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1.
RNA Biol ; : 1-11, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32456538

RESUMO

MicroRNAs are regulators of gene expressionand may be key markers in liquid biopsy.Early diagnosis is an effective means to increase patients' overall survival. We generated genome-wide miRNA profiles from serum of patients and controls from the population-based Janus Serum Bank (JSB) and analysed them by bioinformatics and artificial intelligence approaches. JSB contains sera from 318,628 originally healthy persons, more than 96,000 of whom developed cancer. We selected 210 serum samples from patients with lung, colon or breast cancer at three time points prior to diagnosis (up to 32 years prior to diagnosis with median 5 years interval between TPs), one time-point after diagnosis and from individually matched controls. The controls were matched on age and year of all pre-diagnostic sampling time-points for the corresponding case. Using ANOVA we report 70 significantly deregulated markers (adjusted p-value<0.05). The driver for the significance was the diagnostic time point (miR-575, miR-6821-5p, miR-630 with adjusted p-values<10-10). Further, 91miRNAs were differently expressed in pre-diagnostic samples as compared to controls (nominal p < 0.05). Self-organized maps (SOMs)indicated larges effects in lung cancer samples while breast cancer samples showed the least pronounced changes. SOMsalsohighlighted cancer and time point specific miRNA dys-regulation. Intriguingly, a detailed breakdown of the results highlighted that 51% of all miRNAs were highly specific, either for a time-point or a cancer entity. Pathway analysis highlighted 12 pathways including Hipo signalling and ABC transporters.Our results indicate that tumours may be indicated by serum miRNAs decades prior the clinical manifestation.

2.
Nucleic Acids Res ; 48(W1): W268-W274, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32356893

RESUMO

Arm selection, the preferential expression of a 3' or 5' mature microRNA (miRNA), is a highly dynamic and tissue-specific process. Time-dependent expression shifts or switches between the arms are also relevant for human diseases. We present miRSwitch, a web server to facilitate the analysis and interpretation of arm selection events. Our species-independent tool evaluates pre-processed small non-coding RNA sequencing (sncRNA-seq) data, i.e. expression matrices or output files from miRNA quantification tools (miRDeep2, miRMaster, sRNAbench). miRSwitch highlights potential changes in the distribution of mature miRNAs from the same precursor. Group comparisons from one or several user-provided annotations (e.g. disease states) are possible. Results can be dynamically adjusted by choosing from a continuous range of highly specific to very sensitive parameters. Users can compare potential arm shifts in the provided data to a human reference map of pre-computed arm shift frequencies. We created this map from 46 tissues and 30 521 samples. As case studies we present novel arm shift information in a Alzheimer's disease biomarker data set and from a comparison of tissues in Homo sapiens and Mus musculus. In summary, miRSwitch offers a broad range of customized arm switch analyses along with comprehensive visualizations, and is freely available at: https://www.ccb.uni-saarland.de/mirswitch/.

3.
Nucleic Acids Res ; 48(W1): W521-W528, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32374865

RESUMO

Gene set enrichment analysis has become one of the most frequently used applications in molecular biology research. Originally developed for gene sets, the same statistical principles are now available for all omics types. In 2016, we published the miRNA enrichment analysis and annotation tool (miEAA) for human precursor and mature miRNAs. Here, we present miEAA 2.0, supporting miRNA input from ten frequently investigated organisms. To facilitate inclusion of miEAA in workflow systems, we implemented an Application Programming Interface (API). Users can perform miRNA set enrichment analysis using either the web-interface, a dedicated Python package, or custom remote clients. Moreover, the number of category sets was raised by an order of magnitude. We implemented novel categories like annotation confidence level or localisation in biological compartments. In combination with the miRBase miRNA-version and miRNA-to-precursor converters, miEAA supports research settings where older releases of miRBase are in use. The web server also offers novel comprehensive visualizations such as heatmaps and running sum curves with background distributions. We demonstrate the new features with case studies for human kidney cancer, a biomarker study on Parkinson's disease from the PPMI cohort, and a mouse model for breast cancer. The tool is freely accessible at: https://www.ccb.uni-saarland.de/mieaa2.

