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Results for “Massively parallel sequencing”

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At least 145 records · Page 8Linked to original sources
PubMed1993

Cellular automaton simulation of pulsed field gel electrophoresis.

We describe simulation techniques well suited to detailed investigation of the microscopic behavior of DNA during electrophoretic separation in the diffusive regime. Long polymers moving diffusively in a medium are simulated using microscopic Monte-Carlo steps. Simulations rely upon a recently introduced two-space abstract polymer that enables fine-grained massively parallel simulation. Tests of the two-space polymer dynamics are reviewed. The scaling with polymer length of the size and relaxation time of isolated polymers are shown to agree with universal scaling relations. The relaxation time is found to be significantly faster than the alternative bond-fluctuation method. Simplicity of implementation enables simulation on cellular automaton machines (CAM) including CAM-6, and a prototype of the new CAM-8, as well as other massively parallel architectures. Preliminary simulations of polymers migrating under an external field through a random medium of obstacles in two dimensions are described. Two sequences of simulations are performed, with different obstacle densities corresponding to pore sizes larger and smaller than the polymer radius of gyration. In the dilute medium polymers are characteristically draped on single obstacles. In the denser medium draping across multiple obstacles results in reduced orientation in the field direction. A demonstration of rapid 90 degrees field direction switching results in polymer motion toward the expected intermediate direction.

Algorithms↗
PubMed2000

Acute onset of type I diabetes mellitus after severe echovirus 9 infection: putative pathogenic pathways.

Enterovirus infections have been implicated in the development of type I diabetes mellitus. They may cause beta cell destruction either by cytolytic infection in the pancreas or indirectly by contributing to autoimmune reactivity. We sought evidence for these 2 mechanisms in a case of acute-onset diabetes mellitus that occurred during severe echovirus 9 infection. The virus was isolated and administered to cultured human beta cells. No viral proliferation was observed, and no beta cell death was induced, while parallel exposure to Coxsackie B virus serotype 3 resulted in viral proliferation and massive beta cell death. Although the viral protein 2C exhibited a sequence similar to that of the beta cell autoantigen glutamic acid decarboxylase (GAD(65)), no cross-reactive T cell responses were detected. The patient did not develop antibodies to GAD(65) either. Absence of evidence for direct cytolytic action or an indirect effect through molecular mimicry with GAD(65) in the present case raises the possibility of another indirect pathway through which enteroviruses can cause diabetes mellitus.

Acute Disease↗
PubMed1998

Microarrays: biotechnology's discovery platform for functional genomics.

Advances in microarray technology enable massive parallel mining of biological data, with biological chips providing hybridization-based expression monitoring, polymorphism detection and genotyping on a genomic scale. Microarrays containing sequences representative of all human genes may soon permit the expression analysis of the entire human genome in a single reaction. These 'genome chips' will provide unprecedented access to key areas of human health, including disease prognosis and diagnosis, drug discovery, toxicology, aging, and mental illness. Microarray technology is rapidly becoming a central platform for functional genomics.

Biotechnology↗
PubMed2002

Computational methods for the prediction of protein interactions.

Establishing protein interaction networks is crucial for understanding cellular operations. Detailed knowledge of the 'interactome', the full network of protein-protein interactions, in model cellular systems should provide new insights into the structure and properties of these systems. Parallel to the first massive application of experimental techniques to the determination of protein interaction networks and protein complexes, the first computational methods, based on sequence and genomic information, have emerged.

Artificial Gene Fusion↗
PubMed2005

A genome-wide scalable SNP genotyping assay using microarray technology.

Oligonucleotide probe arrays have enabled massively parallel analysis of gene expression levels from a single cDNA sample. Application of microarray technology to analyzing genomic DNA has been stymied by the sequence complexity of the entire human genome. A robust, single base-resolution direct genomic assay would extend the reach of microarray technology. We developed an array-based whole-genome genotyping assay that does not require PCR and enables effectively unlimited multiplexing. The assay achieves a high signal-to-noise ratio by combining specific hybridization of picomolar concentrations of whole genome-amplified DNA to arrayed probes with allele-specific primer extension and signal amplification. As proof of principle, we genotyped several hundred previously characterized SNPs. The conversion rate, call rate and accuracy were comparable to those of high-performance PCR-based genotyping assays.

