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Illumina human exome genotyping array clustering and quality control

Illumina human exome genotyping array clustering and quality...

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作者： Yan Guo Jing He Shilin Zhao Hui Wu Xue Zhong Quanhu Sheng David C Samuels Yu Shyr Jirong Long

With the rise of high-throughput sequencing technology, traditional genotyping arrays are gradually being replaced by sequencing technology. Against this trend, Illumina has introduced an exome genotyping array that provides an alternative approach to sequencing, especially suited to large-scale genome-wide association studies (GWASs). The exome genotyping array targets the exome plus rare single-nucleotide polymorphisms (SNPs), a feature that makes it substantially more challenging to process than previous genotyping arrays that targeted common SNPs. Researchers have struggled to generate a reliable protocol for processing exome genotyping array data. The Vanderbilt Epidemiology Center, in cooperation with Vanderbilt Technologies for Advanced Genomics Analysis and Research Design (VANGARD), has developed a thorough exome chip–processing protocol. The protocol was developed during the processing of several large exome genotyping array-based studies, which included over 60,000 participants combined. The protocol described herein contains detailed clustering techniques and robust quality control procedures, and it can benefit future exome genotyping array–based GWASs. less

关键词： Genotyping High-Throughput Sequencing Genomics SNP Calling Whole-exome sequencing Principal Component Analysis programming language Python Homo sapiens Computational biology and bioinformatics Genome-wide association studies

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Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications

Quantification of larval zebrafish motor function in multiwe...

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作者： Yangzhong Zhou Richard T Cattley Clinton L Cario Qing Bai Edward A Burton

This article describes a method to quantify the movements of larval zebrafish in multiwell plates, using the open-source MATLAB applications LSRtrack and LSRanalyze. The protocol comprises four stages: generation of high-quality, flatly illuminated video recordings with exposure settings that facilitate object recognition; analysis of the resulting recordings using tools provided in LSRtrack to optimize tracking accuracy and motion detection; analysis of tracking data using LSRanalyze or custom MATLAB scripts; and implementation of validation controls. The method is reliable, automated and flexible, requires <1 h of hands-on work for completion once optimized and shows excellent signal:noise characteristics. The resulting data can be analyzed to determine the following: positional preference; displacement, velocity and acceleration; and duration and frequency of movement events and rest periods. This approach is widely applicable to the analysis of spontaneous or stimulus-evoked zebrafish larval neurobehavioral phenotypes resulting from a broad array of genetic and environmental manipulations, in a multiwell plate format suitable for high-throughput applications. less

关键词： MATLAB ImageJ Image Processing programming language Danio rerio Lens Software Behavioural methods Motor control

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Natural language Processing in Biomedicine: A Unified System Architecture Overview

Natural Language Processing in Biomedicine: A Unified System...

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作者： Son Doan Mike Conway Tu Minh Phuong Lucila Ohno-Machado

In contemporary electronic medical records much of the clinically important data—signs and symptoms, symptom severity, disease status, etc.—are not provided in structured data fields but rather are encoded in clinician-generated narrative text. Natural language processing (NLP) provides a means of unlocking this important data source for applications in clinical decision support, quality assurance, and public health. This chapter provides an overview of representative NLP systems in biomedicine based on a unified architectural view. A general architecture in an NLP system consists of two main components: background knowledge that includes biomedical knowledge resources and a framework that integrates NLP tools to process text. Systems differ in both components, which we review briefly. Additionally, the challenge facing current research efforts in biomedical NLP includes the paucity of large, publicly available annotated corpora, although initiatives that facilitate data sharing, system evaluation, and collaborative work between researchers in clinical NLP are starting to emerge. less

关键词： Natural language Processing Machine Learning Mammography programming language Homo sapiens Biomedicine Electronic medical record Rule-based learning method System architecture Unified Medical language System

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Using TRIP for genome-wide position effect analysis in cultured cells

Using TRIP for genome-wide position effect analysis in cultu...