4.
J Mol Cell Cardiol ; 2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32305360

RESUMO

BACKGROUND: Cardiac troponins are the preferred biomarkers of acute myocardial infarction. Despite superior sensitivity, serial testing of Troponins to identify patients suffering acute coronary syndromes is still required in many cases to overcome limited specificity. Moreover, unstable angina pectoris relies on reported symptoms in the troponin-negative group. In this study, we investigated genome-wide miRNA levels in a prospective cohort of patients with clinically suspected ACS and determined their diagnostic value by applying an in silico neural network. METHODS: PAXgene blood and serum samples were drawn and hsTnT was measured in patients at initial presentation to our Chest-Pain Unit. After clinical and diagnostic workup, patients were adjudicated by senior cardiologists in duty to their final diagnosis: STEMI, NSTEMI, unstable angina pectoris and non-ACS patients. ACS patients and a cohort of healthy controls underwent deep transcriptome sequencing. Machine learning was implemented to construct diagnostic miRNA classifiers. RESULTS: We developed a neural network model which incorporates 34 validated ACS miRNAs, showing excellent classification results. By further developing additional machine learning models and selecting the best miRNAs, we achieved an accuracy of 0.96 (95% CI 0.96-0.97), sensitivity of 0.95, specificity of 0.96 and AUC of 0.99. The one-point hsTnT value reached an accuracy of 0.89, sensitivity of 0.82, specificity of 0.96, and AUC of 0.96. CONCLUSIONS: Here we show the concept of neural network based biomarkers for ACS. This approach also opens the possibility to include multi-modal data points to further increase precision and perform classification of other ACS differential diagnoses.

5.
JAMA Oncol ; 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32134442

RESUMO

Importance: The overall low survival rate of patients with lung cancer calls for improved detection tools to enable better treatment options and improved patient outcomes. Multivariable molecular signatures, such as blood-borne microRNA (miRNA) signatures, may have high rates of sensitivity and specificity but require additional studies with large cohorts and standardized measurements to confirm the generalizability of miRNA signatures. Objective: To investigate the use of blood-borne miRNAs as potential circulating markers for detecting lung cancer in an extended cohort of symptomatic patients and control participants. Design, Setting, and Participants: This multicenter, cohort study included patients from case-control and cohort studies (TREND and COSYCONET) with 3102 patients being enrolled by convenience sampling between March 3, 2009, and March 19, 2018. For the cohort study TREND, population sampling was performed. Clinical diagnoses were obtained for 3046 patients (606 patients with non-small cell and small cell lung cancer, 593 patients with nontumor lung diseases, 883 patients with diseases not affecting the lung, and 964 unaffected control participants). No samples were removed because of experimental issues. The collected data were analyzed between April 2018 and November 2019. Main Outcomes and Measures: Sensitivity and specificity of liquid biopsy using miRNA signatures for detection of lung cancer. Results: A total of 3102 patients with a mean (SD) age of 61.1 (16.2) years were enrolled. Data on the sex of the participants were available for 2856 participants; 1727 (60.5%) were men. Genome-wide miRNA profiles of blood samples from 3046 individuals were evaluated by machine-learning methods. Three classification scenarios were investigated by splitting the samples equally into training and validation sets. First, a 15-miRNA signature from the training set was used to distinguish patients diagnosed with lung cancer from all other individuals in the validation set with an accuracy of 91.4% (95% CI, 91.0%-91.9%), a sensitivity of 82.8% (95% CI, 81.5%-84.1%), and a specificity of 93.5% (95% CI, 93.2%-93.8%). Second, a 14-miRNA signature from the training set was used to distinguish patients with lung cancer from patients with nontumor lung diseases in the validation set with an accuracy of 92.5% (95% CI, 92.1%-92.9%), sensitivity of 96.4% (95% CI, 95.9%-96.9%), and specificity of 88.6% (95% CI, 88.1%-89.2%). Third, a 14-miRNA signature from the training set was used to distinguish patients with early-stage lung cancer from all individuals without lung cancer in the validation set with an accuracy of 95.9% (95% CI, 95.7%-96.2%), sensitivity of 76.3% (95% CI, 74.5%-78.0%), and specificity of 97.5% (95% CI, 97.2%-97.7%). Conclusions and Relevance: The findings of the study suggest that the identified patterns of miRNAs may be used as a component of a minimally invasive lung cancer test, complementing imaging, sputum cytology, and biopsy tests.

