Molecular characterization of cell-free eccDNAs in human plasma.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhu J
PROVIDER: S-EPMC5591271 | biostudies-literature | 2017 Sep
REPOSITORIES: biostudies-literature
Publications
Molecular characterization of cell-free eccDNAs in human plasma.
Zhu Jing J Zhang Fan F Du Meijun M Zhang Peng P Fu Songbin S Wang Liang L
Scientific reports 20170908 1
Extrachromosomal circular DNAs (eccDNAs) have been reported in most eukaryotes. However, little is known about the cell-free eccDNA profiles in circulating system such as blood. To characterize plasma cell-free eccDNAs, we performed sequencing analysis in 26 libraries from three blood donors and negative controls. We identified thousands of unique plasma eccDNAs in the three subjects. We observed proportional eccDNA increase with initial DNA input. The detected eccDNAs were also associated with ...[more]
Similar Datasets
Project description:Many emerging technologies are reliant on circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) applications in the clinic. However, the impact of diurnal cycles or daily meals on circulating analytes are poorly understood and may be confounding factors when developing diagnostic platforms. To begin addressing this knowledge gap, we obtained plasma from four healthy donors serially sampled five times during 12 h in a single day. For all samples, we measured concentrations of cfDNA and cfRNA using both bulk measurements and gene-specific digital droplet PCR. We found no significant variation attributed to blood draw number for the cfDNA or cfRNA. This indicated that natural diurnal cycles and meal consumption do not appear to significantly affect abundance of total cfDNA, total cfRNA, or our two selected cfRNA transcripts. Conversely, we observed significant variation between individual donors for cfDNA and one of the cfRNA transcripts. The results of this work suggest that it will be important to consider patient-specific baselines when designing reliable circulating cfDNA or cfRNA clinical assays.
| S-EPMC7536441 | biostudies-literature
Project description:INTRODUCTION: Schistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR. METHODOLOGY/PRINCIPAL FINDINGS: Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14) showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year. CONCLUSIONS/SIGNIFICANCE: PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.
| S-EPMC2667260 | biostudies-literature
Project description:The utility of cell-free nucleic acids in monitoring cancer has been recognized by both scientists and clinicians. In addition to human transcripts, a fraction of cell-free nucleic acids in human plasma were proven to be derived from microbes and reported to have relevance to cancer. To obtain a better understanding of plasma cell-free RNAs (cfRNAs) in cancer patients, we profiled cfRNAs in ~300 plasma samples of 5 cancer types (colorectal cancer, stomach cancer, liver cancer, lung cancer, and esophageal cancer) and healthy donors (HDs) with RNA-seq. Microbe-derived cfRNAs were consistently detected by different computational methods when potential contaminations were carefully filtered. Clinically relevant signals were identified from human and microbial reads, and enriched Kyoto Encyclopedia of Genes and Genomes pathways of downregulated human genes and higher prevalence torque teno viruses both suggest that a fraction of cancer patients were immunosuppressed. Our data support the diagnostic value of human and microbe-derived plasma cfRNAs for cancer detection, as an area under the ROC curve of approximately 0.9 for distinguishing cancer patients from HDs was achieved. Moreover, human and microbial cfRNAs both have cancer type specificity, and combining two types of features could distinguish tumors of five different primary locations with an average recall of 60.4%. Compared to using human features alone, adding microbial features improved the average recall by approximately 8%. In summary, this work provides evidence for the clinical relevance of human and microbe-derived plasma cfRNAs and their potential utilities in cancer detection as well as the determination of tumor sites.
