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Methylated DNA Immunoprecipitation Genome-Wide Analysis

Methylated DNA Immunoprecipitation Genome-Wide Analysis

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作者： Mattia Pelizzola Annette Molinaro

DNA methylation is an important and potentially heritable component of the epigenetic machinery that has a major role in the control of gene expression and can be deregulated in many diseases. This modification of genomic DNA can be assessed using the methylated DNA immunoprecipitation (MeDIP) method, based on the quantification of methylated DNA fragments enriched using an antibody specific for methyl-cytosines. The relationship between the enrichment level and the real DNA methylation status is complex, and only a few methods have been developed to evaluate MeDIP enrichment measures to estimate the absolute or relative number of methyl-cytosines in a given sample. Two such methods are MEDME and BATMAN. This chapter focuses on the description and use of the former with a brief discussion of the latter. less

关键词： Methylated DNA Immunoprecipitation Methylation Analysis methylated DNA immunoprecipitation microarray (MeDIP-chip) High-Throughput Sequencing programming language Homo sapiens Mus musculus DNA methylation Epigenetics MEDME

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Bioinformatics Procedures for Analysis of Quantitative Proteomics Experiments Using iTRAQ

Bioinformatics Procedures for Analysis of Quantitative Prote...

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作者： Pim Nierop Maarten Loos

The combined use of liquid chromatography followed by tandem mass spectrometry (LC-MS-MS) in proteomics research has proven to be a valuable asset in the success of this field of science. Advances in LC-MS-MS technology have allowed researchers to identify an increasing number of proteins from complex biological preparations in a high-throughput fashion. Moreover, techniques based on the labeling of peptides with stable isotopes have made it possible to determine relative differences in abundance of proteins between biological samples. As has been the case for microarray technology, the newly emerging field of quantitative proteomics is associated with the development of novel bioinformatics and statistical approaches that, within the boundaries of particular aspects and limitations of the technique, allow us to ask biological questions and derive meaningful answers (1–3). In this chapter, we describe a protocol of an integrated bioinformatics workflow that deals with the identification of proteins and the relative quantification using iTRAQ labeling in complex proteomics experiments that also involves comparison of quantitative data obtained in separate LC-MS-MS runs. less

关键词： Proteomics Sequence Clustering Liquid Chromatography (LC) Tandem Mass Spectrometry Liquid Chromatography-tandem Mass Spectrometry Peptide Labeling Microarray ITRAQ MALDI Database Search MALDI-TOF/TOF Mass Spectrometry NCBI BLAST GenBank programming language Protocol Bioinformatics iTRAQ Mascot server

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Microarray Bioinformatics

Microarray Bioinformatics

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作者： Robert P. Loewe Peter J. Nelson

Bioinformatics has become an increasingly important tool for molecular biologists, especially for the analysis of microarray data. Microarrays can produce vast amounts of information requiring a series of consecutive analyses to render the data interpretable. The direct output of microarrays cannot be directly interpreted to show differences in settings, conditions of samples, or time points. To make microarray experiments interpretable, it is necessary that a series of algorithms and approaches be applied. After normalization of generated data, which is necessary to make a comparison feasible, significance analysis, clustering of samples and biological compounds of interest and visualization are generally performed. This chapter will focus on providing a basic understanding of the generally approaches and algorithms currently employed in microarray bioinformatics. less

关键词： Microarray RNA Extraction Gene Expression Profiling Statistical Calculation Simulated Annealing Fuzzy Logic Principal Component Analysis Genomics programming language SAM PCA Normalization Bioinformatics RMA Clustering

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Imaging Calcium Sparks in Cardiac Myocytes

Imaging Calcium Sparks in Cardiac Myocytes

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作者： Silvia Guatimosim Cristina Guatimosim Long-Sheng Song

Calcium ions play fundamental roles in many cellular processes in virtually all type of cells. The use of Ca2+ sensitive fluorescent indicators has proven to be an indispensable tool for studying the spatio-temporal dynamics of intracellular calcium ([Ca2+]i). With the aid of laser scanning confocal microscopy and new generation of Ca2+ indicators, highly localized, short-lived Ca2+ signals, namely Ca2+ sparks, were revealed as elementary Ca2+ release events during excitation–contraction coupling in cardiomyocytes. Since the discovery of Ca2+ sparks in 1993, the demonstration of dynamic Ca2+ micro-domains in living cardiomyocytes has revolutionized our understanding of Ca2+-mediated signal transduction in normal and diseased hearts. In this chapter, we have described a commonly used method for recording local and global Ca2+ signals in cardiomyocytes using the fluorescent indicator fluo-4 acetoxymethyl (AM) and laser scanning confocal microscopy. less