6.
Nucleic Acids Res ; 48(D1): D142-D147, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31691816

RESUMO

Since the initial release of miRPathDB, tremendous progress has been made in the field of microRNA (miRNA) research. New miRNA reference databases have emerged, a vast amount of new miRNA candidates has been discovered and the number of experimentally validated target genes has increased considerably. Hence, the demand for a major upgrade of miRPathDB, including extended analysis functionality and intuitive visualizations of query results has emerged. Here, we present the novel release 2.0 of the miRNA Pathway Dictionary Database (miRPathDB) that is freely accessible at https://mpd.bioinf.uni-sb.de/. miRPathDB 2.0 comes with a ten-fold increase of pre-processed data. In total, the updated database provides putative associations between 27 452 (candidate) miRNAs, 28 352 targets and 16 833 pathways for Homo sapiens, as well as interactions of 1978 miRNAs, 24 898 targets and 6511 functional categories for Mus musculus. Additionally, we analyzed publications citing miRPathDB to identify common use-cases and further extensions. Based on this evaluation, we added new functionality for interactive visualizations and down-stream analyses of bulk queries. In summary, the updated version of miRPathDB, with its new custom-tailored features, is one of the most comprehensive and advanced resources for miRNAs and their target pathways.

8.
Genomics Proteomics Bioinformatics ; 17(4): 430-440, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31809862

RESUMO

Blood-borne small non-coding (sncRNAs) are among the prominent candidates for blood-based diagnostic tests. Often, high-throughput approaches are applied to discover biomarker signatures. These have to be validated in larger cohorts and evaluated by adequate statistical learning approaches. Previously, we published high-throughput sequencing based microRNA (miRNA) signatures in Alzheimer's disease (AD) patients in the United States (US) and Germany. Here, we determined abundance levels of 21 known circulating miRNAs in 465 individuals encompassing AD patients and controls by RT-qPCR. We computed models to assess the relation between miRNA expression and phenotypes, gender, age, or disease severity (Mini-Mental State Examination; MMSE). Of the 21 miRNAs, expression levels of 20 miRNAs were consistently de-regulated in the US and German cohorts. 18 miRNAs were significantly correlated with neurodegeneration (Benjamini-Hochberg adjusted P < 0.05) with highest significance for miR-532-5p (Benjamini-Hochberg adjusted P = 4.8 × 10-30). Machine learning models reached an area under the curve (AUC) value of 87.6% in differentiating AD patients from controls. Further, ten miRNAs were significantly correlated with MMSE, in particular miR-26a/26b-5p (adjusted P = 0.0002). Interestingly, the miRNAs with lower abundance in AD were enriched in monocytes and T-helper cells, while those up-regulated in AD were enriched in serum, exosomes, cytotoxic t-cells, and B-cells. Our study represents the next important step in translational research for a miRNA-based AD test.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Aprendizado de Máquina , MicroRNAs/sangue , Área Sob a Curva , Biomarcadores/sangue , Exossomos , Feminino , Alemanha , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , MicroRNAs/genética , Monócitos/citologia , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T Auxiliares-Indutores/citologia , Regulação para Cima
9.
Brief Bioinform ; 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31792536

