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Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing

Rapid identification of causal mutations in tomato EMS popul...

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作者： Virginie Garcia Cécile Bres Daniel Just Lucie Fernandez Fabienne Wong Jun Tai Jean-Philippe Mauxion Marie-Christine Le Paslier Aurélie Bérard Dominique Brunel Koh Aoki Saleh Alseekh Alisdair R Fernie Paul D Fraser Christophe Rothan

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6–12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping. less

关键词： Genetic Mapping Library Preparation Whole-genome Sequencing Genotyping Agarose Gel Electrophoresis Illumina Sequencing Variant Calling Genome Annotation Mutagenesis DNA Sequencing TruSeq DNA Ligation SAMtools Python programming language Solanum lycopersicum Homo sapiens Next-generation sequencing Plant genetics Plant breeding

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Computational Biology Methods for Characterization of Pluripotent Cells

Computational Biology Methods for Characterization of Plurip...

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作者： Marcos J. Araúzo-Bravo

Pluripotent cells are a powerful tool for regenerative medicine and drug discovery. Several techniques have been developed to induce pluripotency, or to extract pluripotent cells from different tissues and biological fluids. However, the characterization of pluripotency requires tedious, expensive, time-consuming, and not always reliable wet-lab experiments; thus, an easy, standard quality-control protocol of pluripotency assessment remains to be established. Here to help comes the use of high-throughput techniques, and in particular, the employment of gene expression microarrays, which has become a complementary technique for cellular characterization. Research has shown that the transcriptomics comparison with an Embryonic Stem Cell (ESC) of reference is a good approach to assess the pluripotency. Under the premise that the best protocol is a computer software source code, here I propose and explain line by line a software protocol coded in R-Bioconductor for pluripotency assessment based on the comparison of transcriptomics data of pluripotent cells with an ESC of reference. I provide advice for experimental design, warning about possible pitfalls, and guides for results interpretation. less

关键词： Transcriptomics Principal Component Analysis Statistical Calculation Next Generation Sequencing MATLAB Microarray programming language Data analysis Mus musculus R iPSC Cellular reprogramming Computational biology Bioconductor Hierarchical clustering Scatterplots

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Ensembl Plants: Integrating Tools for Visualizing, Mining, and Analyzing Plant Genomics Data

Ensembl Plants: Integrating Tools for Visualizing, Mining, a...

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作者： Dan Bolser Daniel M. Staines Emily Pritchard Paul Kersey

Ensembl Plants (http://*** ) is an integrative resource presenting genome-scale information for a growing number of sequenced plant species (currently 33). Data provided includes genome sequence, gene models, functional annotation, and polymorphic loci. Various additional information are provided for variation data, including population structure, individual genotypes, linkage, and phenotype data. In each release, comparative analyses are performed on whole genome and protein sequences, and genome alignments and gene trees are made available that show the implied evolutionary history of each gene family. Access to the data is provided through a genome browser incorporating many specialist interfaces for different data types, and through a variety of additional methods for programmatic access and data mining. These access routes are consistent with those offered through the Ensembl interface for the genomes of non-plant species, including those of plant pathogens, pests, and pollinators. Ensembl Plants is updated 4–5 times a year and is developed in collaboration with our international partners in the Gramene (http://*** ) and transPLANT projects (http://*** ). less

关键词： Genomics Transcriptomics Data Mining Genome Visualization RNA-seq Multiple Sequence Alignment Genome Assembly Genome Alignment Ensembl Gramene Database Management System programming language Arabidopsis lyrata Arabidopsis thaliana Chlamydomonas reinhardtii Zea mays Medicago truncatula Setaria italica Physcomitrella patens Populus trichocarpa Populus (cottonwood) Oryza brachyantha Oryza glaberrima Oryza sativa Indica Group Oryza sativa Japonica Group Selaginella moellendorffii Solanum lycopersicum Oryza barthii Oryza punctata Oryza nivara Oryza rufipogon Brassica rapa Solanum tuberosum Prunus Rosaceae Glycine max Triticum aestivum Databases Genome Cereals Phenotype Genetic Crops Comparative genomic Functional genomic

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Introduction to Computer-Assisted Analysis in Hydrocarbon and Lipid Microbiology

Introduction to Computer-Assisted Analysis in Hydrocarbon an...

