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Results for “reduced representation sequencing”

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PubMed2006

Assessment of representational difference analysis (RDA) to construct informative cDNA microarrays for gene expression analysis of species with limited transcriptome information, using red and green tomatoes as a model.

Microarray technology makes it feasible to analyse the expression of thousands of different gene elements in a single experiment. Most informative are 'whole genome' arrays, where all gene expression products of a single species or variety are represented. Such arrays are now available for a limited number of model species. However, for other, less well-documented species other routes are still necessary to obtain informative arrays. This includes the use of cDNA libraries. To enhance the amount of information that can be obtained from cDNA libraries, redundancy needs to be minimised, and the number of cDNAs relevant for the conditions of interest needs to be increased. Here, we used representational difference analysis (RDA), a mRNA subtraction procedure, as a tool to enhance the efficiency of cDNA libraries to be used to generate microarrays. Tomato was chosen as a model system for a less well-documented species. cDNA libraries for two distinct physiological conditions of tomato fruits, red and green, were made. The libraries were characterized by sequencing and hybridisation analysis. The RDA procedure was shown to be effective in selecting for genes of relevance for the physiological conditions under investigation, and against constitutively expressed genes. At the same time, redundancy was reduced, but complete normalisation was not obtained, and subsequent sequence analysis will be required to obtain non-redundant arrays. Further, known and putative ripening-related cDNAs were identified in hybridisation experiments on the basis of RNA populations as isolated from the green and red stage of ripening.

Fruit↗

PubMed2006

Core set approach to reduce uncertainty of gene trees.

BACKGROUND: A genealogy based on gene sequences within a species plays an essential role in the estimation of the character, structure, and evolutionary history of that species. Because intraspecific sequences are more closely related than interspecific ones, detailed information on the evolutionary process may be available by determining all the node sequences of trees and provide insight into functional constraints and adaptations. However, strong evolutionary correlations on a few lineages make this determination difficult as a whole, and the maximum parsimony (MP) method frequently allows a number of topologies with a same total branching length. RESULTS: Kitazoe et al. developed multidimensional vector-space representation of phylogeny. It converts additivity of evolutionary distances to orthogonality among the vectors expressing branches, and provides a unified index to measure deviations from the orthogoality. In this paper, this index is used to detect and exclude sequences with large deviations from orthogonality, and then selects a maximum subset ("core set") of sequences for which MP generates a single solution. Once the core set tree is formed whose all the node sequences are given, the excluded sequences are found to have basically two phylogenetic positions on this tree, respectively. Fortunately, since multiple substitutions are rare in intra-species sequences, the variance of nucleotide transitions is confined to a small range. By applying the core set approach to 38 partial env sequences of HIV-1 in a single patient and also 198 mitochondrial COI and COII DNA sequences of Anopheles dirus, we demonstrate how consistently this approach constructs the tree. CONCLUSION: In the HIV dataset, we confirmed that the obtained core set tree is the unique maximum set for which MP proposes a single tree. In the mosquito data set, the fluctuation of nucleotide transitions caused by the sequences excluded from the core set was very small. We reproduced this core-set tree by simulation based on random process, and applied our approach to many sets of the obtained endpoint sequences. Consequently, the ninety percent of the endpoint sequences was identified as the core sets and the obtained node sequences were perfectly identical to the true ones.

Animals↗

PubMed2004

Universal Reference RNA as a standard for microarray experiments.

BACKGROUND: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. RESULTS: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97). CONCLUSION: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.

Animals↗

PubMed1994

The topography of visual evoked response properties across the visual field.

Visual evoked potentials (VEPs) to luminance and pattern reversal stimulation were derived for a large number of small areas throughout the central visual field. In one study, the field was tested with a stimulus array consisting of 64 equal-area patches. Local response components were extracted by independent m-sequence modulation of the patches. Field topographies were compared between and within subjects using different electrode placements. The subject-dependent local variability observed in response characteristics is attributed to contributions from two or more cortical representations of the visual field and to inter-subject variations in gross cortical anatomy. The second study used luminance modulation of 56 patches across a 15 degrees field, scaled to activate approximately equal cortical areas in area V1. This produced many robust signals at all eccentricities. Bipolar and double differential ("1-dimensional Laplacian") signals were compared. The double differencing reduced contributions from distant or distributed sources, enhancing nearby current source activity, and greatly improved S/N for many stimulus locations. The high-resolution visual field maps demonstrated that clinical field testing using the VEP is not feasible because of effects of cortical convolutions on responses. However, the vast improvement in data quality and quantity make it a useful tool for VEP source localization and identification.