RESUMO

MOTIVATION: Since the initial discovery of microRNAs as post-transcriptional, regulatory key players in the 1990s, a total number of $2656$ mature microRNAs have been publicly described for Homo sapiens. As discovery of new miRNAs is still on-going, target identification remains to be an essential and challenging step preceding functional annotation analysis. One key challenge for researchers seems to be the selection of the most appropriate tool out of the larger multiverse of published solutions for a given research study set-up. RESULTS: In this review we collectively describe the field of in silico target prediction in the course of time and point out long withstanding principles as well as recent developments. By compiling a catalog of characteristics about the 98 prediction methods and identifying common and exclusive traits, we signpost a simplified mechanism to address the problem of application selection. Going further we devised interpretation strategies for common types of output as generated by frequently used computational methods. To this end, our work specifically aims to make prospective users aware of common mistakes and practical questions that arise during the application of target prediction tools. AVAILABILITY: An interactive implementation of our recommendations including materials shown in the manuscript is freely available at https://www.ccb.uni-saarland.de/mtguide.

10.
Clin Res Cardiol ; 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31686209

RESUMO

AIMS: We aimed at developing a structured study protocol utilizing the bibliographic web-application science performance evaluation (SciPE) to perform comprehensive scientometric analyses. METHODS AND RESULTS: Metadata related to publications derived from online databases were processed and visualized by transferring the information to an undirected multipartite graph and distinct partitioned sets of nodes. Also, institution-specific data were normalized and merged allowing precise geocoordinate positioning, to enable heatmapping and valid identification. As a result, verified, processed data regarding articles, institutions, journals, authors gender, nations and subject categories can be obtained. We recommend including the total number of publications, citations, the population, research institutions, gross domestic product, and the country-specific modified Hirsch Index and to form corresponding ratios (e.g., population/publication). Also, our approach includes implementation of bioinformatical methods such as heatmapping based on exact geocoordinates, simple chord diagrams, and the central implementation of specific ratios with plain visualization techniques. CONCLUSION: This protocol allows precise conduction of contemporaneous scientometric analyses based on bioinformatic and meta-analytical techniques, allowing to evaluate and contextualize scientific efforts. Data presentation with the depicted visualization techniques is mandatory for transparent and consistent analyses of research output across different nations and topics. Research performance can then be discussed in a synopsis of all findings.

11.
Clin Chem ; 65(12): 1581-1591, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31645340

RESUMO

BACKGROUND: Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria. METHODS: We compared 4 different manipulations of RNA for library preparation: (a) a ligation-based procedure allowing only 5'-mono-phosphorylated RNA to enter the library, (b) a ligation-based procedure allowing additional 5'-triphosphates and Cap structures, (c) a ligation-independent, template-switch-based library preparation, and (d) a template-switch-based library preparation allowing 3'-phosphorylated RNAs to enter the library. RESULTS: Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species. CONCLUSIONS: Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.


Assuntos
Técnicas de Química Combinatória/métodos , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Viés , DNA Complementar , Biblioteca Gênica , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs , RNA Interferente Pequeno , RNA de Transferência , Análise de Sequência de RNA/métodos
12.
Cells ; 8(10)2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569706

RESUMO

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk of death, reducing life expectancy on average between 5 and 7 years. The survival time after diagnosis, however, varies considerably as a result of the heterogeneity of COPD. Therefore, markers that predict individual survival of COPD patients are of great value. We analyzed baseline molecular profiles and collected 54 months of follow-up data of the cohort study "COPD and SYstemic consequences-COmorbidities NETwork" (COSYCONET). Genome-wide microRNA signatures from whole blood collected at time of the inclusion in the study were generated for 533 COPD patients including patients that deceased during the 54-month follow-up period (n = 53) and patients that survived this period (n = 480). We identified two blood-born microRNAs (miR-150-5p and miR-320b) that were highly predictive for survival of COPD patients. The expression change was then confirmed by RT-qPCR in 245 individuals. Ninety percent of patients with highest expression of miR-150-5p survived the 54-month period in contrast to only 50% of patients with lowest expression intensity. Moreover, the abundance of the oncogenic miR-150-5p in blood of COPD patients was predictive for the development of cancer. Thus, molecular profiles measured at the time of a COPD diagnosis have a high predictive power for the survival of patients.

