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Using RuleBuilder to Graphically Define and Visualize BioNetGen-language Patterns and Reaction Rules

Using RuleBuilder to Graphically Define and Visualize BioNet...

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作者： Ryan Suderman G. Matthew Fricke William S. Hlavacek

RuleBuilder is a tool for drawing graphs that can be represented by the BioNetGen language (BNGL), which is used to formulate mathematical, rule-based models of biochemical systems. BNGL provides an intuitive plain text, or string, representation of such systems, which is based on a graphical formalism. Reactions are defined in terms of graph-rewriting rules that specify the necessary intrinsic properties of the reactants, a transformation, and a rate law. Rules also contain contextual constraints that restrict application of the rule. In some cases, the specification of contextual constraints can be verbose, making a rule difficult to read. RuleBuilder is designed to ease the task of reading and writing individual reaction rules or other BNGL patterns required for model formulation. The software assists in the reading of existing models by converting BNGL strings of interest into a graph-based representation composed of nodes and edges. RuleBuilder also enables the user to construct de novo a visual representation of BNGL strings using drawing tools available in its interface. As objects are added to the drawing canvas, the corresponding BNGL string is generated on the fly, and objects are similarly drawn on the fly as BNGL strings are entered into the application. RuleBuilder thus facilitates construction and interpretation of rule-based models. less

关键词： Mathematical Modeling Rearrangement programming language Visualization Software Systems biology Dynamical systems Rule-based modeling Chemical kinetics Graph rewriting Drawing tool

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Atomistic Molecular Dynamics Simulations of DNA Minicircle Topoisomers: A Practical Guide to Setup, Performance, and Analysis

Atomistic Molecular Dynamics Simulations of DNA Minicircle T...

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作者： Thana Sutthibutpong Agnes Noy Sarah Harris

While DNA supercoiling is ubiquitous in vivo, the structure of supercoiled DNA is more challenging to study experimentally than simple linear sequences because the DNA must have a closed topology in order to sustain superhelical stress. DNA minicircles, which are closed circular double-stranded DNA sequences typically containing between 60 and 500 base pairs, have proven to be useful biochemical tools for the study of supercoiled DNA mechanics. We present detailed protocols for constructing models of DNA minicircles in silico, for performing atomistic molecular dynamics (MD) simulations of supercoiled minicircle DNA, and for analyzing the results of the calculations. These simulations are computationally challenging due to the large system sizes. However, improvements in parallel computing software and hardware promise access to improve conformational sampling and simulation timescales. Given the concurrent improvements in the resolution of experimental techniques such as atomic force microscopy (AFM) and cryo-electron microscopy, the study of DNA minicircles will provide a more complete understanding of both the structure and the mechanics of supercoiled DNA. less

关键词： Molecular Dynamics Simulation Atomic Force Microscopy (AFM) Cryo-EM NMR Gel Electrophoresis Cryo-electron Tomography Python Data analysis programming language DNA supercoiling

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Methods to Identify and Study the Evolution of Pseudogenes Using a Phylogenetic Approach

Methods to Identify and Study the Evolution of Pseudogenes U...

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作者： Jacques Dainat Pierre Pontarotti

The discovery that pseudogenes are involved in important biological processes has excited enthusiasm and increased the research interest on them. An accurate detection and analysis of pseudogenes can be achieved using comparative methods, but only the use of phylogenetic tools can provide accurate information about their birth, their evolution and their death, hence about the impact that they have on genes and genomes. Here, phylogenetic methods that allow for studying pseudogene history are described. less

关键词： Phylogenetic Tree Analysis Genome Assembly Genome Annotation Ensembl programming language Speciation Phylogeny Paralogy Pseudogene Orthology

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Imaging Single mRNA Molecules in Mammalian Cells Using an Optimized MS2-MCP System

Imaging Single mRNA Molecules in Mammalian Cells Using an Op...