Computational Biology↗
PubMed1999

Structural genomics: beyond the human genome project.

With access to whole genome sequences for various organisms and imminent completion of the Human Genome Project, the entire process of discovery in molecular and cellular biology is poised to change. Massively parallel measurement strategies promise to revolutionize how we study and ultimately understand the complex biochemical circuitry responsible for controlling normal development, physiologic homeostasis and disease processes. This information explosion is also providing the foundation for an important new initiative in structural biology. We are about to embark on a program of high-throughput X-ray crystallography aimed at developing a comprehensive mechanistic understanding of normal and abnormal human and microbial physiology at the molecular level. We present the rationale for creation of a structural genomics initiative, recount the efforts of ongoing structural genomics pilot studies, and detail the lofty goals, technical challenges and pitfalls facing structural biologists.

Computational Biology↗
PubMed1994

High speed pattern matching in genetic data base with reconfigurable hardware.

Homology detection in large data bases is probably the most time consuming operation in molecular genetic computing systems. Moreover, the progresses made all around the world concerning the mapping and sequencing of the genome of Homo Sapiens and other species have increased the size of data bases exponentially. Therefore even the best workstation would not be able to reach the scanning speed required. In order to answer this need we propose an algorithm, A2R2, and its implementation on a massively parallel system. Basically, two kinds of algorithms are used to search in molecular genetic data bases. The first kind is based on dynamic programming and the second on word processing, A2R2 belongs to the second kind. The structure of the motif (pattern) searched by A2R2 can support those from FAST, BLAST and FLASH algorithms. After a short presentation of the reconfigurable hardware concept and technology used in our massively parallel accelerator we present the A2R2 implementation. This parallel implementation outperforms any kind of previously published genetic data base scanning hardware or algorithms. We report up to 25 million nucleotides per scanning seconds as our best results.

Algorithms↗
PubMed1999

Acinar-ductal-carcinoma sequence in transforming growth factor-alpha transgenic mice.

Transgenic mice overexpressing transforming growth factor-alpha (TGF-alpha) display an expansion of intrapancreatic fibroblasts and a progressive accumulation of extracellular matrix. This massive fibrosis is associated with an increase in pancreatic size and weight. In parallel, tubular complexes appear that are composed of acinar cells with a decreased height. These acinar cell lose zymogen granules and become transitional cells, which subsequently gain duct cell features. In animals older than one year dysplastic lesions develop, which originate from tubular complexes. Occasionally these dysplastic foci transform to papillary and cystic pancreatic carcinoma. These tumors are positive for the duct-specific antigen Duct-1 and carbonic anhydrase activity indicative of ductal differentiation. Tumors overexpress the epidermal growth factor (EGF)-receptor and p53, but lack K-ras mutations. These data suggest an acinar-ductal-carcinoma sequence in TGF-alpha transgenic mice.

3T3 Cells↗
PubMed2002

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology.

An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA-protein fusion to create an addressable protein microarray that self-assembles via hybridization to surface-bound DNA capture probes. The nucleic acid component not only directs the mRNA-protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA-protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub-attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.

DNA Primers↗
PubMed1991

Efficient detection of three-dimensional structural motifs in biological macromolecules by computer vision techniques.