关键词： Confocal Microscopy Calcium Imaging Intraperitoneal Injection Image Processing programming language fluorescence calcium indicators Calcium sparks ventricular myocytes

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Helicos Single-Molecule Sequencing of Bacterial Genomes

Helicos Single-Molecule Sequencing of Bacterial Genomes

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作者： Kathleen E. Steinmann Christopher E. Hart John F. Thompson Patrice M. Milos

With the advent of high-throughput sequencing technologies, multiple bacterial genomes can be sequenced in days. While the ultimate goal of de novo assembly of bacterial genomes is progressing, changes in the genomic sequence of closely related bacterial strains and isolates are now easily monitored by comparison of their sequences to those of a reference genome. Such studies can be applied to the fields of bacterial evolution, epidemiology, and diagnostics. We present a protocol for single-molecule sequencing of bacterial DNA whose end result is the identification of single nucleotide variants, and various size insertions and deletions relative to a reference genome. The protocol is characterized by the simplicity of sample preparation and the lack of amplification-related sequencing bias. less

关键词： DNA Extraction High-Throughput Sequencing DNA Purification Degassing Electrophoresis PCR Genetic Analysis Data analysis Python programming language Single-molecule sequencing SNV Bacterial genome Sequencing bias PolyA tail HeliScopeTM sequencer

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Omics Technologies, Data and Bioinformatics Principles

Omics Technologies, Data and Bioinformatics Principles

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作者： Maria V. Schneider Sandra Orchard

We provide an overview on the state of the art for the Omics technologies, the types of omics data and the bioinformatics resources relevant and related to Omics. We also illustrate the bioinformatics challenges of dealing with high-throughput data. This overview touches several fundamental aspects of Omics and bioinformatics: data standardisation, data sharing, storing Omics data appropriately and exploring Omics data in bioinformatics. Though the principles and concepts presented are true for the various different technological fields, we concentrate in three main Omics fields namely: genomics, transcriptomics and proteomics. Finally we address the integration of Omics data, and provide several useful links for bioinformatics and Omics. less

关键词： Genomics Transcriptomics Proteomics Protein Structure Prediction De Novo Sequencing Genome Annotation De-novo Assembly Sequence Assembly Gene Prediction Genome Visualization Mass Spectrometry Cytoscape Data Mining Serial Analysis Of Gene Expression Functional Genomics Protein Array Genotyping Metabolomics Swiss-Prot Image Analysis High-Throughput Sequencing Metagenomics Next Generation Sequencing RNA-seq Gene Expression Analysis DNA Sequencing ChIP-seq ChIP Assay Protein Purification Affinity Chromatography Co-immunoprecipitation X-ray Crystallography Harvesting Cells Recombinant Protein Expression UniProtKB Sequence Analysis Phylogenetics Genome Sequencing Ensembl Microarray Data analysis programming language Pull-down Assay Homo sapiens Bioinformatics Data integration High-throughput Interactomics Omics Omics databases Omics tools

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A Computational Method to Search for DNA Structural Motifs in Functional Genomic Elements

A Computational Method to Search for DNA Structural Motifs i...

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作者： Stephen C.J. Parker Aaron Harlap Thomas D. Tullius

The rapidly increasing availability of DNA sequence data from modern high-throughput experimental techniques has created the need for computational algorithms to aid in motif discovery in genomic DNA. Such algorithms are typically used to find a statistical representation of the nucleotide sequence of the target site of a DNA-binding protein within a collection of DNA sequences that are thought to contain segments to which the protein is bound. A major assumption of these algorithms is that the protein recognizes the primary order of nucleotides in the sequence. However, proteins can also recognize the three-dimensional shape and structure of DNA. To account for this, we developed a computational method to predict the local structural profiles of any set of DNA sequences and then to search within these profiles for common DNA structural motifs. Here we describe the details of this method and use it to find a DNA structural motif in the Saccharomyces cerevisiae yeast genome that is associated with binding of the transcription factor RLM1, a component of the protein kinase C-mediated MAP kinase pathway. less

关键词： Protein Structure Analysis Genome Visualization programming language Microarray Saccharomyces cerevisiae Homo sapiens hydroxyl radical transcription factor Motif discovery Gibbs sampling DNA structure RLM1

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DNA Contour Length Measurements as a Tool for the Structural Analysis of DNA and Nucleoprotein Complexes

DNA Contour Length Measurements as a Tool for the Structural...

