13,944 research outputs found
Accurate, precise modeling of cell proliferation kinetics from time-lapse imaging and automated image analysis of agar yeast culture arrays
BACKGROUND: Genome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images. RESULTS: YeastXtract software was developed for kinetic growth curve analysis of spotted agar cultures. The accuracy and precision for image analysis of agar culture arrays was comparable to OD measurements of liquid cultures. Using YeastXtract, image intensity vs. biomass of spot cultures was linearly correlated over two orders of magnitude. Thus cell proliferation could be measured over about seven generations, including four to five generations of relatively constant exponential phase growth. Spot area normalization reduced the variation in measurements of total growth efficiency. A growth model, based on the logistic function, increased precision and accuracy of maximum specific rate measurements, compared to empirical methods. The logistic function model was also more robust against data sparseness, meaning that less data was required to obtain accurate, precise, quantitative growth phenotypes. CONCLUSION: Microbial cultures spotted onto agar media are widely used for genotype-phenotype analysis, however quantitative HTCP methods capable of measuring kinetic growth rates have not been available previously. YeastXtract provides objective, automated, quantitative, image analysis of agar cell culture arrays. Fitting the resulting data to a logistic equation-based growth model yields robust, accurate growth rate information. These methods allow the incorporation of imaging and automated image analysis of cell arrays, grown on solid agar media, into HTCP-driven experimental approaches, such as global, quantitative analysis of gene interaction networks
Microarray sub-grid detection: A novel algorithm
This is the post print version of the article. The official published version can be obtained from the link below - Copyright 2007 Taylor & Francis LtdA novel algorithm for detecting microarray subgrids is proposed. The only input to the algorithm is the raw microarray image, which can be of any resolution, and the subgrid detection is performed with no prior assumptions. The algorithm consists of a series of methods of spot shape detection, spot filtering, spot spacing estimation, and subgrid shape detection. It is shown to be able to divide images of varying quality into subgrid regions with no manual interaction. The algorithm is robust against high levels of noise and high percentages of poorly expressed or missing spots. In addition, it is proved to be effective in locating regular groupings of primitives in a set of non-microarray images, suggesting potential application in the general area of image processing
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Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells
Thermal error modelling of machine tools based on ANFIS with fuzzy c-means clustering using a thermal imaging camera
Thermal errors are often quoted as being the largest contributor to CNC machine tool errors, but they can be effectively reduced using error compensation. The performance of a thermal error compensation system depends on the accuracy and robustness of the thermal error model and the quality of the inputs to the model. The location of temperature measurement must provide a representative measurement of the change in temperature that will affect the machine structure. The number of sensors and their locations are not always intuitive and the time required to identify the optimal locations is often prohibitive, resulting in compromise and poor results.
In this paper, a new intelligent compensation system for reducing thermal errors of machine tools using data obtained from a thermal imaging camera is introduced. Different groups of key temperature points were identified from thermal images using a novel schema based on a Grey model GM (0, N) and Fuzzy c-means (FCM) clustering method. An Adaptive Neuro-Fuzzy Inference System with Fuzzy c-means clustering (FCM-ANFIS) was employed to design the thermal prediction model. In order to optimise the approach, a parametric study was carried out by changing the number of inputs and number of membership functions to the FCM-ANFIS model, and comparing the relative robustness of the designs. According to the results, the FCM-ANFIS model with four inputs and six membership functions achieves the best performance in terms of the accuracy of its predictive ability. The residual value of the model is smaller than ± 2 μm, which represents a 95% reduction in the thermally-induced error on the machine. Finally, the proposed method is shown to compare favourably against an Artificial Neural Network (ANN) model
Methods to improve gene signal : Application to cDNA microarrays
Microarrays are high throughput biological assays that allow the screening of thousands of genes for their expression. The main idea behind microarrays is to compute for each gene a unique signal that is directly proportional to the quantity of mRNA that was hybridized on the chip. A large number of steps and errors associated with each step make the generated expression signal noisy. As a result, microarray data need to be carefully pre-processed before their analysis can be assumed to lead to reliable and biologically relevant conclusions.
