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作者： Alex J. Dumbrell Robert M. Ferguson Dave R. Clark

Quantifying the functional and taxonomic diversity of microbial assemblages is essential to understanding almost all aspects of microbial ecology. In recent years, the advent of Next Generation Sequencing (NGS) technology has accelerated this process. It is now common practice to target and amplify phylogenetic and/or functional marker genes, and use NGS approaches to characterise their diversity across multiple samples. However, all NGS approaches contain inherent methodological biases, producing both the high-quality data required by researchers, and in addition, erroneous sequences and noise. Careful bioinformatic analysis of NGS data is therefore required to quality filter and process sequences in order to avoid misleading inferences from artifactual results. A similar consideration must also be given to any downstream statistical analysis, as an incorrect choice of approach can also produce false conclusions. These various analytical steps and considerations can appear daunting to the uninitiated and may be perceived as a hurdle to completing research. In this chapter, we aim to provide the methods and guidance required to overcome this hurdle, and impart the skills required for a novice bioinformatician to produce a basic analysis of their NGS amplicon data. We focus on data produced using two common NGS technologies, the historically more widely used 454-pyrosequencer, and the currently more widely used Illumina platform. We cover methods for quality filtering and denoising data, picking Operational Taxonomic Units (OTUs), assigning taxonomy to sequences and basic statistical analyses required for hypothesis testing. Implementation of these methods is demonstrated for both of two commonly used pipelines (QIIME and mothur), and additional stand-alone packages (including R), providing the reader with maximum flexibility when analysing their data. less

关键词： Next Generation Sequencing Statistical Calculation PCR Illumina Sequencing Library Preparation Mothur Data analysis programming language Homo sapiens Bioinformatics Microbial ecology 16S rRNA Amplicon Illumina 454-Pyrosequencing BioLinux QIIME

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Genotype Specification language

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ACS SYNTHETIC BIOLOGY 2016年第6期5卷 471-478页

作者： Wilson, Erin H. Sagawa, Shiori Weis, James W. Schubert, Max G. Bissell, Michael Hawthorne, Brian Reeves, Christopher D. Dean, Jed Platt, Darren Amyris Inc 5885 Hollis StSuite 100 Emeryville CA 94608 USA

We describe here the Genotype Specification language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.

关键词： programming language bio-design automation DNA assembly

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Protein Multiplexed Immunoassay Analysis with R

Protein Multiplexed Immunoassay Analysis with R

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作者： Edmond J. Breen

Plasma samples from 177 control and type 2 diabetes patients collected at three Australian hospitals are screened for 14 analytes using six custom-made multiplex kits across 60 96-well plates. In total 354 samples were collected from the patients, representing one baseline and one end point sample from each patient. R methods and source code for analyzing the analyte fluorescence response obtained from these samples by Luminex Bio-Plex® xMap multiplexed immunoassay technology are disclosed. Techniques and R procedures for reading Bio-Plex® result files for statistical analysis and data visualization are also presented. The need for technical replicates and the number of technical replicates are addressed as well as plate layout design strategies. Multinomial regression is used to determine plate to sample covariate balance. Methods for matching clinical covariate information to Bio-Plex® results and vice versa are given. As well as methods for measuring and inspecting the quality of the fluorescence responses are presented. Both fixed and mixed-effect approaches for immunoassay statistical differential analysis are presented and discussed. A random effect approach to outlier analysis and detection is also shown. The bioinformatics R methodology present here provides a foundation for rigorous and reproducible analysis of the fluorescence response obtained from multiplexed immunoassays. less

关键词： Proteomics Multiplex Immunoassay Statistical Calculation Regression Analysis Immunoassay programming language Bioinformatics Quality Mixed-effects model xMap

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A Gene Ontology Tutorial in Python

A Gene Ontology Tutorial in Python

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作者： Alex Warwick Vesztrocy Christophe Dessimoz

This chapter is a tutorial on using Gene Ontology resources in the Python programming language. This entails querying the Gene Ontology graph, retrieving Gene Ontology annotations, performing gene enrichment analyses, and computing basic semantic similarity between GO terms. An interactive version of the tutorial, including solutions, is available at http://*** . less

关键词： Python programming language Gene Ontology Tutorial

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Databases for Wheat Genomics and Crop Improvement

Databases for Wheat Genomics and Crop Improvement

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作者： Yuxuan Yuan Armin Scheben Chon-Kit Kenneth Chan David Edwards

The genomics revolution brought on by advances in high-throughput sequencing has led to the production of vast amounts of data. Databases play an essential role in storing and managing this information to make it available to researchers and crop breeders. This chapter provides an outline of how to use databases and tools for wheat genome research. less

关键词： Genome Visualization Genomics High-Throughput Sequencing PCR Genome-Wide Association Study Ensembl programming language Triticum aestivum Crop breeding TAGdb WheatIS

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Genome Sequencing of Steroid Producing Bacteria Using Ion Torrent Technology and a Reference Genome

Genome Sequencing of Steroid Producing Bacteria Using Ion To...