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作者： Waseem Akhtar Alexey V Pindyurin Johann de Jong Ludo Pagie Jelle ten Hoeve Anton Berns Lodewyk F A Wessels Bas van Steensel Maarten van Lohuizen

The influence of local chromatin context on gene expression can be explored by integrating a transcription reporter at different locations in the genome as a sensor. Here we provide a detailed protocol for analyzing thousands of reporters integrated in parallel (TRIP) at a genome-wide level. TRIP is based on tagging each reporter with a unique barcode, which is used for independent reporter expression analysis and integration site mapping. Compared with previous methods for studying position effects, TRIP offers a 100–1,000-fold higher throughput in a faster and less-labor-intensive manner. The entire experimental protocol takes ∼42 d to complete, with high-throughput sequencing and data analysis requiring an additional ∼11 d. TRIP was developed by using transcription reporters in mouse embryonic stem (mES) cells, but because of its flexibility the method can be used to probe the influence of chromatin context on a variety of molecular processes in any transfectable cell line. less

关键词： PCR Transfection High-Throughput Sequencing Restriction Enzyme Digestion DNA Purification Electrotransformation Electroporation Library Preparation Cycle Sequencing Sanger Sequencing Cell And Tissue Culture Flow Cytometry Copy Number Estimation DNA Extraction RNA Extraction Nested PCR Illumina Sequencing Inverse PCR Bowtie Capillary Electrophoresis (CE) DNA Sequencing Real-time PCR DNA Ligation Data analysis programming language Escherichia coli Rattus norvegicus BRL Next-generation sequencing Chromatin analysis Gene regulation Epigenetics

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Bioinformatics Identification of Coevolving Residues

Bioinformatics Identification of Coevolving Residues

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作者： Russell J. Dickson Gregory B. Gloor

Positions in a protein are thought to coevolve to maintain important structural and functional interactions over evolutionary time. The detection of putative coevolving positions can provide important new insights into a protein family in the same way that knowledge is gained by recognizing evolutionarily conserved characters and characteristics. Putatively coevolving positions can be detected with statistical methods that identify covarying positions. However, positions in protein alignments can covary for many other reasons than coevolution; thus, it is crucial to create high-quality multiple sequence alignments for coevolution inference. Furthermore, it is important to understand common signs and sources of error. When confounding factors are accounted for, coevolution is a rich resource for protein engineering information. less

关键词： Structure Prediction Protein Engineering Statistical Calculation Protein Structure Analysis Structure Alignment Sequence Alignment Tools programming language Protein design Coevolution Covariation Protein alignment

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FX Cloning: A Simple and Robust High-Throughput Cloning Method for Protein Expression

FX Cloning: A Simple and Robust High-Throughput Cloning Meth...

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作者： Eric R. Geertsma

The immense amount of gene sequences available nowadays allows scientist to screen broadly for extraordinary proteins. Reliable cloning tools that allow the parallel processing of many targets are vital for the success of this strategy. The FX cloning procedure detailed here is such a straightforward and efficient tool. It is dedicated to the cloning of open reading frames (ORFs) with the final aim of expressing the corresponding proteins. FX cloning combines attractive features of established high-throughput cloning methods that were thus far not unified in one single method. It facilitates the subcloning of a sequence-verified ORF to a variety of expression vectors, but is sufficiently versatile to accept PCR products as well. Moreover, the common, but undesirable feature of extending target ORFs with long cloning-related sequences is avoided. It leads to the addition of only one amino acid to each side of the protein. As a consequence, only one primer pair or PCR product suffices to generate expression vectors for both N- and C-terminal translational fusions. FX cloning is highly efficient and economical in its use. The method is suited for high-throughput cloning projects and also for everyday cloning of single targets. FX cloning is based on the use of type IIS restriction enzymes and negative selection markers. The full procedure takes place in one pot in less than 3 h and does not require intermediate purification steps nor extensive handling. The method has proven to be very robust and suitable for all common expression systems. less

关键词： Recombinant Protein Expression PCR Gel Electrophoresis DNA Sequencing DNA Ligation Python programming language Escherichia coli Saccharomyces cerevisiae Subcloning High-throughput cloning Type IIS restriction enzymes Counterselection marker sacB ccdB

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Multiple Protein Sequence Alignment with MSAProbs

Multiple Protein Sequence Alignment with MSAProbs

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作者： Yongchao Liu Bertil Schmidt

Multiple sequence alignment (MSA) generally constitutes the foundation of many bioinformatics studies involving functional, structural, and evolutionary relationship analysis between sequences. As a result of the exponential computational complexity of the exact approach to producing optimal multiple alignments, the majority of state-of-the-art MSA algorithms are designed based on the progressive alignment heuristic. In this chapter, we outline MSAProbs, a parallelized MSA algorithm for protein sequences based on progressive alignment. To achieve high alignment accuracy, this algorithm employs a hybrid combination of a pair hidden Markov model and a partition function to calculate posterior probabilities. Furthermore, we provide some practical advice on the usage of the algorithm.