Macromolecules carrying biological information often consist of independent modules containing recurring structural motifs. Detection of a specific structural motif within a protein (or DNA) aids in elucidating the role played by the protein (DNA element) and the mechanism of its operation. The number of crystallographically known structures at high resolution is increasing very rapidly. Yet, comparison of three-dimensional structures is a laborious time-consuming procedure that typically requires a manual phase. To date, there is no fast automated procedure for structural comparisons. We present an efficient O(n3) worst case time complexity algorithm for achieving such a goal (where n is the number of atoms in the examined structure). The method is truly three-dimensional, sequence-order-independent, and thus insensitive to gaps, insertions, or deletions. This algorithm is based on the geometric hashing paradigm, which was originally developed for object recognition problems in computer vision. It introduces an indexing approach based on transformation invariant representations and is especially geared toward efficient recognition of partial structures in rigid objects belonging to large data bases. This algorithm is suitable for quick scanning of structural data bases and will detect a recurring structural motif that is a priori unknown. The algorithm uses protein (or DNA) structures, atomic labels, and their three-dimensional coordinates. Additional information pertaining to the structure speeds the comparisons. The algorithm is straightforwardly parallelizable, and several versions of it for computer vision applications have been implemented on the massively parallel connection machine. A prototype version of the algorithm has been implemented and applied to the detection of substructures in proteins.

Algorithms↗
PubMed2001

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips.

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T(m)) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T(m). The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

Bacterial Proteins↗
PubMed2003

Primer-design for multiplexed genotyping.

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Mutation analysis by polymerase-mediated single-base primer extension (minisequencing) can be massively parallelized using DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5' end of the minisequencing primer and attaching the complementary antitag to the array or bead surface, the assay can be 'demultiplexed'. Such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. We present a computer program to automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/antitag system is generated, and the pairing of primers and DNA tags is automatically done in a way to avoid any crossreactivity. We report results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping. The software is available to academic users on request.

Algorithms↗
PubMed2006

High-density haplotyping with microarray-based expression and single feature polymorphism markers in Arabidopsis.

Expression microarrays hybridized with RNA can simultaneously provide both phenotypic (gene expression) and genotypic (marker) data. We developed two types of genetic markers from Affymetrix GeneChip expression data to generate detailed haplotypes for 148 recombinant inbred lines (RILs) derived from Arabidopsis thaliana accessions Bayreuth and Shahdara. Gene expression markers (GEMs) are based on differences in transcript levels that exhibit bimodal distributions in segregating progeny, while single feature polymorphism (SFP) markers rely on differences in hybridization to individual oligonucleotide probes. Unlike SFPs, GEMs can be derived from any type of DNA-based expression microarray. Our method identifies SFPs independent of a gene's expression level. Alleles for each GEM and SFP marker were ascertained with GeneChip data from parental accessions as well as RILs; a novel algorithm for allele determination using RIL distributions capitalized on the high level of genetic replication per locus. GEMs and SFP markers provided robust markers in 187 and 968 genes, respectively, which allowed estimation of gene order consistent with that predicted from the Col-0 genomic sequence. Using microarrays on a population to simultaneously measure gene expression variation and obtain genotypic data for a linkage map will facilitate expression QTL analyses without the need for separate genotyping. We have demonstrated that gene expression measurements from microarrays can be leveraged to identify polymorphisms across the genome and can be efficiently developed into genetic markers that are verifiable in a large segregating RIL population. Both marker types also offer opportunities for massively parallel mapping in unsequenced and less studied species.

Arabidopsis↗
PubMed2026

Deciphering the prodrome of inflammatory bowel disease up to 10 years before disease onset by massively parallel serology.