Brain↗

PubMed2005

Enhancing BOLD response in the auditory system by neurophysiologically tuned fMRI sequence.

Auditory neuroscience has not tapped fMRI's full potential because of acoustic scanner noise emitted by the gradient switches of conventional echoplanar fMRI sequences. The scanner noise is pulsed, and auditory cortex is particularly sensitive to pulsed sounds. Current fMRI approaches to avoid stimulus-noise interactions are temporally inefficient. Since the sustained BOLD response to pulsed sounds decreases with repetition rate and becomes minimal with unpulsed sounds, we developed an fMRI sequence emitting continuous rather than pulsed gradient sound by implementing a novel quasi-continuous gradient switch pattern. Compared to conventional fMRI, continuous-sound fMRI reduced auditory cortex BOLD baseline and increased BOLD amplitude with graded sound stimuli, short sound events, and sounds as complex as orchestra music with preserved temporal resolution. Response in subcortical auditory nuclei was enhanced, but not the response to light in visual cortex. Finally, tonotopic mapping using continuous-sound fMRI demonstrates that enhanced functional signal-to-noise in BOLD response translates into improved spatial separability of specific sound representations.

Acoustic Stimulation↗

PubMed2004

Age-structured population growth rates in constant and variable environments: a near equilibrium approach.

General measures summarizing the shapes of mortality and fecundity schedules are proposed. These measures are derived from moments of probability distributions related to mortality and fecundity schedules. Like moments, these measures form infinite sequences, but the first terms of these sequences are of particular value in approximating the long-term growth rate of an age- structured population that is growing slowly. Higher order terms are needed for approximating faster growing populations. These approximations offer a general nonparametric approach to the study of life-history evolution in both constant and variable environments. These techniques provide simple quantitative representations of the classical findings that, with fixed expected lifetime and net reproductive rate, type I mortality and early peak reproduction increase the absolute magnitude of the population growth rate, while type III mortality and delayed peak reproduction reduce this absolute magnitude.

Environment↗

PubMed2007

Establishing a liquid-phase IEF in combination with 2-DE for the analysis of Leishmania proteins.

The recent completion of genome sequencing projects for Leishmania major and near completion for two other species, L. infantum and L. braziliensis, has provided the needed genomic information for investigating the proteomes of Leishmania parasites. However, the design of effective 2-DE-based proteome mapping for complex protozoan parasites like Leishmania has proven to be severely compromised due to extensive overcrowding of spots especially in the acidic regions, coupled to a relatively low representation of basic proteins. In the present study, we optimized a liquid-phase IEF in combination with 2-DE for L. amazonensis promastigote as a way of reducing protein complexity and enhancing representation for low-abundance proteins on gels. Of 20 pH-based fractions eluted from Rotofor cells, 5 representative fractions selected from acidic, basic or neutral regions of the proteome and with adequate protein concentration were further analyzed by 2-DE using medium-range IPG strips. On this basis, we were able to generate high-resolution 2-DE maps encompassing both the acidic and basic ends of the proteome with enhanced spot representation.

Animals↗

PubMed2003

Normally functioning murF is essential for the optimal expression of methicillin resistance in Staphylococcus aureus.