| S-EPMC9273212 | biostudies-literature
Project description:Conventional next generation sequencing analysis has provided important insights into cancer genetics. However, the detection of rare (low allele fraction) variants remains difficult because of the error-prone nucleotide changes derived from sequencing/PCR errors. To eliminate the false-positive variants and detect genuine rare variants, sequencing technology combined with molecular barcodes will be useful. Here, we used the newly developed dual-molecular barcode technology (Ion AmpliSeq HD) to analyze somatic mutations in 24 samples (12 tumor tissues and 12 plasma) from 12 patients with biliary-pancreatic and non-small cell lung cancers. We compared the results between next generation sequencing analysis with or without molecular barcode technologies. The variant allele fraction (VAF) between non-molecular barcode and molecular barcode sequencing was correlated in plasma DNA (R2?=?0.956) and tumor (R2?=?0.935). Both methods successfully detected high VAF mutations, however, rare variants were only identified by molecular barcode sequencing and not by non-molecular barcode sequencing. Some of these rare variants in tumors were annotated as pathogenic, and therefore subclonal driver mutations could be observed. Furthermore, the very low VAF down to 0.17% were identified in cell free DNA in plasma. These results demonstrate that the dual molecular barcode sequencing technologies can sensitively detect rare somatic mutations, and will be important in the investigation of the clonal and subclonal architectures of tumor heterogeneity.
| S-EPMC7042261 | biostudies-literature
Project description:Circulating tumor DNA (ctDNA) carries genetic information consistent with tumor cells and has potential value for molecular diagnosis of tumors. The present study analysed the gene mutations of plasma circulating cell-free DNA (cfDNA) and tumor tissue DNA in hepatocellular carcinoma (HCC) patients and explored the clinical application value of plasma cfDNA as a tumor marker in HCC molecular diagnosis. Samples from 29 patients with primary HCC were collected. Hotspot mutations in 50 tumor-associated genes were analysed using amplicon sequencing technology and gene loci with a mutant allele frequency (MAF) >1% were analysed. 35 mutant genes in total were detected by deep sequencing method of which the genes with maximum mutation frequencies were TP53, ATM, and ALK. In addition, a total of 21 patients were found to have a consistent gene mutation in plasma cfDNA and tumor tissue DNA and 17 cases had consistent gene mutations in the paracancerous tissue and tumor tissue DNA. Further analysis showed that the MAFs in the TP53, CTNNB1, PIK3CA, and CDKN2A genes were higher in patients with tumor diameters >5 cm than those with tumor diameters <5 cm. And the MAFs in the TP53, RET, FGFR3 and APC genes were significantly higher in patients with multiple tumors or with metastasis than in single tumor patients. In conclusion, amplicon sequencing technology is highly sensitive for the detection of mutant genes in the plasma cfDNA of HCC patients. Plasma cfDNA might be an effective molecular marker for HCC molecular diagnosis.
| S-EPMC6456549 | biostudies-literature
Project description:BACKGROUND:Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established. METHODS:Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n?=?91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine. RESULTS:Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n?=?22) improved detection to ~?50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n?=?16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings. CONCLUSIONS:These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.
| S-EPMC7048087 | biostudies-literature
Project description:Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma. CMV reads were identified in 120 (5.4%) samples. Median cfDNA fragment size derived from CMV was significantly shorter than cfDNA derived from human chromosomes (103 vs 172?bp, p?<?0.0001), corresponding to the 3rd percentile of human cfDNA. Sequencing of cfDNA from seven plasma samples from transplant patients positive for CMV confirmed the extraordinarily short nature of CMV cfDNA fragment size with a median length of 149?bp. We further show that these high-resolution measurements of CMV DNA fragment size accurately predict measured discrepancies in serum viral load measurements by different qPCR assays. These results highlight the exceptionally fragmented nature of CMV cfDNA and illustrate the promise of plasma cfDNA sequencing for quantitating viral loads through detection of fragments that would be unrecoverable by qPCR.
