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1.
Article in English | MEDLINE | ID: mdl-30917985

ABSTRACT

We developed a rapid high-throughput PCR test and evaluated highly antibiotic-resistant clinical isolates of Escherichia coli (n = 2,919), Klebsiella pneumoniae (n = 1,974), Proteus mirabilis (n = 1,150), and Pseudomonas aeruginosa (n = 1,484) for several antibiotic resistance genes for comparison with phenotypic resistance across penicillins, cephalosporins, carbapenems, aminoglycosides, trimethoprim-sulfamethoxazole, fluoroquinolones, and macrolides. The isolates originated from hospitals in North America (34%), Europe (23%), Asia (13%), South America (12%), Africa (7%), or Oceania (1%) or were of unknown origin (9%). We developed statistical methods to predict phenotypic resistance from resistance genes for 49 antibiotic-organism combinations, including gentamicin, tobramycin, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, ertapenem, imipenem, cefazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, ampicillin, and aztreonam. Average positive predictive values for genotypic prediction of phenotypic resistance were 91% for E. coli, 93% for K. pneumoniae, 87% for P. mirabilis, and 92% for P. aeruginosa across the various antibiotics for this highly resistant cohort of bacterial isolates.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Microbial/genetics , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/genetics , Gram-Negative Bacterial Infections/drug therapy , Africa , Asia , Cross Infection/drug therapy , Cross Infection/microbiology , Europe , Gram-Negative Bacterial Infections/microbiology , Humans , North America , Polymerase Chain Reaction/methods , South America
2.
Genomics ; 96(5): 290-302, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20654709

ABSTRACT

Here we report the use of a multi-genome DNA microarray to elucidate the genomic events associated with the emergence of the clonal variants of Haemophilus influenzae biogroup aegyptius causing Brazilian Purpuric Fever (BPF), an important pediatric disease with a high mortality rate. We performed directed genome sequencing of strain HK1212 unique loci to construct a species DNA microarray. Comparative genome hybridization using this microarray enabled us to determine and compare gene complements, and infer reliable phylogenomic relationships among members of the species. The higher genomic variability observed in the genomes of BPF-related strains (clones) and their close relatives may be characterized by significant gene flux related to a subset of functional role categories. We found that the acquisition of a large number of virulence determinants featuring numerous cell membrane proteins coupled to the loss of genes involved in transport, central biosynthetic pathways and in particular, energy production pathways to be characteristics of the BPF genomic variants.


Subject(s)
Fever/microbiology , Genetic Variation , Genome, Bacterial , Haemophilus influenzae/classification , Phylogeny , Purpura/microbiology , Bacterial Proteins/genetics , Brazil , Comparative Genomic Hybridization , Haemophilus Infections/microbiology , Haemophilus influenzae/genetics , Haemophilus influenzae/pathogenicity , Humans , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Sequence Analysis, DNA , Virulence Factors/genetics
3.
BMC Bioinformatics ; 9: 529, 2008 Dec 09.
Article in English | MEDLINE | ID: mdl-19068132

ABSTRACT

BACKGROUND: Mass spectrometry (MS) based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS/MS) data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS/MS instrument. Peptide spectral counting techniques attempt to quantify protein abundance by counting the number of detected tryptic peptides and their corresponding MS spectra. However, spectral counting is confounded by the fact that peptide physicochemical properties severely affect MS detection resulting in each peptide having a different detection probability. Lu et al. (2007) described a modified spectral counting technique, Absolute Protein Expression (APEX), which improves on basic spectral counting methods by including a correction factor for each protein (called Oi value) that accounts for variable peptide detection by MS techniques. The technique uses machine learning classification to derive peptide detection probabilities that are used to predict the number of tryptic peptides expected to be detected for one molecule of a particular protein (Oi). This predicted spectral count is compared to the protein's observed MS total spectral count during APEX computation of protein abundances. RESULTS: The APEX Quantitative Proteomics Tool, introduced here, is a free open source Java application that supports the APEX protein quantitation technique. The APEX tool uses data from standard tandem mass spectrometry proteomics experiments and provides computational support for APEX protein abundance quantitation through a set of graphical user interfaces that partition th parameter controls for the various processing tasks. The tool also provides a Z-score analysis for identification of significant differential protein expression, a utility to assess APEX classifier performance via cross validation, and a utility to merge multiple APEX results into a standardized format in preparation for further statistical analysis. CONCLUSION: The APEX Quantitative Proteomics Tool provides a simple means to quickly derive hundreds to thousands of protein abundance values from standard liquid chromatography-tandem mass spectrometry proteomics datasets. The APEX tool provides a straightforward intuitive interface design overlaying a highly customizable computational workflow to produce protein abundance values from LC-MS/MS datasets.


Subject(s)
Chromatography, Liquid , Proteome/analysis , Proteomics/methods , Software , Tandem Mass Spectrometry , Databases, Protein , Sequence Analysis, Protein/methods , User-Computer Interface
4.
Methods Mol Biol ; 353: 265-300, 2007.
Article in English | MEDLINE | ID: mdl-17332646

ABSTRACT

Gene expression microarrays are being used widely to address a myriad of complex biological questions. To gather meaningful expression data, it is crucial to have a firm understanding of the steps involved in the application of microarrays. The available microarray platforms are discussed along with their advantages and disadvantages. Additional considerations include study design, quality control and systematic assessment of microarray performance, RNA-labeling strategies, sample allocation, signal amplification schemes, defining the number of appropriate biological replicates, data normalization, statistical approaches to identify differentially regulated genes, and clustering algorithms for data visualization. In this chapter, the underlying principles regarding microarrays are reviewed, to serve as a guide when navigating through this powerful technology.