This thesis focuses on developing methods for improving gene signal and further utilizing this improved signal for higher level analysis. To achieve this, first, approaches for designing microarray experiments using various optimality criteria, considering both biological and technical replicates, are described. A carefully designed experiment leads to signal with low noise, as the effect of unwanted variations is minimized and the precision of the estimates of the parameters of interest are maximized. Second, a system for improving the gene signal by using three scans at varying scanner sensitivities is developed. A novel Bayesian latent intensity model is then applied on these three sets of expression values, corresponding to the three scans, to estimate the suitably calibrated true signal of genes. Third, a novel image segmentation approach that segregates the fluorescent signal from the undesired noise is developed using an additional dye, SYBR green RNA II. This technique helped in identifying signal only with respect to the hybridized DNA, and signal corresponding to dust, scratch, spilling of dye, and other noises, are avoided. Fourth, an integrated statistical model is developed, where signal correction, systematic array effects, dye effects, and differential expression, are modelled jointly as opposed to a sequential application of several methods of analysis.
The methods described in here have been tested only for cDNA microarrays, but can also, with some modifications, be applied to other high-throughput technologies.
Keywords: High-throughput technology, microarray, cDNA, multiple scans, Bayesian hierarchical models, image analysis, experimental design, MCMC, WinBUGS.Tarkastellaan menetelmiä, joilla voidaan parantaa geneetisiä signaaleja ja hyödyntää vahvistetun signaalin käyttöä myöhemmissä analyyseissä
Isolating and Quantifying the Role of Developmental Noise in Generating Phenotypic Variation
Genotypic variation, environmental variation, and their interaction may produce variation in the developmental process and cause phenotypic differences among individuals. Developmental noise, which arises during development from stochasticity in cellular and molecular processes when genotype and environment are fixed, also contributes to phenotypic variation. While evolutionary biology has long focused on teasing apart the relative contribution of genes and environment to phenotypic variation, our understanding of the role of developmental noise has lagged due to technical difficulties in directly measuring the contribution of developmental noise. The influence of developmental noise is likely underestimated in studies of phenotypic variation due to intrinsic mechanisms within organisms that stabilize phenotypes and decrease variation. Since we are just beginning to appreciate the extent to which phenotypic variation due to stochasticity is potentially adaptive, the contribution of developmental noise to phenotypic variation must be separated and measured to fully understand its role in evolution. Here, we show that variation in the component of the developmental process corresponding to environmental and genetic factors (here treated together as a unit called the LALI-type) versus the contribution of developmental noise, can be distinguished for leopard gecko (Eublepharis macularius) head color patterns using mathematical simulations that model the role of random variation (corresponding to developmental noise) in patterning. Specifically, we modified the parameters of simulations corresponding to variation in the LALI-type to generate the full range of phenotypic variation in color pattern seen on the heads of eight leopard geckos. We observed that over the range of these parameters, variation in color pattern due to LALI-type variation exceeds that due to developmental noise in the studied gecko cohort. However, the effect of developmental noise on patterning is also substantial. Our approach addresses one of the major goals of evolutionary biology: to quantify the role of stochasticity in shaping phenotypic variation
HST Spectral Mapping of L/T Transition Brown Dwarfs Reveals Cloud Thickness Variations
Most directly imaged giant exoplanets are fainter than brown dwarfs with
similar spectra. To explain their relative underluminosity unusually cloudy
atmospheres have been proposed. However, with multiple parameters varying
between any two objects, it remained difficult to observationally test this
idea. We present a new method, sensitive time-resolved Hubble Space Telescope
near-infrared spectroscopy, to study two rotating L/T transition brown dwarfs
(2M2139 and SIMP0136). The observations provide spatially and spectrally
resolved mapping of the cloud decks of the brown dwarfs. The data allow the
study of cloud structure variations while other parameters are unchanged. We
find that both brown dwarfs display variations of identical nature: J- and
