Analysis Of Sequencing Datasets Research Articles

CircRNF10 triggers a positive feedback loop to facilitate progression of glioblastoma via redeploying the ferroptosis defense in GSCs

BackgroundGlioma exhibit heterogeneous susceptibility for targeted ferroptosis. How circRNAs alterations in glioma promote iron metabolism and ferroptosis defense remains unclarified.MethodsThe highly enriched circRNAs in glioblastoma (GBM) were obtained through analysis of sequencing datasets. Quantitative real-time PCR (qRT–PCR) was used to determine the expression of circRNF10 in glioma and normal brain tissue. Both gain-of-function and loss-of-function studies were used to assess the effects of circRNF10 on ferroptosis using in vitro and in vivo assays. The hypothesis that ZBTB48 promotes ferroptosis defense was established using bioinformatics analysis and functional assays. RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to examine the interaction between circRNF10 and target proteins including ZBTB48, MKRN3 and IGF2BP3. The posttranslational modification mechanism of ZBTB48 was verified using coimmunoprecipitation (co-IP) and ubiquitination assays. The transcription activation of HSPB1 and IGF2BP3 by ZBTB48 was confirmed through luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays. The stabilizing effect of IGF2BP3 on circRNF10 was explored by actinomycin D assay. Finally, a series of in vivo experiments were performed to explore the influences of circRNF10 on the glioma progression.ResultsA novel circular RNA, hsa_circ_0028912 (named circRNF10), which is significantly upregulated in glioblastoma tissues and correlated with patients’ poor prognosis. Through integrated analysis of the circRNA-proteins interaction datasets and sequencing results, we reveal ZBTB48 as a transcriptional factor binding with circRNF10, notably promoting upregulation of HSPB1 and IGF2BP3 expression to remodel iron metabolism and facilitates the launch of a circRNF10/ZBTB48/IGF2BP3 positive feedback loop in GSCs. Additionally, circRNF10 can competitively bind to MKRN3 and block E3 ubiquitin ligase activity to enhance ZBTB48 expression. Consequently, circRNF10-overexpressed glioma stem cells (GSCs) display lower Fe2+ accumulation, selectively priming tumors for ferroptosis evading.ConclusionOur research presents abnormal circRNAs expression causing a molecular and metabolic change of glioma, which we leverage to discover a therapeutically exploitable vulnerability to target ferroptosis.

Challenges and opportunities for whole-genome sequencing-based surveillance of antibiotic resistance.

Infections caused by drug-resistant bacteria are increasingly reported across the planet, and drug-resistant bacteria are recognized to be a major threat to public health and modern medicine. In this review, we discuss how whole-genome sequencing (WGS)-based approaches can contribute to the surveillance of the emergence and spread of antibiotic resistance. We outline the characteristics of sequencing technologies that are currently most used for WGS (Illumina short-read technologies and the long-read sequencing platforms developed by Pacific Biosciences and Oxford Nanopore). The challenges posed by the analysis of sequencing data sets for antimicrobial-resistance determinants and the solutions offered by modern bioinformatics tools are discussed. Finally, we illustrate the power of WGS-based surveillance of antimicrobial resistance by summarizing recent studies on the spread of the multidrug-resistant opportunistic pathogen Klebsiella pneumoniae and the transferable colistin-resistance gene mcr-1, in which high-throughput WGS analyses played essential roles. The implementation of WGS for surveillance of antibiotic-resistant bacteria is technically feasible and cost effective and provides actionable results with reference to infection control. Consequently, the time has come for laboratories to implement routine genome sequencing as part of their surveillance programs for antibiotic-resistant bacteria.