A carboxy-terminal fragment of murF was used to construct and insert a suicide plasmid into the chromosomal copy of the gene in the highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain COL by Campbell type integration. The plasmid insertion generated a mutant in which the MIC value for oxacillin was reduced from 400 microg/ml of the parental strain to 0.75 microg/ml in 90% of the cells of the mutant cultures that were heterogeneous: they contained subpopulations of bacteria with a frequency of 10(-3) that were capable of expressing resistance at nearly the parental level. The impact of the murF mutation on antibiotic resistance was selective for beta-lactam antibiotics: there was no change in the susceptibility of the mutant to D-cycloserine, fosfomycin, beta-D-chloro-alanine, moenomycin, bacitracin, or vancomycin. Analysis of the mutant peptidoglycan showed decrease in the percentage of oligomeric components in rough proportion to the accumulation of several abnormal muropeptide components, which were identified as structural variants of the disaccharide tripeptide monomer. An abnormal cell wall precursor identified as UDP MurNac tripeptide was also detected in the cytoplasmic pool of the mutant strain. A normal proportion of oligomers and a greatly reduced representation of the disaccharide tripeptide were demonstrated in the cell wall of the murF mutant's subpopulation that has retained the parental level of resistance. Northern analysis demonstrated a drastic reduction in the transcription rate of mecA in mutant F9 whereas mecA transcription increased in the subpopulation of bacteria that retained high-level resistance.

Amino Acid Sequence↗

PubMed2003

Effects of recombination rate and gene density on transposable element distributions in Arabidopsis thaliana.

Transposable elements (TEs) comprise a major component of eukaryotic genomes, and exhibit striking deviations from random distribution across the genomes studied, including humans, flies, nematodes, and plants. Although considerable progress has been made in documenting these patterns, the causes are subject to debate. Here, we use the genome sequence of Arabidopsis thaliana to test for the importance of competing models of natural selection against TE insertions. We show that, despite TE accumulation near the centromeres, recombination does not generally correlate with TE abundance, suggesting that selection against ectopic recombination does not influence TE distribution in A. thaliana. In contrast, a consistent negative correlation between gene density and TE abundance, and a strong under-representation of TE insertions in introns suggest that selection against TE disruption of gene expression is playing a more important role in A. thaliana. High rates of self-fertilization may reduce the importance of recombination rate in genome structuring in inbreeding organisms such as A. thaliana and Caenorhabditis elegans.

Arabidopsis↗

PubMed2004

Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes.

Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.

Animals↗

PubMed2004

Algorithms for association study design using a generalized model of haplotype conservation.

There is considerable interest in computational methods to assist in the use of genetic polymorphism data for locating disease-related genes. Haplotypes, contiguous sets of correlated variants, may provide a means of reducing the difficulty of the data analysis problems involved. The field to date has been dominated by methods based on the "haplotype block" hypothesis, which assumes discrete population-wide boundaries between conserved genetic segments, but there is strong reason to believe that haplotype blocks do not fully capture true haplotype conservation patterns. In this paper, we address the computational challenges of using a more flexible, block-free representation of haplotype structure called the "haplotype motif" model for downstream analysis problems. We develop algorithms for htSNP selection and missing data inference using this more generalized model of sequence conservation. Application to a dataset from the literature demonstrates the practical value of these block-free methods.

Algorithms↗

PubMed2004

MR visualization of the inner ear structures: comparison of 1.5 Tesla and 3 Tesla images.

AIM OF THE STUDY: To compare high resolution MRI examinations of inner ear structures at 1.5 T and at 3 T. METHOD: Temporal bones were measured bilaterally in 3 healthy volunteers in a 1.5 T and in a 3 T MR-scanner using the respective one channel head coil (quadrature detection) of the manufacturer. The same steady-state gradient echo sequence (3D-CISS) was employed at a voxel size of 0.4 x 0.4 x 0.4 mm(3). The signal-to-noise ratio (SNR) was determined quantitatively. RESULTS: An SNR of 8 could be achieved for the measurements at 3 T in 7:37 min. The SNR at 3 T was, on average, a factor of 1.34 higher than that at 1.5 T despite the fact that the excitation angle had to be drastically reduced (alpha = 42 degrees instead of alpha = 70 degrees at 1.5 T) due to the limit of the specific absorption rate (SAR). DISCUSSION: The MR representation of the inner ear is clearly improved at 3 T. To obtain the same SNR at 1.5 T approximately the double measuring time would be required, connected with reduced patient comfort and an increased risk for a displacement of the head during the high resolution measurement.

Cochlea↗

PubMed2026

A token-pruning framework enables efficient representation of the human genome for RNA modification analysis.