H-band brightness variations with minimal color and spectral changes. Our light
curve models show that even the simplest surface brightness distributions
require at least three elliptical spots. We show that for each source the
spectral changes can be reproduced with a linear combination of only two
different spectra, i.e. the entire surface is covered by two distinct types of
regions. Modeling the color changes and spectral variations together reveal
patchy cloud covers consisting of a spatially heterogenous mix of
low-brightness, low-temperature thick clouds and brighter, thin and warm
clouds. We show that the same thick cloud patches seen in our varying brown
dwarf targets, if extended to the entire photosphere, predict near-infrared
colors/magnitudes matching the range occupied by the directly imaged exoplanets
that are cooler and less luminous than brown dwarfs with similar spectral
types. This supports the models in which thick clouds are responsible for the
near infrared properties of these underluminous exoplanets.Comment: Astrophysical Journal, in pres
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Microarray image processing: A novel neural network framework
This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Due to the vast success of bioengineering techniques, a series of large-scale analysis tools has been developed to discover the functional organization of cells. Among them, cDNA microarray has emerged as a powerful technology that enables biologists to cDNA microarray technology has enabled biologists to study thousands of genes simultaneously within an entire organism, and thus obtain a better understanding of the gene interaction and regulation mechanisms involved. Although microarray technology has been developed so as to offer high tolerances, there exists high signal irregularity through the surface of the microarray image. The imperfection in the microarray image generation process causes noises of many types, which contaminate the resulting image. These errors and noises will propagate down through, and can significantly affect, all subsequent processing and analysis. Therefore, to realize the potential of such technology it is crucial to obtain high quality image data that would indeed reflect the underlying biology in the samples. One of the key steps in extracting information from a microarray image is segmentation: identifying which pixels within an image represent which gene. This area of spotted microarray image analysis has received relatively little attention relative to the advances in proceeding analysis stages. But, the lack of advanced image analysis, including the segmentation, results in sub-optimal data being used in all downstream analysis methods.
Although there is recently much research on microarray image analysis with many methods have been proposed, some methods produce better results than others. In general, the most effective approaches require considerable run time (processing) power to process an entire image. Furthermore, there has been little progress on developing sufficiently fast yet efficient and effective algorithms the segmentation of the microarray image by using a highly sophisticated framework such as Cellular Neural Networks (CNNs). It is, therefore, the aim of this thesis to investigate and develop novel methods processing microarray images. The goal is to produce results that outperform the currently available approaches in terms of PSNR, k-means and ICC measurements.Aleppo University, Syri
Bioinformatics framework for genotyping microarray data analysis
Functional genomics is a flourishing science enabled by recent technological breakthroughs in high-throughput instrumentation and microarray data analysis. Genotyping microarrays establish the genotypes of DNA sequences containing single nucleotide polymorphisms (SNPs), and can help biologists probe the functions of different genes and/or construct complex gene interaction networks. The enormous amount of data from these experiments makes it infeasible to perform manual processing to obtain accurate and reliable results in daily routines. Advanced algorithms as well as an integrated software toolkit are needed to help perform reliable and fast data analysis.
The author developed a MatlabTM based software package, called TIMDA (a Toolkit for Integrated Genotyping Microarray Data Analysis), for fully automatic, accurate and reliable genotyping microarray data analysis. The author also developed new algorithms for image processing and genotype-calling. The modular design of TIMDA allows satisfactory extensibility and maintainability. TIMDA is open source (URL: http://timda.SF.net and can be easily customized by users to meet their particular needs. The quality and reproducibility of results in image processing and genotype-calling and the ease of customization indicate that TIMDA is a useful package for genomics research
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