MIPHENO: data normalization for high throughput metabolite analysis

Background  

High throughput methodologies such as microarrays, mass spectrometry and plate-based small molecule screens are increasingly used to facilitate discoveries from gene function to drug candidate identification. These large-scale experiments are typically carried out over the course of months and years, often without the controls needed to compare directly across the dataset. Few methods are available to facilitate comparisons of high throughput metabolic data generated in batches where explicit in-group controls for normalization are lacking.     

A computer program for on-line measurement, storage, analysis and retrieval of urodynamic data

A computer program is presented which allows for direct connection of a minicomputer to a urodynamic set-up. The program stores measured pressure and flow data in a random access disc file with minimal intervention of the urodynamicist, and enables the direct application of a number of methods of analysis to the data. The program is modular, and other analysis methods are easily added. Results of analyses are stored in the same disc file, and both results and measured data can be quickly and easily retrieved. The program is written in FORTRAN; hardware-dependent functions (analog input, graphics display, and random access disc storage) are implemented in subroutines (partly assembler) which can easily be replaced.     

Optimizing transformations for automated,high throughput analysis of flow cytometry data

Background  

In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations.     

Statistical considerations for high throughput screening data

High throughput screening (HTS) is a widely used effective approach in genome-wide association and large scale protein expression studies, drug discovery, and biomedical imaging research. How to accurately identify candidate ‘targets’ or biologically meaningful features with a high degree of confidence has led to extensive statistical research in an effort to minimize both false-positive and false-negative rates. A large body of literature on this topic with in-depth statistical contents is available. We examine currently available statistical methods on HTS and aim to summarize some selected methods into a concise, easy-tofollow introduction for experimental biologists.     

A computer system for the storage and retrieval of clinical pharmacokinetic data

A computer system is described which stores patient data relating to clinical pharmacokinetic assessments made by clinical pharmacists, who are participating in a clinical pharmacokinetics service. The system was developed to assist in the documentation of service activities and storage of patients' pharmacokinetic data. An additional component of the system is the ability for retrospective review of the stored data. Applications of this system to the derivation of new information on drug pharmacokinetics and drug efficacy/toxicity in various patient groups is discussed. The implications for Phase IV drug studies and toxicity screening studies is also described.     

Design and analysis of experiments with high throughput biological assay data

The design and analysis of experiments using gene expression microarrays is a topic of considerable current research, and work is beginning to appear on the analysis of proteomics and metabolomics data by mass spectrometry and NMR spectroscopy. The literature in this area is evolving rapidly, and commercial software for analysis of array or proteomics data is rarely up to date, and is essentially nonexistent for metabolomics data. In this paper, I review some of the issues that should concern any biologists planning to use such high-throughput biological assay data in an experimental investigation. Technical details are kept to a minimum, and may be found in the referenced literature, as well as in the many excellent papers which space limitations prevent my describing. There are usually a number of viable options for design and analysis of such experiments, but unfortunately, there are even more non-viable ones that have been used even in the published literature. This is an area in which up-to-date knowledge of the literature is indispensable for efficient and effective design and analysis of these experiments. In general, we concentrate on relatively simple analyses, often focusing on identifying differentially expressed genes and the comparable issues in mass spectrometry and NMR spectroscopy (consistent differences in peak heights or areas for example). Complex multivariate and pattern recognition methods also need much attention, but the issues we describe in this paper must be dealt with first. The literature on analysis of proteomics and metabolomics data is as yet sparse, so the main focus of this paper will be on methods devised for analysis of gene expression data that generalize to proteomics and metabolomics, with some specific comments near the end on analysis of metabolomics data by mass spectrometry and NMR spectroscopy.     

A high throughput method for genome-wide analysis of retroviral integration

Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20–25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.     

MiMiR: a comprehensive solution for storage,annotation and exchange of microarray data

Background  

The generation of large amounts of microarray data presents challenges for data collection, annotation, exchange and analysis. Although there are now widely accepted formats, minimum standards for data content and ontologies for microarray data, only a few groups are using them together to build and populate large-scale databases. Structured environments for data management are crucial for making full use of these data.     

A design for computer nucleic-acid-sequence storage, retrieval, and manipulation

We have designed and built a data-base system for the storage of nucleic-acid sequences. The system consists of a data base (“the library”) and software that manages and provides access to that data base (“the Librarian”).     