13.
Cells ; 8(9)2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500139

RESUMO

Multiple studies endorsed the positive effect of regular exercise on mental and physical health. However, the molecular mechanisms underlying training-induced fitness in combination with personal life-style remain largely unexplored. Circulating biomarkers such as microRNAs (miRNAs) offer themselves for studying systemic and cellular changes since they can be collected from the bloodstream in a low-invasive manner. In Homo sapiens miRNAs are known to regulate a substantial number of protein-coding genes in a post-transcriptional manner and hence are of great interest to understand differential gene expression profiles, offering a cost-effective mechanism to study molecular training adaption, and connecting the dots from genomics to observed phenotypes. Here, we investigated molecular expression patterns of 2549 miRNAs in whole-blood samples from 23 healthy and untrained adult participants of a cross-over study, consisting of eight weeks of endurance training, with several sessions per week, followed by 8 weeks of washout and another 8 weeks of running, using microarrays. Participants were randomly assigned to one of the two study groups, one of which administered carbohydrates before each session in the first training period, and switching the treatment group for the second training period. During running sessions clinical parameters as heartbeat frequency were recorded. This information was extended with four measurements of maximum oxygen uptake (VO 2 max) for each participant. We observed that multiple circulating miRNAs show expression changes after endurance training, leveraging the capability to separate the blood samples by training status. To this end, we demonstrate that most of the variance in miRNA expression can be explained by both common and known biological and technical factors. Our findings highlight six distinct clusters of miRNAs, each exhibiting an oscillating expression profile across the four study timepoints, that can effectively be utilized to predict phenotypic VO 2 max levels. In addition, we identified miR-532-5p as a candidate marker to determine personal alterations in physical training performance on a case-by-case analysis taking the influence of a carbohydrate-rich nutrition into account. In literature, miR-532-5p is known as a common down-regulated miRNA in diabetes and obesity, possibly providing a molecular link between cellular homeostasis, personal fitness levels, and health in aging. We conclude that circulating miRNA expression can be altered due to regular endurance training, independent of the carbohydrate (CHO) availability in the training timeframe. Further validation studies are required to confirm the role of exercise-affected miRNAs and the extraordinary function of miR-532-5p in modulating the metabolic response to a high availability of glucose.

14.
Ann Surg Oncol ; 26(11): 3765-3773, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31270716

RESUMO

BACKGROUND: In order to improve individual prognostication as well as stratification for adjuvant therapy in patients with clinically localized clear cell renal cell carcinoma (ccRCC), reliable prognostic biomarkers are urgently needed. In this study, microRNAs (miRNAs) have emerged as promising candidates. We investigated whether a combination of differently expressed miRNAs in primary tumors can predict the individual metastatic risk. METHODS: Using two prospectively collected biobanks of academic centers, 108 ccRCCs were selected, including 57 from patients with metastatic disease at diagnosis or during follow-up and 51 without evidence of metastases. Fourteen previously identified candidate miRNAs were tested in 20 representative formalin-fixed and paraffin embedded samples in order to select the best discriminators between metastatic and nonmetastatic ccRCC. These miRNAs were approved in 108 tumor samples. We evaluated the association of altered miRNA expression with the metastatic potential of tumors using quantitative polymerase chain reaction. A prognostic 4-miRNA model has been established using a random forest classifier. Cox regression analyses were performed for correlation of the miRNA model and clinicopathological parameters to metastasis-free and overall survival. RESULTS: Nine miRNAs indicated significant expression alterations in the small cohort. These miRNAs were validated in the whole cohort. The established 4-miRNA score (miR-30a-3p/-30c-5p/-139-5p/-144-5p) has been identified as a superior predictor for metastasis-free survival (hazard ratio 12.402; p = 7.0E-05) and overall survival (p = 1.1E-04) compared with clinicopathological parameters, and likewise in the Leibovich score subgroups. CONCLUSIONS: We identified a 4-miRNA model that was found to be superior to clinicopathological parameters in accurately predicting individual metastatic risk and can support patient selection for risk-stratified follow-up and adjuvant therapy studies.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/secundário , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , MicroRNAs/genética , Nefrectomia/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/cirurgia , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida
15.
RNA Biol ; 16(8): 1034-1043, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31035857