Functional and genetic characterization of hydrocarbon biodegrader and exopolymer-producing clones from a petroleum reservoir metagenomic library

ABSTRACTMicrobial degradation of petroleum is a worldwide issue, which causes physico-chemical changes in its compounds, diminishing its commercial value. Biosurfactants are chemically diverse molecules that can be produced by several microorganisms and can enable microbial access to hydrocarbons. In order to investigate both microbial activities, function-driven screening assays for biosurfactant production and hydrocarbon biodegradation were carried out from a metagenomic fosmid library. It was constructed from the total DNA extracted from aerobic and anaerobic enrichments from a Brazilian biodegraded petroleum sample. A sum of 10 clones were selected in order to evaluate their ability to produce exopolymers (EPS) with emulsifying activity, as well as to characterize the gene sequences, harbored by the fosmid clones, through 454 pyrosequencing. Functional analyses confirmed the ability of some clones to produce surfactant compounds. Regarding hydrocarbon as microbial carbon sources, n-alkane (in mixture or not) and naphthalene were preferentially consumed as substrates. Analysis of sequence data set revealed the presence of genes related to xenobiotics biodegradation and carbohydrate metabolism. These data were corroborated by the results of hydrocarbon biodegradation and biosurfactant production detected in the evaluated clones.

Online resources for genomic analysis using high-throughput sequencing.

The availability of high-throughput sequencing has created enormous possibilities for scientific discovery. However, the massive amount of data being generated has resulted in a severe informatics bottleneck. A large number of tools exist for analyzing next-generation sequencing (NGS) data, yet often there remains a disconnect between these research tools and the ability of many researchers to use them. As a consequence, several online resources and communities have been developed to assist researchers with both the management and the analysis of sequencing data sets. Here we describe the use and applications of common file formats for coding and storing genomic data, consider several web-accessible open-source resources for the visualization and analysis of NGS data, and provide examples of typical analyses with links to further detailed exercises.

A Truncation Mutant of CSF3R, Identified As a Novel Driver in De Novo Myeloid Leukemia, Signals Through TNK2, and Is Responsive to Dasatinib.

Abstract 2412 Background:The use of of kinase inhibitors for personalized medicine holds great promise for the treatment of patients with hematologic malignancies. In order to accomplish this goal, matching kinase inhibitor efficacy to the underlying causative genetic lesions in all patients will be required. One challenge in this process arises from the observation that kinase mutations are much less frequent than kinase dysregulation, indicating that mutations in non-kinases must result in downstream kinase activation. Hence a functional genomics approach is required to understand the genetic etiology of kinase pathway dependence and to match effective kinase inhibitors with genetic biomarkers. Using this functional genomics approach, we have identified TNK2 as a kinase upon which a chronic neutrophilic leukemia patient sample showed dependence for growth and viability. Deep sequencing also identified that this patient sample possessed a mutation in the GCSF receptor, CSF3R. This mutation leads to an increase in the levels of TNK2 and confers sensitivity to the TNK2/SRC inhibitor, dasatinib. Methods:To understand the underlying tyrosine kinase sensitivities in patients with leukemia we have employed siRNA and inhibitor screens in parallel with deep sequencing of ∼1800 kinase-associated genes. Genetic variants were prioritized for validation based on proximity to functional targets in protein interaction network databases and subsequently confirmed via Sanger sequencing. Oncogenic capacity of these mutations was assessed in an interleukin-3 (IL-3) dependent Ba/F3 murine pro-B cell system. Expression levels of CSF3R and TNK2 were assessed by immunoblot analysis in Ba/F3 and HEK293 cells. A mouse bone marrow colony assay was performed to assess the sensitivity of CSF3R-mutant cells to dasatinib. Results:Using this functional genomics approach we examined cells from a patient with chronic neutrophilic leukemia using siRNA and kinase inhibitor screens and observed hyper-sensitivity to siRNA directed against the kinase TNK2 and to the TNK2/SRC inhibitor dasatinib. Sequencing of ∼1800 kinase associated genes in this patient sample revealed a one-base pair insertion that results in a premature stop and truncation of the cytoplasmic domain of the GCSF receptor, CSF3R. This CSF3R mutation transformed Ba/F3 cells to IL-3 independence, and induced an upregulation of TNK2. The CSF3R truncation mutation also led to increased formation of colonies compared with wild type in a mouse bone marrow colony assay. Furthermore, colony formation was inhibited by dasatinib, indicating that dasatinib is a potential therapeutic for CSF3R truncation mutations. CSF3R is known to promote neutrophil proliferation and differentiation and similar truncation mutations in CSF3R have been previously found in patients with severe congenital neutropenia, a disease that can transform to acute myeloid leukemia. Published reports of CSF3R mutations in de novo leukemia have been scarce, however, subsequent analysis of sequencing data sets has identified recurrent CSF3R truncation mutations in patients with neutrophilic/myeloid leukemia. Conclusion:This work identifies CSF3R truncation mutations as a leukemogenic mechanism in patients with de novo myeloid leukemia. CSF3R truncation mutations elevate TNK2 levels and confer sensitivity to the TNK2/SRC inhibitor, dasatinib. CSF3R truncation mutations are therefore promising novel biomarkers for dasatinib treatment in patients with myeloid leukemia. Disclosures:Off Label Use: Dasatinib, for off-label use in MPN. Fleischman:Incyte: Speakers Bureau. Druker:Bristol-Myers Squibb: Clinical Trial Funding Other.