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作者： Wilfred F. Röling

The large datasets generated in the immense complex field of hydrocarbon and lipid microbiology cannot be addressed and understood without computer-assisted analysis. Key components in computer-assisted analysis are bioinformatics, databases, statistics, and mathematical modeling. Computer-assisted analysis contributes to the entire scientific process: experimental design, data analysis, data storage, and utilization of these stored data. A wide range of relevant protocols are presented in this volume: protocols dealing with subjects that are also popular in other research areas and protocols addressing important topics specific for hydrocarbon and lipid microbiology. The basics of a major open-source programming language, Python, will be introduced. less

关键词： Statistical Calculation Mathematical Modeling Data analysis programming language Python Bioinformatics Systems biology Computational biology Mathematical biology

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Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry

Analysis of bacterial-surface-specific antibodies in body fl...

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作者： Kathrin Moor Jehane Fadlallah Albulena Toska Delphine Sterlin Maria L Balmer Andrew J Macpherson Guy Gorochov Martin Larsen Emma Slack

Antibacterial antibody responses that target surfaces of live bacteria or secreted toxins are likely to be relevant in controlling bacterial pathogenesis. The ability to specifically quantify bacterial-surface-binding antibodies is therefore highly attractive as a quantitative correlate of immune protection. Here, binding of antibodies from various body fluids to pure-cultured live bacteria is made visible with fluorophore-conjugated secondary antibodies and measured by flow cytometry. We indicate the necessary controls for excluding nonspecific binding and also demonstrate a cross-adsorption technique for determining the extent of cross-reactivity. This technique has numerous advantages over standard ELISA and western blotting techniques because of its independence from scaffold binding, exclusion of cross-reactive elements from lysed bacteria and ability to visualize bacterial subpopulations. In addition, less than 105 bacteria and less than 10 μg of antibody are required per sample. The technique requires 3–4 h of hands-on experimentation and analysis. Moreover, it can be combined with automation and mutliplexing for high-throughput applications. less

关键词： Flow Cytometry ELISA Western Blot Microbial Culture Anaerobic Culture Titration Lavage FACS Statistical Calculation PCR Data analysis GraphPad Prism programming language Homo sapiens Ovis aries Mus musculus Equus asinus Capra hircus Saccharomyces cerevisiae Glycine max Immunological techniques Antibodies Bacterial pathogenesis Anti-human Anti-mouse Anti-mouse IgM Anti-mouse IgA

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Agent-Based Modeling of Cancer Stem Cell Driven Solid Tumor Growth

Agent-Based Modeling of Cancer Stem Cell Driven Solid Tumor ...

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作者： Jan Poleszczuk Paul Macklin Heiko Enderling

Computational modeling of tumor growth has become an invaluable tool to simulate complex cell–cell interactions and emerging population-level dynamics. Agent-based models are commonly used to describe the behavior and interaction of individual cells in different environments. Behavioral rules can be informed and calibrated by in vitro assays, and emerging population-level dynamics may be validated with both in vitro and in vivo experiments. Here, we describe the design and implementation of a lattice-based agent-based model of cancer stem cell driven tumor growth. less

关键词： Microscopy programming language Python Calibration Cancer stem cell Agent-based model Tumor growth Domain Search order High performance Digital cell line

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Review and Literature Mining on Proteostasis Factors and Cancer

Review and Literature Mining on Proteostasis Factors and Can...

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作者： Ana Sofia Carvalho Manuel S. Rodríguez Rune Matthiesen

Automatic analysis of increasingly growing literature repositories including data integration to other databases is a powerful tool to propose hypothesis that can be used to plan experiments to validate or disprove the hypothesis. Furthermore, it provides means to evaluate the redundancy of research line in comparison to the published literature. This is potentially beneficial for those developing research in a specific disease which are interested in exploring a particular pathway or set of genes/proteins. In the scope of the integrating book a case will be made addressing proteostasis factors in cancer. The maintenance of proteome homeostasis, known as proteostasis, is a process by which cells regulate protein translation, degradation, subcellular localization, and protein folding and consists of an integrated network of proteins. The ubiquitin-proteasome system plays a key role in essential biological processes such as cell cycle, DNA damage repair, membrane trafficking, and maintaining protein homeostasis. Cells maintain proteostasis by regulating protein translation, degradation, subcellular localization, and protein folding. Aberrant proteostasis leads to loss-of-function diseases (cystic fibrosis) and gain-of-toxic-function diseases (Alzheimer’s, Parkinson’s, and Huntington’s disease). Cancer therapy on the other hand explores inhibition of proteostasis factors to trigger endoplasmic reticulum stress with subsequent apoptosis. Alternatively therapies target deubiquitinases and thereby regulate tumor promoters or suppressors. Furthermore, mutations in specific proteostasis factors are associated with higher risk for specific cancers, e.g., BRCA mutations in breast cancer. This chapter discusses proteostasis protein factors’ association with cancer from a literature mining perspective. less