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作者： Maria Vera Evelina Tutucci Robert H. Singer

Visualization of single mRNAs in their native cellular environment provides key information to study gene expression regulation. This fundamental biological question triggered the development of the MS2-MCP (MS2-Capsid Protein) system to tag mRNAs and image their life cycle using widefield fluorescence microscopy. The last two decades have evolved toward improving the qualitative and quantitative characteristics of the MS2-MCP system. Here, we provide a protocol to use the latest versions, MS2V6 and MS2V7, to tag and visualize mRNAs in mammalian cells in culture. The motivation behind engineering MS2V6 and MS2V7 was to overcome a degradation caveat observed in S. cerevisiae with the previous MS2-MCP systems. While for yeast we recommend the use of MS2V6, we found that for live-cell imaging experiments in mammalian cells, the MS2V7 has improved reporter properties. less

关键词： Lentivirus Production Live Cell Imaging Fluorescence Microscopy Wide-field Microscopy FISH PCR DNA Extraction Cell And Tissue Culture Transient Transfection CRISPR-Cas9 Genome Editing Imaging Techniques MetaMorph Recombinant Protein Expression Fiji Single-molecule FISH MATLAB CellProfiler DNA Purification Cell Separation And Isolation Transfection Microscopy Image Analysis DNA Ligation programming language Data analysis Mus musculus Escherichia coli Lentivirus HEK293T Single-molecule imaging Single cell MS2 system MS2V6 MS2V7 Short-lived mRNAs

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The SQUAD Method for the Qualitative Modeling of Regulatory Networks

The SQUAD Method for the Qualitative Modeling of Regulatory ...

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作者： Akram Méndez Carlos Ramírez Mauricio Pérez Martínez Luis Mendoza

The wealth of molecular information provided by high-throughput technologies has enhanced the efforts dedicated to the reconstruction of regulatory networks in diverse biological systems. This information, however, has proven to be insufficient for the construction of quantitative models due to the absence of sufficiently accurate measurements of kinetic constants. As a result, there have been efforts to develop methodologies that permit the use of qualitative information about patterns of expression to infer the regulatory networks that generate such patterns. One of these approaches is the SQUAD method, which approximates a Boolean network with the use of a set of ordinary differential equations. The main benefit of the SQUAD method over purely Boolean approaches is the possibility of evaluating the effect of continuous external signals, which are pervasive in biological phenomena. A brief description and code on how to implement this method can be found at the following link: https://***/caramirezal/SQUADBookChapter . less

关键词： Fuzzy Logic programming language Cell fate Regulatory networks Expression pattern Network modeling

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CRISPR/Cas9-Based Chemogenomic Profiling in Mammalian Cells

CRISPR/Cas9-Based Chemogenomic Profiling in Mammalian Cells

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作者： Dominic Hoepfner Gregory McAllister Gregory R. Hoffman

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. It is based on identifying cellular hypersensitivity and resistance caused by individual gene modulations with genome-wide coverage. Due to the requirement of bar-coded, genome-wide deletion collections, high-resolution experiments of this nature have historically been limited to fungal systems. Pooled RNAi reagents have enabled similar attempts in mammalian cells but efforts have been hampered by significant off-target effects and experimental noise. The CRISPR/Cas9 system for the first time enables precise DNA editing at defined loci in a genome-wide fashion. Here we present the detailed protocol that leverages the CRISPR/Cas9 system for chemogenomic profiling and target identification of diverse chemical probes. less

关键词： CRISPR Genomics DNA Extraction Next Generation Sequencing RNA Interference CRISPR-Cas9 Genome Editing Liquid Handling Cell And Tissue Culture Regression Analysis Transfection Virus Production Real-time PCR Immunofluorescence Western Blot Flow Cytometry FACS Illumina Sequencing Bowtie Statistical Calculation Principal Component Analysis Data analysis programming language Python HCT 116 HEK293T Target identification Genetics Resistance Cas9 Gene editing Haploinsufficiency Chemogenomic profiling NAMPT Signal peptidase

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Using Deep Sequencing Data for Identification of Editing Sites in Mature miRNAs

Using Deep Sequencing Data for Identification of Editing Sit...

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作者： Shahar Alon Eli Eisenberg

Deep sequencing has many possible applications; one of them is the identification and quantification of RNA editing sites. The most common type of RNA editing is adenosine to inosine (A-to-I) editing. A prerequisite for this editing process is a double-stranded RNA (dsRNA) structure. Such dsRNAs are formed as part of the microRNA (miRNA) maturation process, and it is therefore expected that miRNAs are affected by A-to-I editing. Indeed, tens of editing sites were found in miRNAs, some of which change the miRNA binding specificity. Here, we describe a protocol for the identification of RNA editing sites in mature miRNAs using deep sequencing data. less

关键词： Deep Sequencing Genomics Statistical Calculation Bowtie Illumina Sequencing programming language Mus musculus Bioinformatics miRNA A-to-I editing

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RNA Sequencing and Quantitation Using the Helicos Genetic Analysis System

RNA Sequencing and Quantitation Using the Helicos Genetic An...