Subject(s)
Oligonucleotide Array Sequence Analysis/methods , Animals , Cluster Analysis , DNA Probes , Fluorescent Dyes , Gene Expression Profiling , Humans , Nucleic Acid Amplification Techniques , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Polymerase Chain Reaction , RNA Probes
5.
Methods Enzymol ; 411: 134-93, 2006.
Article in English | MEDLINE | ID: mdl-16939790

ABSTRACT

Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data management and reporting, image analysis, normalization and pipeline control, and data mining and visualization. An integrated MIAME-compliant MySQL database is included. This chapter describes each component of the suite and includes a sample analysis walk-through.


Subject(s)
Oligonucleotide Array Sequence Analysis/methods , Software , Algorithms , Animals , Gene Expression Profiling/methods , Gene Expression Profiling/statistics & numerical data , Humans , Oligonucleotide Array Sequence Analysis/statistics & numerical data
6.
Nat Genet ; 38(2): 234-9, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16415889

ABSTRACT

Cardiovascular disorders are influenced by genetic and environmental factors. The TIGR rodent expression web-based resource (TREX) contains over 2,200 microarray hybridizations, involving over 800 animals from 18 different rat strains. These strains comprise genetically diverse parental animals and a panel of chromosomal substitution strains derived by introgressing individual chromosomes from normotensive Brown Norway (BN/NHsdMcwi) rats into the background of Dahl salt sensitive (SS/JrHsdMcwi) rats. The profiles document gene-expression changes in both genders, four tissues (heart, lung, liver, kidney) and two environmental conditions (normoxia, hypoxia). This translates into almost 400 high-quality direct comparisons (not including replicates) and over 100,000 pairwise comparisons. As each individual chromosomal substitution strain represents on average less than a 5% change from the parental genome, consomic strains provide a useful mechanism to dissect complex traits and identify causative genes. We performed a variety of data-mining manipulations on the profiles and used complementary physiological data from the PhysGen resource to demonstrate how TREX can be used by the cardiovascular community for hypothesis generation.


Subject(s)
Databases, Genetic , Disease Models, Animal , Genomics , Heart Diseases/genetics , Hematologic Diseases/genetics , Lung Diseases/genetics , Animals , Gene Expression Profiling , Genetic Variation , Genomics/methods , Heart Diseases/physiopathology , Hematologic Diseases/physiopathology , Hypoxia/chemically induced , Internet , Lung Diseases/physiopathology , Male , Microarray Analysis , Myocardium/metabolism , Rats , Rats, Inbred BN , Rats, Inbred Dahl , Regulatory Sequences, Nucleic Acid/genetics
7.
Bioinformatics ; 21(15): 3308-11, 2005 Aug 01.
Article in English | MEDLINE | ID: mdl-15905276

ABSTRACT

SUMMARY: This synopsis provides an overview of array-based comparative genomic hybridization data display, abstraction and analysis using CGHAnalyzer, a software suite, designed specifically for this purpose. CGHAnalyzer can be used to simultaneously load copy number data from multiple platforms, query and describe large, heterogeneous datasets and export results. Additionally, CGHAnalyzer employs a host of algorithms for microarray analysis that include hierarchical clustering and class differentiation. AVAILABILITY: CGHAnalyzer, the accompanying manual, documentation and sample data are available for download at http://acgh.afcri.upenn.edu. This is a Java-based application built in the framework of the TIGR MeV that can run on Microsoft Windows, Macintosh OSX and a variety of Unix-based platforms. It requires the installation of the free Java Runtime Environment 1.4.1 (or more recent) (http://www.java.sun.com).


Subject(s)
Algorithms , Chromosome Mapping/methods , DNA Mutational Analysis/methods , Gene Dosage , Neoplasms/genetics , Oligonucleotide Array Sequence Analysis/methods , Software , User-Computer Interface , Animals , Computer Graphics , Humans
8.
Genome Biol ; 3(11): research0062, 2002 Oct 24.
Article in English | MEDLINE | ID: mdl-12429061

ABSTRACT

BACKGROUND: 'Fold-change' cutoffs have been widely used in microarray assays to identify genes that are differentially expressed between query and reference samples. More accurate measures of differential expression and effective data-normalization strategies are required to identify high-confidence sets of genes with biologically meaningful changes in transcription. Further, the analysis of a large number of expression profiles is facilitated by a common reference sample, the construction of which must be carefully addressed. RESULTS: We carried out a series of 'self-self' hybridizations in which aliquots of the same RNA sample were labeled separately with Cy3 and Cy5 fluorescent dyes and co-hybridized to the same microarray. From this, we can analyze the intensity-dependent behavior of microarray data, define a statistically significant measure of differential expression that exploits the structure of the fluorescent signals, and measure the inherent reproducibility of the technique. We also devised a simple procedure for identifying and eliminating low-quality data for replicates within and between slides. We examine the properties required of a universal reference RNA sample and show how pooling a small number of samples with a diverse representation of expressed genes can outperform more complex mixtures as a reference sample. CONCLUSION: Analysis of cell-line samples can identify systematic structure in measured gene-expression levels. A general procedure for analyzing cDNA microarray data is proposed and validated. We show that pooled reference samples should be based not only on the expression of individual genes in each cell line but also on the expression levels of genes within cell lines.


Subject(s)
Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis/methods , Brain Neoplasms/genetics , Carcinoma/genetics , Colonic Neoplasms/genetics , DNA, Complementary/analysis , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Male , Nucleic Acid Hybridization/methods , Organ Specificity/genetics , Ovarian Neoplasms/genetics , Pancreatic Neoplasms/genetics , RNA, Neoplasm/analysis , Reference Values , Testicular Neoplasms/genetics , Tumor Cells, Cultured
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