关键词： Text Mining Recombinant Protein Expression Data Mining programming language Cancer Proteostasis Wordcloud

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TUBEs-Mass Spectrometry for Identification and Analysis of the Ubiquitin-Proteome

TUBEs-Mass Spectrometry for Identification and Analysis of t...

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作者： Mikel Azkargorta Iraide Escobes Felix Elortza Rune Matthiesen Manuel S. Rodríguez

Mass spectrometry (MS) has become the method of choice for the large-scale analysis of protein ubiquitylation. There exist a number of proposed methods for mapping ubiquitin sites, each with different pros and cons. We present here a protocol for the MS analysis of the ubiquitin-proteome captured by TUBEs and subsequent data analysis. Using dedicated software and algorithms, specific information on the presence of ubiquitylated peptides can be obtained from the MS search results. In addition, a quantitative and functional analysis of the ubiquitylated proteins and their interacting partners helps to unravel the biological and molecular processes they are involved in. less

关键词： Collision-induced Dissociation Mass Spectrometry Statistical Calculation Spectroscopy Gel Staining Database Search Spectral Counting Proteomics Liquid Chromatography-mass Spectrometry Functional Enrichment Data analysis programming language Escherichia coli Ubiquitin Posttranslational modification (PTM) Tandem Ubiquitin Binding Entities (TUBEs) Gene Ontology (GO) Iodoacetamide (IAA) Chloroacetamide (CAA)

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A scored human protein–protein interaction network to catalyze genomic interpretation

A scored human protein–protein interaction network to catal...

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作者： Taibo Li Rasmus Wernersson Rasmus B Hansen Heiko Horn Johnathan Mercer Greg Slodkowicz Christopher T Workman Olga Rigina Kristoffer Rapacki Hans H Stærfeldt Søren Brunak Thomas S Jensen Kasper Lage

Genome-scale human protein–protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein–protein interaction network (InWeb_InBioMap, or InWeb_IM) with severalfold more interactions (>500,000) and better functional biological relevance than comparable resources. We illustrate that InWeb_InBioMap enables functional interpretation of >4,700 cancer genomes and genes involved in autism. less

关键词： Functional Genomics Statistical Calculation Proteomics Immunoprecipitation Ensembl programming language Pull-down Assay Homo sapiens Protein–protein interaction networks Systems biology Literature mining Protein databases

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In Vitro and In Vivo Imaging of Fluorescent Aptamers

In Vitro and In Vivo Imaging of Fluorescent Aptamers

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作者： Ioanna Théodorou Nam Nguyen Quang Karine Gombert Benoit Thézé Benoit Lelandais Frédéric Ducongé

Fluorescence imaging techniques could be used in different ways to study the interaction of aptamers with biological systems from cell culture to animal models. Here, we present the methods developed in our laboratory for fluorescently labeled aptamers, study their internalization inside living cells using time-lapse microscopy, and monitor their biodistribution in mice bearing subcutaneous xenograft tumors using planar fluorescence imaging and fluorescence diffuse optical tomography (fDOT). less

关键词： Time Lapse Microscopy Optical Tomography In Vivo Imaging Fluorescence Labeling Fluorescence Microscopy Cell And Tissue Culture Titration Spectroscopy Freeze-thaw Method Transillumination ImageJ Image Analysis Computed Tomography MRI Nucleic Acid Labeling Subcutaneous Injection 3D Imaging DNA hybridization programming language Rattus norvegicus Mus musculus MCF-7 Oligonucleotide Fluorescence Aptamer

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