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作者： Tal Raz Marie Causey Daniel R. Jones Alix Kieu Stan Letovsky Doron Lipson Edward Thayer John F. Thompson Patrice M. Milos

The recent transition in gene expression analysis technology to ultra high-throughput cDNA sequencing provides a means for higher quantitation sensitivity across a wider dynamic range than previously possible. Sensitivity of detection is mostly a function of the sheer number of sequence reads generated. Typically, RNA is converted to cDNA using random hexamers and the cDNA is subsequently sequenced (RNA-Seq). With this approach, higher read numbers are generated for long transcripts as compared to short ones. This length bias necessitates the generation of very high read numbers to achieve sensitive quantitation of short, low-expressed genes. To eliminate this length bias, we have developed an ultra high-throughput sequencing approach where only a single read is generated for each transcript molecule (single-molecule sequencing Digital Gene Expression (smsDGE)). So, for example, equivalent quantitation accuracy of the yeast transcriptome can be achieved by smsDGE using only 25% of the reads that would be required using RNA-Seq. For sample preparation, RNA is first reverse-transcribed into single-stranded cDNA using oligo-dT as a primer. A poly-A tail is then added to the 3′ ends of cDNA to facilitate the hybridization of the sample to the Helicos® single-molecule sequencing Flow-Cell to which a poly dT oligo serves as the substrate for subsequent sequencing by synthesis. No PCR, sample-size selection, or ligation steps are required, thus avoiding possible biases that may be introduced by such manipulations. Each tailed cDNA sample is injected into one of 50 flow-cell channels and sequenced on the Helicos® Genetic Analysis System. Thus, 50 samples are sequenced simultaneously generating 10–20 million sequence reads on average for each sample channel. The sequence reads can then be aligned to the reference of choice such as the transcriptome, for quantitation of known transcripts, or the genome for novel transcript discovery. This chapter provides a summary of the met

关键词： RNA-seq Genetic Analysis Gene Expression Analysis High-Throughput Sequencing PCR Spectroscopy Gel Electrophoresis Titration Data analysis cDNA sequencing DNA Ligation Python programming language Expression analysis Single-molecule sequencing smsDGE

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Gene Regulatory Networks from Single Cell Data for Exploring Cell Fate Decisions

Gene Regulatory Networks from Single Cell Data for Exploring...

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作者： Thalia E. Chan Michael P. Stumpf Ann C. Babtie

Single cell experimental techniques now allow us to quantify gene expression in up to thousands of individual cells. These data reveal the changes in transcriptional state that occur as cells progress through development and adopt specialized cell fates. In this chapter we describe in detail how to use our network inference algorithm (PIDC)—and the associated software package ***—to infer functional interactions between genes from the observed gene expression patterns. We exploit the large sample sizes and inherent variability of single cell data to detect statistical dependencies between genes that indicate putative (co-)regulatory relationships, using multivariate information measures that can capture complex statistical relationships. We provide guidelines on how best to combine this analysis with other complementary methods designed to explore single cell data, and how to interpret the resulting gene regulatory network models to gain insight into the processes regulating cell differentiation. less

关键词： Real-time PCR RNA-seq MATLAB Neuronal Differentiation Statistical Calculation programming language Python Gene regulation Network inference Single cell Information theory Cell development

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Constructing Computational Pipelines

Constructing Computational Pipelines

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作者： Mark Halling-Brown Adrian J. Shepherd

Many bioinformatics tasks involve creating a computational pipeline from existing software components and algorithms. The traditional approach is to glue components together using scripts written in a programming language such as Perl. However, a new, more powerful approach is emerging that promises to revolutionise the way bioinformaticians create applications from existing components, an approach based on the concept of the scientific workflow. Scientific workflows are created in graphical environments known as workflow management systems. They have many benefits over traditional program scripts, including speed of development, portability, and their suitability for developing complex, distributed applications. This chapter explains how to design and implement bioinformatics workflows using free, Open Source software tools, such as the Tav-erna workflow management system. We also demonstrate how new and existing tools can be deployed as Web services so that they can be easily integrated into novel computational pipelines using the scientific workflow paradigm. less

关键词： programming language Web service Computational pipeline scientific workflow workflow management system applications server

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