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检索条件"主题词=Programming language"

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PELDOR Measurements on Nitroxide-Labeled Oligonucleotides

PELDOR Measurements on Nitroxide-Labeled Oligonucleotides

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作者： Tobias Hett Olav Schiemann

In the past decades, pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) has emerged as a powerful tool in biophysical chemistry to study the structure, dynamics, and function of biomolecules like oligonucleotides and proteins. Structural information is obtained from PDS methods in form of a distribution of distances between spin centers. Such spin centers can either be intrinsically present paramagnetic metal ions and organic radicals or may be attached to the biomolecule by means of site-directed spin labeling. The most common PDS experiment for probing interspin distances in the nanometer range is pulsed electron–electron double resonance (PELDOR or DEER). In the protocol presented here, we provide a step-by-step workflow on how to set up a PELDOR experiment on a commercially available pulsed EPR spectrometer, outline the data analysis, and highlight potential pitfalls. We suggest PELDOR measurements on nitroxide-labeled oligonucleotides to study the structure of either RNA-cleaving DNAzymes in complex with their RNA targets or modified DNAzymes with different functions and targets, in which deoxynucleotides are substituted by nitroxide-labeled nucleotides. less

关键词： Spectroscopy Electron Spin Resonance Spectroscopy Two-pulse Electron Spin-echo Envelope Modulation Electron Spin Echo Envelope Modulation Spectroscopy Phase Cycle Rutherford Backscattering Spectroscopy Deep learning MATLAB Data analysis programming language Electron paramagnetic resonance (EPR) Spin label Electron spin resonance (ESR) Double electron–electron resonance (DEER) Pulsed electron–electron double resonance (PELDOR)

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Data Sharing and Reuse: A Method by the AIRR Community

Data Sharing and Reuse: A Method by the AIRR Community

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作者： Brian D. Corrie Scott Christley Christian E. Busse Lindsay G. Cowell Kira C. Neller Florian Rubelt Nicholas Schwab

High-throughput sequencingHigh-throughput sequencing of adaptive immune receptor repertoires (AIRR, i.e., IGImmunoglobulin (IG) and TRT cell receptor (TR)) has revolutionized the ability to study the adaptive immune response via large-scale experiments. Since 2009, AIRR sequencing (AIRR-seq) has been widely applied to survey the immune state of individuals (see “The AIRR Community Guide to Repertoire Analysis” chapter for details). One of the goals of the AIRR Community is to make the resulting AIRR-seq data FAIR (Findable, Accessible, Interoperable, and Reusable) (Wilkinson et al. Sci Data 3:1–9, 2016), with a primary goal of making it easy for the research community to reuse AIRR-seq data (Breden et al. Front Immunol 8:1418, 2017; Scott and Breden. Curr Opin Syst Biol 24:71–77, 2020). The basis for this is the MiAIRR data standard (Rubelt et al. Nat Immunol 18:1274–1278, 2017). For long-term preservation, it is recommended that researchers store their sequence read data in an INSDC repository. At the same time, the AIRR Community has established the AIRR Data Commons (Christley et al. Front Big Data 3:22, 2020), a distributed set of AIRR-compliant repositories that store the critically important annotated AIRR-seq data based on the MiAIRR standard, making the data findable, interoperable, and, because the data are annotated, more valuable in its reuse. Here, we build on the other AIRR Community chapters and illustrate how these principles and standards can be incorporated into AIRR-seq data analysis workflows. We discuss the importance of careful curation of metadata to ensure reproducibility and facilitate data sharing and reuse, and we illustrate how data can be shared via the AIRR Data Commons. less

关键词： Data analysis Sequence Annotation Rearrangement Python Statistical Calculation programming language B-cell receptor T-cell receptor Immunoglobulin Data sharing AIRR-seq FAIR data Data reuse

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Analysis of mRNA Dynamics Using RNA Sequencing Data

Analysis of mRNA Dynamics Using RNA Sequencing Data

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作者： Rached Alkallas Hamed S. Najafabadi

The RNA abundance of each gene is determined by its rates of transcription and RNA decay. Biochemical experiments that measure these rates, including transcription inhibition and metabolic labelling, are challenging to perform and are largely limited to in vitro settings. Most transcriptomic studies have focused on analyzing changes in RNA abundances without attributing those changes to transcriptional or posttranscriptional regulation. Estimating differential transcription and decay rates of RNA molecules would enable the identification of regulatory factors, such as transcription factors, RNA binding proteins, and microRNAs, that govern large-scale shifts in RNA expression. Here, we describe a protocol for estimating differential stability of RNA molecules between conditions using standard RNA-sequencing data, without the need for transcription inhibition or metabolic labeling. We apply this protocol to in vivo RNA-seq data from individuals with Alzheimer’s disease and demonstrate how estimates of differential stability can be leveraged to infer the regulatory factors underlying them. less

