986636 ◽  
Author(s):  
Abel Jacobus Bronkhorst ◽  
Vida Ungerer ◽  
Stefan Holdenrieder
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Culture Supernatant ◽  
Quantitative Polymerase Chain Reaction ◽  
High Sensitivity ◽  
Cell Types ◽  
Cell Culture Supernatant ◽  
Cell Free Dna ◽  
Free Dna

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA–based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

scholarly journals Remote immune processes revealed by immune-derived circulating cell-free DNA

2021 ◽  
Author(s):  
Ilana Fox-Fisher ◽  
Sheina Piyanzin ◽  
Agnes Klochendler ◽  
Bracha Lea Ochana ◽  
Judith Magenheim ◽  
...  
Keyword(s):  
Blood Cell ◽  
B Cell ◽  
Immune Cell ◽  
B Cell Lymphoma ◽  
Cell Types ◽  
Cell Free Dna ◽  
Cell Counts ◽  
Free Dna ◽  
Blood Cell Counts ◽  
Circulating Cell Free Dna

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N=242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N=92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with Eosinophilic Esophagitis (N=21) and B-cell lymphoma (N=27) showed selective elevation of eosinophil and B-cell cfDNA respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.

scholarly journals Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA

2020 ◽  
Author(s):  
Christa Caggiano ◽  
Barbara Celona ◽  
Fleur Garton ◽  
Joel Mefford ◽  
Brian Black ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Pregnant Women ◽  
Reference Data ◽  
Cell Types ◽  
The Body ◽  
Cell Type ◽  
Cell Free Dna ◽  
Free Dna ◽  
Als Patients ◽  
Low Coverage

AbstractCirculating cell-free DNA (cfDNA) in the bloodstream originates from dying cells and is a promising non-invasive biomarker for cell death. Here, we develop a method to accurately estimate the relative abundances of cell types contributing to cfDNA. We leverage the distinct DNA methylation profile of each cell type throughout the body. Decomposing the cfDNA mixture is difficult, as fragments from relevant cell types may only be present in a small amount. We propose an algorithm, CelFiE, that estimates cell type proportion from both whole genome cfDNA input and reference data. CelFiE accommodates low coverage data, does not rely on CpG site curation, and estimates contributions from multiple unknown cell types that are not available in reference data. In simulations we show that CelFiE can accurately estimate known and unknown cell type of origin of cfDNA mixtures in low coverage and noisy data. Simulations also demonstrate that we can effectively estimate cfDNA originating from rare cell types composing less than 0.01% of the total cfDNA. To validate CelFiE, we use a positive control: cfDNA extracted from pregnant and non-pregnant women. CelFiE estimates a large placenta component specifically in pregnant women (p = 9.1 × 10−5). Finally, we use CelFiE to decompose cfDNA from ALS patients and age matched controls. We find increased cfDNA concentrations in ALS patients (p = 3.0 × 10−3). Specifically, CelFiE estimates increased skeletal muscle component in the cfDNA of ALS patients (p = 2.6 × 10−3), which is consistent with muscle impairment characterizing ALS. Quantification of skeletal muscle death in ALS is novel, and overall suggests that CelFiE may be a useful tool for biomarker discovery and monitoring of disease progression.

scholarly journals Baseline mutation profiling of 1134 samples of circulating cell-free DNA and blood cells from healthy individuals

2016 ◽  
Author(s):  
Ligang Xia ◽  
Zhoufang Li ◽  
Bo Zhou ◽  
Geng Tian ◽  
Lidong Zeng ◽  
...  
Keyword(s):  
Blood Cells ◽  
Somatic Mutations ◽  
White Blood Cells ◽  
Clinical Diagnostics ◽  
Healthy Individuals ◽  
Sequencing Data ◽  
Cell Free Dna ◽  
Early Cancer Diagnosis ◽  
Free Dna ◽  
Circulating Cell Free Dna

AbstractThe molecular alteration in circulating cell-free DNA (cfDNA) in plasma can reflect the status of the human body in a timely manner. Hence, cfDNA has emerged as important biomarkers in clinical diagnostics, particularly in cancer. However, somatic mutations are also commonly found in healthy individuals, which extensively interfere with the diagnostic results in cancer. This study was designed to examine the background somatic mutations in white blood cells (WBC) and cfDNA for healthy controls based on the sequencing data from 1134 samples, to understand the patterns and origin of mutations detected in cfDNA. We determined the mutation frequencies in both the WBC and cfDNA groups of the samples by a panel of 50 cancer-associated genes which covered 20K nucleotide regions using ultra-deep sequencing with average depth >40000 folds. Our results showed that most of mutations in cfDNA originated from WBC. We also observed that NPM1 gene was the most frequently mutant gene in both WBC and cfDNA. Our study highlighted the importance of sequencing both cfDNA and WBC, to improve the sensitivity and accuracy for calling cancer-related mutations from circulating tumor DNA, and shielded light on developing the early cancer diagnosis by cfDNA sequencing.

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