The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed ...the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.
We report the complete nucleotide sequence of a plasmid, pA31-12, carrying blaCTX-M-55 and mcr-1 from a chicken Escherichia coli isolate. pA31-12 has an IncI2 replicon that displays extensive ...sequence similarity with pHN1122-1-borne blaCTX-M-55 and pHNSHP45-borne mcr-1 Insertion sequences ISEcp1 and ISApl1 are responsible for the mobilization of blaCTX-M-55 and mcr-1, respectively. The colocalization of mcr-1 with an extended-spectrum β-lactamase gene on a conjugative plasmid may accelerate the dissemination of both genes by coselection.
RNA-sequencing (RNA-seq) is an essential technique for transcriptome studies, hundreds of analysis tools have been developed since it was debuted. Although recent efforts have attempted to assess the ...latest available tools, they have not evaluated the analysis workflows comprehensively to unleash the power within RNA-seq. Here we conduct an extensive study analysing a broad spectrum of RNA-seq workflows. Surpassing the expression analysis scope, our work also includes assessment of RNA variant-calling, RNA editing and RNA fusion detection techniques. Specifically, we examine both short- and long-read RNA-seq technologies, 39 analysis tools resulting in ~120 combinations, and ~490 analyses involving 15 samples with a variety of germline, cancer and stem cell data sets. We report the performance and propose a comprehensive RNA-seq analysis protocol, named RNACocktail, along with a computational pipeline achieving high accuracy. Validation on different samples reveals that our proposed protocol could help researchers extract more biologically relevant predictions by broad analysis of the transcriptome.RNA-seq is widely used for transcriptome analysis. Here, the authors analyse a wide spectrum of RNA-seq workflows and present a comprehensive analysis protocol named RNACocktail as well as a computational pipeline leveraging the widely used tools for accurate RNA-seq analysis.
Studies in mice and humans have revealed intriguing associations between host genetics and the microbiome. Here we report a 16S rRNA-based analysis of the gut microbiome in 1,126 twin pairs, a subset ...of which was previously reported. Tripling the sample narrowed the confidence intervals around heritability estimates and uncovered additional heritable taxa, some of which are validated in other studies. Repeat sampling of subjects showed heritable taxa to be temporally stable. A candidate gene approach uncovered associations between heritable taxa and genes related to diet, metabolism, and olfaction. We replicate an association between Bifidobacterium and the lactase (LCT) gene locus and identify an association between the host gene ALDH1L1 and the bacteria SHA-98, suggesting a link between formate production and blood pressure. Additional genes detected are involved in barrier defense and self/non-self recognition. Our results indicate that diet-sensing, metabolism, and immune defense are important drivers of human-microbiome co-evolution.
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•16S rRNA-based analysis of the gut microbiome in 1,126 twin pairs•Heritable bacterial taxa are temporally stable•Bifidobacterium associates with lactase gene variants; formate production links to blood pressure•Gene-microbe links involve genes related to diet, metabolism, olfaction, and defense
Does host genotype shape the microbiome? Goodrich et al. present a gut microbiome analysis of 1,126 twin pairs, which extends the association between host genetics and select bacterial taxa. Lactase nonpersistence was linked to higher levels of Bifidobacteria. Other gene/microbe links relate to diet and barrier defense.