高通量测序数据的基因组拷贝数变异检测方法综述

拷贝数变异是指基因组中发生大片段的DNA序列的拷贝数增加或者减少。根据现有的研究可知,拷贝数变异是多种人类疾病的成因,与其发生与发展机制密切相关。高通量测序技术的出现为拷贝数变异检测提供了技术支持,在人类疾病研究、临床诊疗等领域,高通量测序技术已经成为主流的拷贝数变异检测技术。虽然不断有新的基于高通量测序技术的算法和软件被人们开发出来,但是准确率仍然不理想。本文全面地综述基于高通量测序数据的拷贝数变异检测方法,包括基于reads深度的方法、基于双末端映射的方法、基于拆分read的方法、基于从头拼接的方法以及基于上述4种方法的组合方法,深入探讨了每类不同方法的原理,代表性的软件工具以及每类方法适用的数据以及优缺点等,并展望未来的发展方向。     

Applications of ion mobility mass spectrometry for high throughput,high resolution glycan analysis

BackgroundDiverse varieties of often heterogeneous glycans are ubiquitous in nature. They play critical roles in recognition events, act as energy stores and provide structural stability at both molecular and cellular levels. Technologies capable of fully elucidating the structures of glycans are far behind the other ‘-omic’ fields. Liquid chromatography (LC) and mass spectrometry (MS) are currently the most useful techniques for high-throughput analysis of glycans. However, these techniques do not provide full unambiguous structural information and instead the gap in full sequence assignment is frequently filled by a priori knowledge of the biosynthetic pathways and the assumption that these pathways are highly conserved.Scope of the reviewThis comprehensive review details the rise of the emerging analytical technique ion mobility spectrometry (IMS) (coupled to MS) to facilitate the determination of three-dimensional shape: the separation and characterization of isobaric glycans, glyco(peptides/proteins), glycolipids, glycosaminoglycans and other polysaccharides; localization of sites of glycosylation; or interpretation of the conformational change to proteins upon glycan binding.Major conclusionsIMS is a highly promising new analytical route, able to provide rapid isomeric separation (ms timescale) of either precursor or product ions facilitating MS characterization. This additional separation also enables the deconvolution of carbohydrate MS(/MS) information from contaminating ions, improving sensitivity and reducing chemical noise. Derivation of collision cross sections (CCS) from IM-MS(/MS) data and subsequent calculations validate putative structures of carbohydrates from ab initio derived candidates. IM-MS has demonstrated that amounts of specific glycan isomers vary between disease states, which would be challenging to detect using standard analytical approaches.General significanceIM-MS is a promising technique that fills an important gap within the Glycomics toolbox, namely identifying and differentiating the three-dimensional structure of chemically similar carbohydrates and glycoconjugates. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

A colorimetric microplate assay method for high throughput analysis of lipase activity

The present work describes a colorimetric microplate assay for lipase activity based on the reaction between 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) and the hydrolysis product of 2,3-dimercapto-1-propanol tributyrate (DMPTB). Reaction mixtures containing DTNB, DMPTB, and lipase were prepared in microplate wells, and the absorbance at 405nm was recorded after incubation at 37 degrees C for 30 min. A linear relationship was obtained in the range of 0.1-1 U of lipase activity by this method. The reaction conditions were also optimized for the range of 0.01-0.1 U or 1-10 U. When assaying crude tissue extracts, the reaction of DTNB with non-specific reducing agents created a major source of error. However, this error was corrected by the use of blank samples that did not contain DMPTB.     

Microfluidic techniques for high throughput single cell analysis

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A reproducible, high throughput method for fabricating fibrin gels

ABSTRACT: BACKGROUND: Fibrin gels are a promising biomaterial for tissue engineering. However, current fabrication methods are time intensive with inherent variation. There is a pressing need to develop new and consistent approaches for producing fibrin-based hydrogels for examination. RESULTS: We developed a high throughput method for creating fibrin gels using molds fabricated from polydimethylsiloxane (PDMS). Fibrin gels were produced by adding solutions of fibrinogen and thrombin to cylindrical defects in a PDMS sheet. Undisturbed gels were collected by removing the sheet, and fibrin gels were characterized. The characteristics of resulting gels were compared to published data by measuring compressive stiffness and osteogenic response of entrapped human mesenchymal stem cells (MSCs). Gels exhibited compressive moduli nearly identical to our previously reported fabrication method. Trends in alkaline phosphatase activity, an early marker of osteogenic differentiation in MSCs, were also consistent with previous data. CONCLUSIONS: These findings demonstrate a streamlined approach to fibrin gel production that drastically reduces the time required to make fibrin gels, while also reducing variability between gel batches. This fabrication technique provides a valuable tool for generating large numbers of gels in a cost-effective manner.     

A nonradioactive,high throughput assay for chitin synthase activity

Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.     

Structural genomics for Caenorhabditis elegans: high throughput protein expression analysis

The structural genomics initiatives have begun with the aim to create a so-called "basic set library" of protein folds that will be used to improve protein prediction methods. Such a library is thought to require the determination of up to 10,000 new structures, including representative structures of several sequence variants from each protein fold. To meet this goal in a reasonable time frame and cost, automated systems must be utilized to clone and to identify the soluble recombinant proteins contained in multiple genomes. This paper presents such a system, developed using the genome of Caenorhabditis elegans (19,099 genes) as a model eukaryotic organism for structural genomics. This system successfully automates nearly all aspects of recombinant protein expression analysis including subcloning, bacterial growth, recombinant protein expression, protein purification, and scoring protein solubility.