MOTIVATION: Modelling long genomic sequences remains challenging due to extreme sequence length, high redundancy, and the need for biological interpretability. Although Transformer-based architectures have achieved strong performance across genomic tasks, their high computational cost and reliance on fixed tokenization strategies limit their scalability and ability to focus on biologically informative regions. RESULTS: We propose ATSFormer, a token-pruning Transformer framework for efficient and biologically informed genomic sequence modelling. ATSFormer incorporates an attention-guided and parameter-free Adaptive Token Sampling (ATS) module into Transformer layers. Guided by attention-derived importance scores, ATS dynamically retains informative tokens while probabilistically discarding redundant ones, thereby reducing sequence length, FLOPs, and memory usage without introducing additional learnable parameters or extra training procedures. Importantly, the retained tokens correspond to key contributors to model predictions, enabling ATSFormer to highlight biologically meaningful sites and sequence motifs. We evaluated ATSFormer on four benchmark RNA modification datasets derived from RMVar 2.0, covering A-to-I, m1A, m5C, and m7G. Experimental results show that ATSFormer consistently outperforms existing state-of-the-art methods while achieving substantial computational savings. Furthermore, structural analysis using AlphaFold3 supports the biological relevance of the motifs identified by ATSFormer. AVAILABILITY AND IMPLEMENTATION: The source data and code are freely available at GitHub (https://github.com/1gao2/ATSFormer) and Zenodo (https://doi.org/10.5281/zenodo.21813541).

Humans↗

PubMed2006

A dimensionless number analysis of the hybridization process in diffusion- and convection-driven DNA microarray systems.

The present theoretical analysis aims at providing a general understanding of the combined effect the many different process variables have on the hybridization rate in diffusion- and convection-driven DNA microarray systems. It is shown that all process variables can be grouped into only four different dimensionless numbers (the Damkohler number Da, the dimensionless association constant kappa(A), the dimensionless initial concentration C'(0) and a geometrical ratio alpha). These four numbers have a straightforward physical meaning and only contain easily measurable parameters. Reducing the solution space from 7D to 4D, the dimensionless number representation greatly facilitates the insight in the conditions leading to the occurrence of diffusion-limited hybridization rates in both diffusion- and convection-driven DNA microarray systems. This in turn simplifies their design and the interpretation of the experimental results that are obtained with these systems.

Diffusion↗

PubMed1999

Control of Expressive and Metronomic Timing in Pianists.

In 12 tasks, each including 10 repetitions, 6 skilled pianists performed or responded to a musical excerpt. In the first 6 tasks, expressive timing was required; in the last 6 tasks, metronomic timing. The pianists first played the music on a digital piano (Tasks 1 and 7), then played it without auditory feedback (Tasks 2 and 8), then tapped on a response key in synchrony with one of their own performances (Tasks 3 and 9), with an imagined performance (Tasks 4 and 10), with a computer-generated performance (Tasks 5 and 11), and with a computer-generated sequence of clicks (Tasks 6 and 12). The results demonstrated that pianists are capable of generating the expressive timing pattern of their performance in the absence of auditory and kinaesthetic (piano keyboard) feedback. They can also synchronize their finger taps quite well with expressively timed music or clicks (while imagining the music), although they tend to underestimate long interonset intervals and to compensate on the following tap. Expressive timing is thus shown to be generated from an internal representation of the music. In metronomic performance, residual expressive timing effects were evident. Those did not depend on auditory feedback, but they were much reduced or absent when kinaesthetic feedback from the piano keyboard was eliminated. Thus, they seemed to arise from the pianist's physical interaction with the instrument. Systematic timing patterns related to expressive timing were also observed in synchronization with a metronomic computer performance and even in synchronization with metronomic clicks. These results shed light on intentional and unintentional, structurally governed processes of timing control in music performance.

Journal Article↗

PubMed2002

Cardiac effects of hypoxia in the neotenous tiger salamander Ambystoma tigrinum.