关键词： RNA-seq Metabolic Labeling Ensembl Genome Visualization HISAT Library Preparation Python programming language RSeQC Data analysis Homo sapiens RNA decay RNA degradation Alzheimer’s disease RNA stability microRNAs Posttranscriptional regulation RNA binding proteins (RBPs)

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High-Throughput DNA FISH (hiFISH)

High-Throughput DNA FISH (hiFISH)

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作者： Elizabeth Finn Tom Misteli Gianluca Pegoraro

High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis. less

关键词： FISH Cell And Tissue Culture In Situ Hybridization Image Analysis Statistical Calculation PCR In Vitro Transcription Pull-down Assay Microscopy programming language Python KNIME Escherichia coli Homo sapiens Saccharomyces cerevisiae High-throughput imaging Nuclear architecture 3D genome

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Investigating Histone Modification Dynamics by Mechanistic Computational Modeling

Investigating Histone Modification Dynamics by Mechanistic C...

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作者： Govind Menon Martin Howard

The maintenance of transcriptional states regulated by histone modifications and controlled switching between these states are fundamental concepts in our understanding of nucleosome-mediated epigenetic memory. Any approach relying on genome-wide bioinformatic analyses alone offers limited scope for dissecting the molecular mechanisms involved in maintenance and switching. Mechanistic mathematical models—describing the dynamics of histone modifications at individual genomic loci—offer an alternative way to investigate these mechanisms. These models, in conjunction with quantitative experimental data—ChIP data, quantification of mRNA levels, and single-cell fluorescence tracking in clonal lineages—can generate predictions that drive more targeted experiments, allowing us to understand mechanisms that would be challenging to unravel by a purely experimental approach. In this chapter, we describe a generic stochastic modeling framework that can be used to capture histone modification dynamics and associated molecular processes—including transcription and read–write feedback by chromatin modifying complexes—at individual genomic loci. Using a specific example—transcriptional silencing by Polycomb-mediated H3K27 methylation—we demonstrate how to construct and simulate a stochastic histone modification model. We provide a step-by-step guide to programming simulations for such a model and discuss how to analyze the simulation output. less

关键词： programming language Python MATLAB Stochastic Simulation ChIP-qPCR ChIP-seq Fluorescence Microscopy Arabidopsis thaliana Arabidopsis halleri Mechanistic modeling Bistability Gillespie algorithm Polycomb Epigenetic memory Gene expression states Maintenance and switching Stochastic histone modification model

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Computational Study on the Dynamics of Mycobacterium Tuberculosis RNA Polymerase Assembly

Computational Study on the Dynamics of Mycobacterium Tubercu...

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作者： Sneha Bheemireddy Narayanaswamy Srinivasan

Gene regulation is an intricate phenomenon involving precise function of many macromolecular complexes. Molecular basis of this phenomenon is highly complex and cannot be fully understood using a single technique. Computational approaches can play a crucial role in overall understanding of functional and mechanistic features of a protein or an assembly. Large amounts of structural data pertaining to these complexes are publicly available. In this project, we took advantage of the availability of the structural information to unravel functional intricacies of Mycobacterium tuberculosis RNA polymerase upon interaction with RbpA. In this article, we discuss how the knowledge on protein structure and dynamics can be exploited to study function using various computational tools and resources. Overall, this article provides an overview of various computational methods which can be efficiently used to understand the role of any protein. We hope especially the nonexperts in the field could benefit from our article. less

关键词： Protein Structure Analysis Structure Determination Cryo-EM Loop Modeling Python NMR Molecular Dynamics Simulation Protein Engineering programming language AlloSite Principal Component Analysis Mycobacterium tuberculosis Allostery Protein dynamics Protein complexes Computational methods Structural features

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High-Throughput Profiling of Metabolic Phenotypes Using High-Resolution GC-MS

High-Throughput Profiling of Metabolic Phenotypes Using High...

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作者： Nishikant Wase Nathan Abshire Toshihiro Obata

Metabolite profiling provides insights into the metabolic signatures, which themselves are considered as phonotypes closely related to the agronomic and phenotypic traits such as yield, nutritional values, stress resistance, and nutrient use efficiency. GC-MS is a sensitive and high-throughput analytical platform and has been proved to be a vital tool for the analysis of primary metabolism to provide an overview of cellular and organismal metabolic status. The potential of GC-MS metabolite profiling as a tool for detecting metabolic changes in plants grown in a high-throughput plant phenotyping platform was explored. In this chapter, we describe an integrated workflow of semi-targeted GC-high-resolution (HR)-time-of-flight (TOF)-MS metabolomics with both the analytical and computational steps, focusing mainly on the sample preparation, GC-HR-TOF-MS analysis part, and data analysis for plant phenotyping efforts. less

关键词： Metabolomics Gas Chromatography-mass Spectrometry Statistical Calculation Gas Chromatography-mass Spectrometry Metabolite Profiling Data analysis Vacuum Drying Principal Component Analysis programming language Decision Tree NMR Liquid Chromatography-mass Spectrometry Phenomics Plant phenotyping GC-HR-TOF-MS

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Locus-Specific DNA Methylation Profiling of Human LINE-1 Retrotransposons

Locus-Specific DNA Methylation Profiling of Human LINE-1 Ret...