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作者： Claudio Rivetti

The atomic force microscope (AFM) is a widely used tool to image DNA and nucleoprotein complexes at the molecular level. This is because the AFM is relatively easy to operate, has the capability to image biomolecules under aqueous solutions, and, most importantly, can image mesoscopic macromolecular structures that are too complex to be studied by X-ray or NMR and too small to be visualized with the optical microscope. Although there are many AFM studies about the structure and the physical properties of DNA, only in few cases a rigorous method has been applied to analyze AFM images. This chapter describes procedures to prepare DNA and nucleoprotein complexes for AFM imaging and methods used to carry out simple image measurements to obtain structural data. In particular, methods to measure DNA contour length and the volume of free or DNA-bound proteins are presented and *** atomic force microscope (AFM) is a widely used tool to image DNA and nucleoprotein complexes at the molecular level. This is because the AFM is relatively easy to operate, has the capability to image biomolecules under aqueous solutions, and, most importantly, can image mesoscopic macromolecular structures that are too complex to be studied by X-ray or NMR and too small to be visualized with the optical microscope. Although there are many AFM studies about the structure and the physical properties of DNA, only in few cases a rigorous method has been applied to analyze AFM images. This chapter describes procedures to prepare DNA and nucleoprotein complexes for AFM imaging and methods used to carry out simple image measurements to obtain structural data. In particular, methods to measure DNA contour length and the volume of free or DNA-bound proteins are presented and discussed.

关键词： Atomic Force Microscopy AFM Image Analysis DNA Extraction NMR Restriction Enzyme Digestion PCR Electroelution DNA Purification Cross-linking Image Processing DNA Electrophoresis programming language Data analysis DNA Nucleoprotein complex Contour length Volume

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In Silico Knowledge and Content Tracking

In Silico Knowledge and Content Tracking

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作者： Herman Haagen Barend Mons

This chapter gives a brief overview of text-mining techniques to extract knowledge from large text collections. It describes the basis pipeline of how to come from text to relationships between biological concepts and the problems that are encountered at each step in the pipeline. We first explain how words in text are recognized as concepts. Second, concepts are associated with each other using 2×2 contingency tables and test statistics. Third, we explain that it is possible to extract indirect links between concepts using the direct links taken from 2×2 table analyses. This we call implicit information extraction. Fourth, the validation techniques to evaluate a text-mining system such as ROC curves and retrospective studies are discussed. We conclude by examining how text information can be combined with other non-textual data sources such as microarray expression data and what the future directions are for text-mining within the Internet. less

关键词： Data Mining Statistical Calculation Microarray STRING programming language information retrieval ontology Text-mining ROC curve disambiguation retrospective analysis prioritizer semantic web

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Flow-Based Combinatorial Antibody Profiling: An Integrated Approach to Cell Characterization

Flow-Based Combinatorial Antibody Profiling: An Integrated A...

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作者： Shane Bruckner Ling Wang Ruiling Yuan Perry Haaland Amitabh Gaur

BD FACS™ CAP (CAP = combinatorial antibody profile) is a screening tool for rapid characterization of human cell surface protein expression profiles using semi-automated high-throughput flow cytometry. The current configuration consists of 229 directly conjugated antibodies arrayed in a 96-well plate as three-color cocktails, which enables the characterization of each of the 229 individual surface markers. Each individual cell type of interest is analyzed on the 96-well screening plates and the data are acquired on a flow cytometer equipped with a high-throughput sampler. The expression level of each marker for each cell type is then calculated using semiautomated custom flow cytometry software. The process of characterizing these surface markers in a highly efficient manner using BD FACS™ CAP is enabled by automated liquid handling for staining, automated flow cytometry for data acquisition, and standardized algorithms for automated data *** FACS™ CAP (CAP = combinatorial antibody profile) is a screening tool for rapid characterization of human cell surface protein expression profiles using semi-automated high-throughput flow cytometry. The current configuration consists of 229 directly conjugated antibodies arrayed in a 96-well plate as three-color cocktails, which enables the characterization of each of the 229 individual surface markers. Each individual cell type of interest is analyzed on the 96-well screening plates and the data are acquired on a flow cytometer equipped with a high-throughput sampler. The expression level of each marker for each cell type is then calculated using semiautomated custom flow cytometry software. The process of characterizing these surface markers in a highly efficient manner using BD FACS™ CAP is enabled by automated liquid handling for staining, automated flow cytometry for data acquisition, and standardized algorithms for automated data analysis.

关键词： Protein Expression Profiling Flow Cytometry Recombinant Protein Expression Liquid Handling Statistical Calculation FACS Data analysis programming language Homo sapiens HPAF-II

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