The aquatic form of the tiger salamander Ambystoma tigrinum lives in high-altitude ponds and is exposed to a hypoxic environment that may be either chronic or intermittent. In many animal species, exposure to hypoxia stimulates cardiac output and is followed by an increase in cardiac mass. The working hypothesis of the present study was that the hearts of these aquatic salamanders exposed to 10-14 days of 5 % oxygen in a laboratory setting would become larger and would differentially express proteins that would help confer tolerance to hypoxia. During exposure to hypoxia, cardiac output increased, as did hematocrit. Cardiac mass also increased, but mitotic figures were not detected in the cardiac myocytes of colchicine-injected animals. The mass increase was probably due to hypertrophy, although a very slow rate of hyperplasia cannot be ruled out. Representational difference analysis indicated that at least 14 mRNAs were expressed in hearts from the hypoxic animals that were not expressed in hearts from normoxic animals. The differentially expressed genes were cloned and sequenced and confirmed as coming from the ventricles of the hypoxic salamanders. Genes differentially expressed include mitochondrial genes and genes for elongation factor 2, a protein synthesis gene. The mechanical performance of buffer-perfused hearts isolated from normoxic and hypoxic animals did not differ. Acute responses to hypoxia were also measured. The rate of oxygen consumption of unanesthetized salamanders in metabolism chambers decreased when chamber oxygen concentration was reduced below 12 % oxygen. At a chamber oxygen concentration of 4-6 %, the rate of oxygen consumption of the salamanders was reduced to approximately one-third of the normoxic rate.

Altitude↗

PubMed1998

A shared system for learning serial and temporal structure of sensori-motor sequences? Evidence from simulation and human experiments.

This research investigates the influences of temporal structure on the representation of serial order. Experiments are performed in a neural network model of sequence learning and in human subjects. In the sequence learning model, a recurrent network of leaky integrator neurons encodes a succession of internal states that become associated, by reinforcement learning, with the correct sequential responses. First, the model is shown to learn a simple temporal discrimination task. The model is then exposed to two novel serial reaction time (SRT) experiments. In the standard SRT task (M.J. Nissen, P. Bullemer, Attentional requirements of learning: evidence from performance measures, Cogn. Psychol. 19 (1987) 1-32 [16]), reaction times for stimuli presented in a repeating sequence are reduced with respect to those for random stimuli, providing a measure of sequence learning. The novelty of the current experiments is that imbedded in the serial order of the sequences, there is a temporal structure of delays. The model is sensitive to both the serial structure and the temporal structure of the sequences. This observation is then confirmed in human subjects. These results demonstrate how a novel recurrent architecture encodes the interaction of temporal and serial structure and provide insight into related aspects of human sensori-motor sequence learning.

Analysis of Variance↗

PubMed1989

The zinc finger region of simian virus 40 large T antigen.

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence Cys-302LeuLysCys-305IleLysLysGluGlnProSerHisTyrLysTyrHis- 317GluLysHis-320. In a conventional representation, the binding of zinc to the cysteines and histidines at positions 302, 305, 317, and 320 would form two minor loops and one major loop from the intervening amino acids. We made single amino acid substitutions at every position in the finger to identify possible functional elements within the putative metal-binding domain. Amino acids in the zinc finger could be divided into three classes characterized by distinct roles in DNA replication and transformation. Class 1 consisted of amino acids in the two minor loops of the finger and in the amino-terminal part of the major loop. Mutations here did not affect either replication or transformation. Class 2 consisted of the SerHisTyrLysTyr amino acids located in the carboxy terminus of the major loop of the finger. Mutations in this contiguous region reduced replication of the mutant viruses to different degrees. This clustering suggested that the region is an active site important for a specific function in DNA replication. With the exception of a mutation in the histidine at position 313, these mutations had no effect on transformation. Class 3 consisted of the proposed zinc-binding amino acids at positions 302, 305, 317, and 320 and the histidine at position 313 in the major loop of the finger. Mutations in these amino acids abolished the viability of the virus completely and had a distinctive effect on the transforming functions of the protein. Thus, the five cysteines and histidines of class 3 may play an important role in determining the overall structure of the protein. The histidine at position 313 may function both in the active site where it is located and in cooperation with the proposed zinc-binding ligands.

Amino Acid Sequence↗