| S-EPMC7048871 | biostudies-literature
Project description:BACKGROUND:Echinococcosis is a life-threatening parasitic disease caused by Echinococcus spp. tapeworms with over one million people affected globally at any time. The Echinococcus spp. tapeworms in the human body release DNA to the circulatory system, which can be a biomarker for echinococcosis. Cell-free DNA (cfDNA) is widely used in medical research and has been applied in various clinical settings. As for echinococcosis, several PCR-based tests had been trialed to detect cell-free Echinococcus spp. DNA in plasma or serum, but the sensitivity was about 20% to 25%. Low sensitivity of PCR-based methods might be related to our limited understanding of the features of cell-free Echinococcus spp. DNA in plasma, including its concentration, fragment pattern and release source. In this study, we applied ultra-high-throughput sequencing to comprehensively investigate the characteristics of cell-free Echinococcus spp. DNA in plasma of echinococcosis patients. METHODOLOGY/PRINCIPAL FINDINGS:We collected plasma samples from 23 echinococcosis patients. Total plasma cfDNA was extracted and sequenced with a high-throughput sequencing platform. An average of 282 million read pairs were obtained for each plasma sample. Sequencing data were analyzed with bioinformatics workflow combined with Echinococcus spp. sequence database. After identification of cell-free Echinococcus spp. reads, we found that the cell-free Echinococcus spp. reads accounted for 1.8e-5 to 4.0e-9 of the total clean reads. Comparing fragment length distribution of cfDNA between Echinococcus spp. and humans showed that cell-free Echinococcus spp. DNA of cystic echinococcosis (CE) had a broad length range, while that of alveolar echinococcosis (AE) had an obvious peak at about 135 bp. We found that most of the cell-free Echinococcus spp. DNA reads were from the nuclear genome with an even distribution, which might indicate a random release pattern of cell-free Echinococcus spp. DNA. CONCLUSIONS/SIGNIFICANCE:With ultra-high-throughput sequencing technology, we analyzed the concentration, fragment length, release source, and other characteristics of cell-free Echinococcus spp. DNA in the plasma of echinococcosis patients. A better understanding of the characteristics of cell-free Echinococcus spp. DNA in plasma may facilitate their future application as a biomarker for diagnosis.
| S-EPMC7209354 | biostudies-literature
Project description:Blastocystis is an anaerobic unicellular protozoal parasite infecting the gastrointestinal tract of humans and a wide range of animals. It is one of the most common enteric microorganisms with higher prevalence rates in developing than in developed countries. Feco-oral is the main route of transmission where low socioeconomic conditions, poor hygienic practices, close contact with animals, and drinking contaminated water act as major risk factors. Infection with Blastocystis was demonstrated in both symptomatic and asymptomatic people. For a long period, Blastocystis was considered a commensal organism with no pathogenic role, but recently, many studies linked it to different gastrointestinal symptoms such as nausea, diarrhea, and abdominal pain. Association with irritable bowel syndrome and colorectal cancer was also reported.
This study aims to:
- Identify subtypes of human Blastocystis isolates in Sohag by using RFLP-PCR and provide additional information on the molecular epidemiology of this parasite in our locality.
| 2751436 | ecrin-mdr-crc
Project description:Intact cell-free mitochondria have been reported in microparticles (MPs) in murine and human bodily fluids under disease conditions. However, cellular origins of circulating extracellular mitochondria have not been characterized. We hypothesize that intact, cell-free mitochondria from heterogeneous cellular sources are present in the circulation under physiological conditions. To test this, circulating MPs were analyzed using flow cytometry and proteomics. Murine and human platelet-depleted plasma showed a cluster of MPs positive for the mitochondrial probe MitoTracker. Transgenic mice expressing mitochondrial-GFP showed GFP positivity in plasma MPs. Murine and human mitochondria-containing MPs were positive for the platelet marker CD41 and the endothelial cell marker CD144, while hematopoietic CD45 labeling was low. Both murine and human circulating cell-free mitochondria maintained a transmembrane potential. Circulating mitochondria were able to enter rho-zero cells, and were visualized using immunoelectron microscopic imaging. Proteomics analysis identified mitochondria specific and extracellular vesicle associated proteins in sorted circulating cell-free human mitochondria. Together the data provide multiple lines of evidence that intact and functional mitochondria originating from several cell types are present in the blood circulation.
| S-EPMC7508808 | biostudies-literature