Performance comparison and evaluation of software tools for microRNA deep-sequencing data analysis

With the development of next-generation sequencing (NGS) techniques, many software tools have emerged for the discovery of novel microRNAs (miRNAs) and for analyzing the miRNAs expression profiles. An overall evaluation of these diverse software tools is lacking. In this study, we evaluated eight software tools based on their common feature and key algorithms. Three deep-sequencing data sets were collected from different species and used to assess the computational time, sensitivity and accuracy of detecting known miRNAs as well as their capacity for predicting novel miRNAs. Our results provide useful information for researchers to facilitate their selection of the optimal software tools for miRNA analysis depending on their specific requirements, i.e. novel miRNAs discovery or miRNA expression profile analysis of sequencing data sets.

Tail Paradox, Partial Identifiability, and Influential Priors in Bayesian Branch Length Inference

Recent studies have observed that Bayesian analyses of sequence data sets using the program MrBayes sometimes generate extremely large branch lengths, with posterior credibility intervals for the tree length (sum of branch lengths) excluding the maximum likelihood estimates. Suggested explanations for this phenomenon include the existence of multiple local peaks in the posterior, lack of convergence of the chain in the tail of the posterior, mixing problems, and misspecified priors on branch lengths. Here, we analyze the behavior of Bayesian Markov chain Monte Carlo algorithms when the chain is in the tail of the posterior distribution and note that all these phenomena can occur. In Bayesian phylogenetics, the likelihood function approaches a constant instead of zero when the branch lengths increase to infinity. The flat tail of the likelihood can cause poor mixing and undue influence of the prior. We suggest that the main cause of the extreme branch length estimates produced in many Bayesian analyses is the poor choice of a default prior on branch lengths in current Bayesian phylogenetic programs. The default prior in MrBayes assigns independent and identical distributions to branch lengths, imposing strong (and unreasonable) assumptions about the tree length. The problem is exacerbated by the strong correlation between the branch lengths and parameters in models of variable rates among sites or among site partitions. To resolve the problem, we suggest two multivariate priors for the branch lengths (called compound Dirichlet priors) that are fairly diffuse and demonstrate their utility in the special case of branch length estimation on a star phylogeny. Our analysis highlights the need for careful thought in the specification of high-dimensional priors in Bayesian analyses.

Taxonomy and DNA sequence databases: A perfect match?

AbstractDespite the declining number of traditional taxonomists, our knowledge of Earth's biodiversity continues to grow in the form of DNA sequence data. Freely available through online databases, analyses of sequence datasets are increasingly used as an alternative for the traditional taxonomic process. Species identifications have become “DNA barcoding,” new species discoveries are characterised by genetic divergences, and traditional classification has been supplanted by molecular phylogenetics. These developments are illustrated through a case study investigating the identities of Taygetis butterflies of Costa Rica. Here I review prospects and problems with the molecularization of taxonomy and the key role of publicly available nucleotide sequence databases in efforts to catalogue diversity of life.