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作者： Alberto Sola-Landa Antonio Rodríguez-García Carlos Barreiro Rosario Pérez-Redondo

The Next-Generation Sequencing technology has enormously eased the bacterial genome sequencing and several tens of thousands of genomes have been sequenced during the last 10 years. Most of the genome projects are published as draft version, however, for certain applications the complete genome sequence is required. In this chapter, we describe the strategy that allowed the complete genome sequencing of Mycobacterium neoaurum NRRL B-3805, an industrial strain exploited for steroid production, using Ion Torrent sequencing reads and the genome of a close strain as the reference. This protocol can be applied to analyze the genetic variations between closely related strains; for example, to elucidate the point mutations between a parental strain and a random mutagenesis-derived mutant. less

关键词： Whole-genome Sequencing Sanger Sequencing Statistical Calculation Variant Calling High-Throughput Sequencing Genotyping Genome Sequencing Ion Torrent Sequencing bedtools programming language SAMtools Genome Analysis Mycobacterium Next-generation sequencing Reference genome Reads Assemble B-3805 Steroid production Analysis of variants

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Mapping the genomic landscape of CRISPR–Cas9 cleavage

Mapping the genomic landscape of CRISPR–Cas9 cleavage

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作者： Peter Cameron Chris K Fuller Paul D Donohoue Brittnee N Jones Matthew S Thompson Matthew M Carter Scott Gradia Bastien Vidal Elizabeth Garner Euan M Slorach Elaine Lau Lynda M Banh Alexandra M Lied Leslie S Edwards Alexander H Settle Daniel Capurso Victor Llaca Stéphane Deschamps Mark Cigan Joshua K Young Andrew P May

RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity. less

关键词： Genomics Statistical Calculation FACS PCR Genome Editing RNA Purification Spectroscopy Affinity Purification Bowtie Nucleic Acid Fragment Analysis Transfection Cell And Tissue Culture Nucleofection DNA Extraction DNA Ligation SAMtools Python programming language DNA Fingerprinting Anti-human

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Automated Analysis of Intracellular Dynamic Processes

Automated Analysis of Intracellular Dynamic Processes

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作者： Yao Yao Ihor Smal Ilya Grigoriev Maud Martin Anna Akhmanova Erik Meijering

The study of intracellular dynamic processes is of fundamental importance for understanding a wide variety of diseases and developing effective drugs and therapies. Advanced fluorescence microscopy imaging systems nowadays allow the recording of virtually any type of process in space and time with super-resolved detail and with high sensitivity and specificity. The large volume and high information content of the resulting image data, and the desire to obtain objective, quantitative descriptions and biophysical models of the processes of interest, require a high level of automation in data analysis. Two key tasks in extracting biologically meaningful information about intracellular dynamics from image data are particle tracking and particle trajectory analysis. Here we present state-of-the-art software tools for these tasks and describe how to use them. less

关键词： Fluorescence Microscopy Image Analysis ImageJ Fiji Time Lapse Microscopy Statistical Calculation Image Processing Data analysis programming language Feature extraction Software tools Particle tracking Trajectory analysis Intracellular dynamics Feature visualization

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Degradation Network Reconstruction Guided by Metagenomic Data

Degradation Network Reconstruction Guided by Metagenomic Dat...

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作者： Rafael Bargiela Manuel Ferrer

Network reconstruction procedures based on meta-“omics” data are an invaluable tool for inferring total and active set of reactions mediated by different members in a microbial community. Within them, network-based methods for automatic analysis of catabolic capacities in metagenomes are currently limited. Here, we describe the complete workflow, scripts, and commands allowing the automatic reconstruction of biodegradation networks using as an input meta-sequences generated by direct DNA sequencing. less

关键词： Metagenomics DNA Sequencing programming language Next-generation sequencing Pathways Biodegradation Pollutants Network reconstruction AromaDeg Aromatics

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Modeling of Receptor Tyrosine Kinase Signaling: Computational and Experimental Protocols

Modeling of Receptor Tyrosine Kinase Signaling: Computationa...

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作者： Dirk Fey Edita Aksamitiene Anatoly Kiyatkin Boris N. Kholodenko

The advent of systems biology has convincingly demonstrated that the integration of experiments and dynamic modelling is a powerful approach to understand the cellular network biology. Here we present experimental and computational protocols that are necessary for applying this integrative approach to the quantitative studies of receptor tyrosine kinase (RTK) signaling networks. Signaling by RTKs controls multiple cellular processes, including the regulation of cell survival, motility, proliferation, differentiation, glucose metabolism, and apoptosis. We describe methods of model building and training on experimentally obtained quantitative datasets, as well as experimental methods of obtaining quantitative dose-response and temporal dependencies of protein phosphorylation and activities. The presented methods make possible (1) both the fine-grained modeling of complex signaling dynamics and identification of salient, course-grained network structures (such as feedback loops) that bring about intricate dynamics, and (2) experimental validation of dynamic models. less

关键词： Mathematical Modeling Electrophoresis Cell And Tissue Culture Protein Purification Western Blot Densitometry Recombinant Protein Expression Cell Fractionation Ultracentrifugation Immunoprecipitation RNA Interference CRISPR Transfection Electroporation Cell Lysis Protein Extraction Subcellular Fractionation Gel Electrophoresis Gel Staining Statistical Calculation programming language Phosphorylation Model validation Kinetics Receptor tyrosine kinase Cell signaling Computational model Cellular networks Semi-quantitative analysis Perturbations

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