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作者： Francisco J. Sanchez-Luque Marie-Jeanne H. Kempen Geoffrey J. Faulkner

Long Interspersed Element 1 (LINE-1 or L1) retrotransposons are the only active and autonomous mobile genetic elements present in the human genome. They are responsible for insertional mutagenesis traced to the germline and early embryo, cancer cells and healthy somatic tissues, such as the brain. L1 insertions can therefore impact both the heritable and somatic genome, with the potential to lead to pathogenesis in either context. The mobility of any given L1 copy is highly dependent on its ability to escape epigenetic repression and be transcribed to produce the intermediary RNA necessary for retrotransposition. L1 transcription is typically driven by an internal promoter encoded within the L1 5′ untranslated region, which contains a CpG island that is in turn subject to epigenetic silencing mediated by DNA methylation. In each of us, there are ~350 transcriptional units of the most recent (and potentially mobile) L1s, with many of these only being found in small human populations. In certain cell types, such as neurons, somatic L1 insertions can arise and incorporate new L1 transcriptional units. The technique presented here allows en masse DNA methylation profiling of multiple L1s in a manner allowing locus-specific resolution of individual L1 copies escaping repression in the brain, other healthy tissues, cancer biopsies, and cultured neural cells, among others. less

关键词： Bisulfite Sequencing DNA Extraction PCR TruSeq Library Preparation High-Throughput Sequencing Insertional Mutagenesis Sanger Sequencing Illumina Sequencing Real-time PCR Cell And Tissue Culture Dissection Electrophoresis DNA Ligation programming language Python Methylation Analysis Statistical Calculation Deionization Method Gel Electrophoresis Sequence Assembly Homo sapiens DNA methylation Epigenetics Retrotransposon LINE-1

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StarMap: a user-friendly workflow for Rosetta-driven molecular structure refinement

StarMap: a user-friendly workflow for Rosetta-driven molecul...

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作者： Wolfgang Lugmayr Vadim Kotov Nikolaus Goessweiner-Mohr Jiri Wald Frank DiMaio Thomas C. Marlovits

Cryogenic electron microscopy (cryo-EM) data represent density maps of macromolecular systems at atomic or near-atomic resolution. However, building and refining 3D atomic models by using data from cryo-EM maps is not straightforward and requires significant hands-on experience and manual intervention. We recently developed StarMap, an easy-to-use interface between the popular structural display program ChimeraX and Rosetta, a powerful molecular modeling engine. StarMap offers a general approach for refining structural models of biological macromolecules into cryo-EM density maps by combining Monte Carlo sampling with local density-guided optimization, Rosetta-based all-atom refinement and real-space B-factor calculations in a straightforward workflow. StarMap includes options for structural symmetry, local refinements and independent model validation. The overall quality of the refinement and the structure resolution is then assessed via analytical outputs, such as magnification calibration (pixel size calibration) and Fourier shell correlations. Z-scores reported by StarMap provide an easily interpretable indicator of the goodness of fit for each residue and can be plotted to evaluate structural models and improve local residue refinements, as well as to identify flexible regions and potentially functional sites in large macromolecular complexes. The protocol requires general computer skills, without the need for coding expertise, because most parts of the workflow can be operated by clicking tabs within the ChimeraX graphical user interface. Time requirements for the model refinement depend on the size and quality of the input data; however, this step can typically be completed within 1 d. The analytical parts of the workflow are completed within minutes. less

关键词： Molecular Modeling Cryo-EM NMR Cross-linking programming language Python X-ray Diffraction Malus domestica Software

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Searching thousands of genomes to classify somatic and novel structural variants using STIX

Searching thousands of genomes to classify somatic and novel...

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作者： Murad Chowdhury Brent S. Pedersen Fritz J. Sedlazeck Aaron R. Quinlan Ryan M. Layer

Structural variants are associated with cancers and developmental disorders, but challenges with estimating population frequency remain a barrier to prioritizing mutations over inherited variants. In particular, variability in variant calling heuristics and filtering limits the use of current structural variant catalogs. We present STIX, a method that, instead of relying on variant calls, indexes and searches the raw alignments from thousands of samples to enable more comprehensive allele frequency estimation. less

关键词： Genomics Variant Calling programming language Genotyping Genome Visualization Software Mutation Genome informatics

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