关键词： Multiple Sequence Alignment programming language Hidden Markov models Progressive alignment Partition function Consistency-based scheme

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Combination of Theoretical and Experimental Approaches for the Design and Study of Fibril-Forming Peptides

Combination of Theoretical and Experimental Approaches for t...

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作者： Phanourios Tamamis Emmanouil Kasotakis Georgios Archontis Anna Mitraki

Self-assembling peptides that can form supramolecular structures such as fibrils, ribbons, and nanotubes are of particular interest to modern bionanotechnology and materials science. Their ability to form biocompatible nanostructures under mild conditions through non-covalent interactions offers a big biofabrication advantage. Structural motifs extracted from natural proteins are an important source of inspiration for the rational design of such peptides. Examples include designer self-assembling peptides that correspond to natural coiled-coil motifs, amyloid-forming proteins, and natural fibrous proteins. In this chapter, we focus on the exploitation of structural information from beta-structured natural fibers. We review a case study of short peptides that correspond to sequences from the adenovirus fiber shaft. We describe both theoretical methods for the study of their self-assembly potential and basic experimental protocols for the assessment of fibril-forming assembly. less

关键词： Molecular Dynamics Simulation TEM Diffraction Method programming language Adenovirus Peptides Self-assembly Amyloid fibrils Beta-structure Implicit solvent Replica-exchange

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Dynamic Clamp in Cardiac and Neuronal Systems Using RTXI

Dynamic Clamp in Cardiac and Neuronal Systems Using RTXI

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作者： Francis A. Ortega Robert J. Butera David J. Christini John A. White Alan D. Dorval

The injection of computer-simulated conductances through the dynamic clamp technique has allowed researchers to probe the intercellular and intracellular dynamics of cardiac and neuronal systems with great precision. By coupling computational models to biological systems, dynamic clamp has become a proven tool in electrophysiology with many applications, such as generating hybrid networks in neurons or simulating channelopathies in cardiomyocytes. While its applications are broad, the approach is straightforward: synthesizing traditional patch clamp, computational modeling, and closed-loop feedback control to simulate a cellular conductance. Here, we present two example applications: artificial blocking of the inward rectifier potassium current in a cardiomyocyte and coupling of a biological neuron to a virtual neuron through a virtual synapse. The design and implementation of the necessary software to administer these dynamic clamp experiments can be difficult. In this chapter, we provide an overview of designing and implementing a dynamic clamp experiment using the Real-Time eXperiment Interface (RTXI), an open-source software system tailored for real-time biological experiments. We present two ways to achieve this using RTXI’s modular format, through the creation of a custom user-made module and through existing modules found in RTXI’s online library. less

关键词： Electrophysiology Patch Clamp Technique Intraperitoneal Injection Microscopy programming language Pull-down Assay Dynamic clamp RTXI Neuronal networks Artificial conductance block Reciprocal neuronal coupling

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Analysis of mRNA Translation States in

Analysis of mRNA Translation States in

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作者： Anamika Missra Albrecht G. Arnim

Gene regulation at the level of translation occurs in response to environmental perturbation and is increasingly recognized as a factor affecting plant development. Despite extensive knowledge of transcriptional control, very little is known about translational regulation of genes in response to the daily light/dark cycles. Here we describe the experimental layout designed to address how the translation states of genes change at various times during a diurnal cycle in Arabidopsis thaliana seedlings. We have adopted a strategy combining sucrose-gradient profiling of ribosomes and high-throughput microarray analysis of the ribosome-associated mRNA to investigate the translational landscape of the Arabidopsis genome. This is a powerful technique that can be easily extended to study translation regulation in different genetic backgrounds and under various environmental conditions. less

关键词： Microarray Real-time PCR RNA Extraction Gel Electrophoresis RNA Purification Sterilization Fractionation Microarray Hybridization Data analysis programming language Arabidopsis Translational regulation Polysome profile Sucrose density gradient

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