BACKGROUND: Defining immune dysregulation during the asymptomatic prodrome of immune-mediated diseases offers opportunities for early disease detection and interception. In inflammatory bowel disease (IBD), prodromal immune changes remain poorly characterised. OBJECTIVE: To define preclinical immunological alterations by characterising longitudinal serum antibody repertoires using high-throughput phage-display immunoprecipitation sequencing (PhIP-Seq). DESIGN: We applied PhIP-Seq to profile antibody responses in 2000 longitudinal serum samples from 200 individuals who developed Crohn's disease (CD), 200 who developed ulcerative colitis (UC) and 100 matched healthy controls within the US military Proteomic Evaluation and Discovery in an IBD Cohort of Tri-service Subjects cohort, collected up to 10 years before diagnosis. Antibody repertoires were profiled against 357 000 microbial-associated, viral-associated, food-associated and immune-associated peptides. RESULTS: Antibody repertoire variability was increased up to ~4 years prediagnosis in pre-CD and pre-UC individuals. Differential analyses revealed elevated herpesvirus-directed responses (notably Epstein-Barr virus) and anti-flagellin antibodies up to 10 years prediagnosis in CD, particularly in individuals who later developed complicated or ileal disease. In contrast, responses to encapsulated bacteria (eg, Streptococcus pneumoniae, Haemophilus, Neisseria) progressively declined towards diagnosis. Pre-UC was characterised by combined antimicrobial, antiviral and autoantibody signatures, including antibodies against the MAP kinase-activating death domain protein. CONCLUSIONS: Large-scale serological profiling of archived prediagnostic samples identified disease-specific immune trajectories years before IBD onset, providing novel insights into disease pathogenesis in its prodromal phase.

ANTIGENS↗
PubMed2001

DNA microarrays in neuropsychopharmacology.

Recent advances in experimental genomics, coupled with the wealth of sequence information available for a variety of organisms, have the potential to transform the way pharmacological research is performed. At present, high-density DNA microarrays allow researchers to quickly and accurately quantify gene-expression changes in a massively parallel manner. Although now well established in other biomedical fields, such as cancer and genetics research, DNA microarrays have only recently begun to make significant inroads into pharmacology. To date, the major focus in this field has been on the general application of DNA microarrays to toxicology and drug discovery and design. This review summarizes the major microarray findings of relevance to neuropsychopharmacology, as a prelude to the design and analysis of future basic and clinical microarray experiments. The ability of DNA microarrays to monitor gene expression simultaneously in a large-scale format is helping to usher in a post-genomic age, where simple constructs about the role of nature versus nurture are being replaced by a functional understanding of gene expression in living organisms.

Aging↗
PubMed2003

Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening.

The generation of the draft human genome sequence has created new possibilities for diagnosis, prevention, and treatment of human disease. One consequence of these new possibilities is an increasing need for methods and technology that can be used for high-throughput screening for mutations in large DNA sample materials. In recent years, a number of mutation screening methods have emerged that are based on the analysis of sequence-dependent changes in the conformation of single- and double-stranded DNA using capillary electrophoresis. Common features of these methods are high sensitivity and reproducibility as well as the possibility for automation and massive parallelization. Thus, at present they are among the most attractive technologies for high-throughput mutation screening. This review describes the recent advances in capillary electrophoresis-based single strand conformation polymorphism (CE-SSCP) for detection of unknown mutations, and assesses its practical usability for high-throughput mutation screening based on the available literature. In addition, future prospects are outlined in light of the recent advances in microchip-based capillary electrophoresis.

DNA Mutational Analysis↗
PubMedDate not supplied

Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH).

This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2).

Bacteria↗
PubMed2002

Restriction landmark genome scanning.

Restriction landmark genome scanning (RLGS) is a quantitative approach that is uniquely suited for simultaneously assessing the methylation status of thousands of CpG islands. RLGS separates radiolabeled NotI fragments (or other CpG-containing restriction enzyme fragments) in two dimensions and allows distinction of single-copy CpG islands from multicopy CpG-rich sequences. The methylation sensitivity of the endonuclease activity of NotI provides the basis for differential methylation analysis, and NotI sites occur primarily in CpG islands and genes. RLGS has been used to identify novel imprinted genes, novel targets of DNA amplification and methylation in human cancer, and to identify deletion, methylation, and gene amplification in a mouse model of tumorigenesis. Such massively parallel analyses are critical for pattern recognition within and between tumor types, and for estimating the overall influence of CpG island methylation on the cancer cell genome. RLGS is also a useful method for integrating methylation analyses with high-resolution gene copy number analyses.

Brain Neoplasms↗