RESUMO

The envisioned application of miRNAs as diagnostic or prognostic biomarkers calls for an in-depth understanding of their distribution and variability in different physiological states. While effects with respect to ethnic origin, age, or gender are known, the inter-individual variability of miRNAs across the four seasons remained largely hidden. We sequentially profiled the complete repertoire of blood-borne miRNAs for 25 physiologically normal individuals in spring, summer, fall, and winter (altogether 95 samples) and validated the results on 292 individuals (919 samples collected with the Mitra home sampling device) by RT-qPCR. Principal variance component analysis suggests that the largest variability observed in miRNA expression is due to individual variability and the individuals' gender. But the results also highlight a deviation of miRNA activity in samples collected during spring time. Following adjustment for multiple testing, remarkable differences are observed between spring and fall (77 miRNAs). The two most dys-regulated miRNAs were miR-181c-5p and miR-106b-5p (adjusted p-value of 0.007). Other significant miRNAs include miR-140-3p, miR-21-3p, and let-7c-5p. The dys-regulation was validated by RT-qPCR. Systems biology analysis further provides strong evidence for the immunological origin of the signals: dys-regulated miRNAs are enriched in CD56 cells and belong to various signalling and immune-system-related pathways. Our data suggest that besides known confounding factors such as age and sex, also the season in which a test is conducted might have a considerable influence on the expression of blood-borne miRNAs and subsequently might interfere with diagnosis based on such signatures.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/sangue , Estações do Ano , Adulto , Antígeno CD56/sangue , Feminino , Humanos , Masculino , Análise de Componente Principal
16.
J Mol Med (Berl) ; 97(8): 1099-1111, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31134286

RESUMO

Gene amplification is an evolutionarily well-conserved and highly efficient mechanism to increase the amount of specific proteins. In humans, gene amplification is a hallmark of cancer and has recently been found during stem cell differentiation. Amplifications in stem cells are restricted to specific tissue areas and time windows, rendering their detection difficult. Here, we report on the performance of deep WGS sequencing (average 82-fold depth of coverage) on the BGISEQ with nanoball technology to detect amplifications in human mesenchymal and neural stem cells. As reference technology, we applied array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and qPCR. Using different in silico strategies for amplification detection, we analyzed the potential of WGS for amplification detection. Our results provide evidence that WGS accurately identifies changes of the copy number profiles in human stem cell differentiation. However, the identified changes are not in all cases consistent between WGS and aCGH. The results between WGS and the validation by qPCR were concordant in 83.3% of all tested 36 cases. In sum, both genome-wide techniques, aCGH and WGS, have unique advantages and specific challenges, calling for locus-specific confirmation by the low-throughput approaches qPCR or FISH. KEY MESSAGES: WGS allows for the identification of dynamic copy number changes in human stem cells. Less stringent threshold setting is crucial for detection of copy number increase. Broad knowledge of dynamic copy number is pivotal to estimate stem cell capabilities.

17.
Nucleic Acids Res ; 47(9): 4431-4441, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30937442

RESUMO

The repertoire of small noncoding RNAs (sncRNAs), particularly miRNAs, in animals is considered to be evolutionarily conserved. Studies on sncRNAs are often largely based on homology-based information, relying on genomic sequence similarity and excluding actual expression data. To obtain information on sncRNA expression (including miRNAs, snoRNAs, YRNAs and tRNAs), we performed low-input-volume next-generation sequencing of 500 pg of RNA from 21 animals at two German zoological gardens. Notably, none of the species under investigation were previously annotated in any miRNA reference database. Sequencing was performed on blood cells as they are amongst the most accessible, stable and abundant sources of the different sncRNA classes. We evaluated and compared the composition and nature of sncRNAs across the different species by computational approaches. While the distribution of sncRNAs in the different RNA classes varied significantly, general evolutionary patterns were maintained. In particular, miRNA sequences and expression were found to be even more conserved than previously assumed. To make the results available for other researchers, all data, including expression profiles at the species and family levels, and different tools for viewing, filtering and searching the data are freely available in the online resource ASRA (Animal sncRNA Atlas) at https://www.ccb.uni-saarland.de/asra/.