Conspectus Metal ions can be beneficial or toxic depending on their identity, oxidation state, and concentration. Therefore, the ability to detect and quantify different types of metal ions using ...portable sensors or in situ imaging agents is important for better environmental monitoring, in vitro medical diagnostics, and imaging of biological systems. While numerous metal ions in different oxidation states are present in the environment and biological systems, only a limited number of them can be detected effectively using current methods. In this Account, we summarize research results from our group that overcome this limitation by the development of a novel class of activity-based sensors based on metal-dependent DNAzymes, which are DNA molecules with enzymatic activity. First, we have developed an in vitro selection method to obtain DNAzymes from a large DNA library of up to 1015 sequences that can carry out cleavage of an oligonucleotide substrate only in the presence of a specific metal ion with high selectivity. Negative selection steps can further be used to improve the selectivity against potentially competing targets by removing sequences that recognize the competing metal ions. Second, we have developed a patented catalytic beacon method to transform the metal-dependent DNAzyme cleavage reaction into a turn-on fluorescent signal by attaching a fluorophore and quenchers to the DNAzyme complex. Because of the difference in the melting temperatures of DNA hybridization before and after metal-ion-dependent cleavage of the DNAzyme substrate, the fluorophore on the DNA cleavage product can be released from its quenchers to create a turn-on fluorescent signal. Because DNAzymes are easy to conjugate with other signaling moieties, such as gold nanoparticles, lanthanide-doped upconversion nanoparticles, electrochemical agents, and gadolinium complexes, these DNAzymes can also readily be converted into colorimetric sensors, upconversion luminescence sensors, electrochemical sensors, or magnetic resonance contrast agents. In addition to describing recent progress in developing and applying these metal ion sensors for environmental monitoring, point-of-care diagnostics, cellular imaging, and in vivo imaging in zebrafish, we summarize major advantages of this class of activity-based sensors. In addition to advantages common to most activity-based sensors, such as enzymatic turnovers that allow for signal amplification and the use of initial rates instead of absolute signals for quantification to avoid interferences from sample matrices, the DNAzyme-based sensors allow for in vitro selection to expand the method to almost any metal ion under a variety of conditions, negative selection to improve the selectivity against competing targets, and reselection of DNAzymes and combination of active and inactive variants to fine-tune the dynamic range of detection. The use of melting temperature differences to separate target binding from signaling moieties in the catalytic beacon method allows the use of different fluorophores and nanomaterials to extend the versatility and modularity of this sensing platform. Furthermore, sensing and imaging artifacts can be minimized by using an inactive mutant DNAzyme as a negative control, while spatiotemporal control of sensing/imaging can be achieved using optical, photothermal, and endogenous orthogonal caging methods. Finally, current challenges, opportunities, and future perspectives for DNAzymes as activity-based sensors are also discussed.
High-throughput genome sequencing continues to grow the need for rapid, accurate genome annotation and tRNA genes constitute the largest family of essential, ever-present non-coding RNA genes. Newly ...developed tRNAscan-SE 2.0 has advanced the state-of-the-art methodology in tRNA gene detection and functional prediction, captured by rich new content of the companion Genomic tRNA Database. Previously, web-server tRNA detection was isolated from knowledge of existing tRNAs and their annotation. In this update of the tRNAscan-SE On-line resource, we tie together improvements in tRNA classification with greatly enhanced biological context via dynamically generated links between web server search results, the most relevant genes in the GtRNAdb and interactive, rich genome context provided by UCSC genome browsers. The tRNAscan-SE On-line web server can be accessed at http://trna.ucsc.edu/tRNAscan-SE/.
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of ...zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
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•One-step generation of mice with reporters in endogenous genes•One-step generation of conditional mutant mice•Off-target analysis suggests high specificity of the CRISPR/Cas9 system
CRISPR/Cas technology is used for insertion of DNA reporter constructs into endogenous genes, derivation of conditional mutant mice and generation of deletions of defined length. Off-target mutations appear only in rare occasions.
We report a graphene oxide (GO)-based fluorescent sensor for Hg(2+) detection in aqueous solutions by using hybridization chain reactions (HCRs). GO is used as an adsorption material for capturing ...single-stranded DNA and an efficient fluorescence quencher for reducing the background signal. In the detection strategy, two hairpin probes and a helper DNA are employed. Without Hg(2+), they are adsorbed by the GO and the fluorescence of one of the hairpin probes is quenched. In the presence of Hg(2+), the HCRs between the two hairpin probes are initiated by Hg(2+) with the aid of the helper DNA through T-Hg(2+)-T coordination chemistry. The double-stranded DNA products of the HCRs are released by the GO and the fluorescence is recovered. The detection limit of the sensing method is 0.3 nM, which is sufficiently sensitive for practical applications. The sensing system also exhibits high selectivity against other divalent metal ions, and the application of the sensor for drinking water shows that the proposed method works well for real samples.
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