Assuntos
Animais de Zoológico/genética , Ácidos Nucleicos Livres/genética , Biologia Computacional , Pequeno RNA não Traduzido/genética , Animais , Ácidos Nucleicos Livres/classificação , Genoma/genética , Alemanha , MicroRNAs/genética , RNA Nucleolar Pequeno/genética , Pequeno RNA não Traduzido/classificação , RNA de Transferência/genética
18.
Nucleic Acids Res ; 47(7): 3353-3364, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30820533

RESUMO

While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.


Assuntos
Simulação por Computador , MicroRNAs/análise , MicroRNAs/genética , Análise de Sequência de RNA , Northern Blotting , Linhagem Celular , Conjuntos de Dados como Assunto , Reações Falso-Positivas , Humanos , MicroRNAs/isolamento & purificação , Anotação de Sequência Molecular , Precursores de RNA/análise , Precursores de RNA/genética , Reprodutibilidade dos Testes
19.
J Chem Inf Model ; 59(4): 1314-1323, 2019 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-30807146

RESUMO

Pharmacophore models in general use a variety of features for distinct chemical characteristics, such as hydrogen-bond properties, lipohilicity, and ionizability. Usually, features have to match onto their identical type. To clarify if this stringent one-to-one assignment is justified, we investigated a set of 581 unique ligands from the BindingDB with known orientation inside the respective binding pockets and conducted a statistical analysis of the likelihood of observed exchanges in between the pharmacophore features, respectively their degree of conservation. To find out if certain features are obsolete, we derived a ranking to determine the most relevant ones. We found that the most conserved one-to-one feature is the negative ionizable (acids), followed by hydrogen-bond donor, positive ionizable (basic nitrogens), hydrogen-bond acceptor, aromatic, nonaromatic π-systems, and other lipophilic characteristics. The most likely exchanges were found between carboxylate groups and hydrogen-bond acceptors and likewise between basic nitrogens and hydrogen-bond donors, which reflects the characteristics of Lewis acids and bases. Exchanges between hydrogen-bond donors and hydrogen-bond acceptors are hardly more likely than by chance. The kind of target (e.g., kinase, phosphatase, protease, phosphodiesterase, nuclear receptor, metal-containing, or transmembrane protein) did not show substantial influence on the degree of conservation. The relevance of the actual pharmacophore features was found to be strongly dependent on the applied ranking scheme. Mutual information ranks all hydrophobic features as least important, whereas the aromatic feature is put into second place by using a geometric series. Both ranking schemes see the negative ionizable feature of higher significance than the positively ionizable feature.

20.
Brief Bioinform ; 20(3): 1011-1020, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29186316

RESUMO

Modern precision medicine comprises the knowledge and understanding of individual differences in the genomic sequence of patients to provide tailor-made treatments. Regularly, such variants are considered in coding regions only, and their effects are predicted based on their impact on the amino acid sequence of expressed proteins. However, assessing the effects of variants in noncoding elements, in particular microRNAs (miRNAs) and their binding sites, is important as well, as a single miRNA can influence the expression patterns of many genes at the same time. To analyze the effects of variants in miRNAs and their target sites, several databases storing variant impact predictions have been published. In this review, we will compare the core functionalities and features of these databases and discuss the importance of up-to-date data resources in the context of web applications. Finally, we will outline some recommendations for future developments in the field.


Assuntos
Bases de Dados Genéticas , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Sítios de Ligação , Humanos , MicroRNAs/metabolismo
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