Computational Analysis of Microbial Flow Cytometry Data.
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ABSTRACT:
SUBMITTER: Rubbens P
PROVIDER: S-EPMC7820666 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Flow cytometry is a widely used analytical technique for examining microscopic particles, such as cells. The Flow Cytometry Standard (FCS) was developed in 1984 for storing flow data and it is supported by all instrument and third party software vendors. However, FCS does not capture the full scope of flow cytometry (FCM)-related data and metadata, and data standards have recently been developed to address this shortcoming. FINDINGS: The Data Standards Task Force (DSTF) of the International Society for the Advancement of Cytometry (ISAC) has developed several data standards to complement the raw data encoded in FCS files. Efforts started with the Minimum Information about a Flow Cytometry Experiment, a minimal data reporting standard of details necessary to include when publishing FCM experiments to facilitate third party understanding. MIFlowCyt is now being recommended to authors by publishers as part of manuscript submission, and manuscripts are being checked by reviewers and editors for compliance. Gating-ML was then introduced to capture gating descriptions - an essential part of FCM data analysis describing the selection of cell populations of interest. The Classification Results File Format was developed to accommodate results of the gating process, mostly within the context of automated clustering. Additionally, the Archival Cytometry Standard bundles data with all the other components describing experiments. Here, we introduce these recent standards and provide the very first example of how they can be used to report FCM data including analysis and results in a standardized, computationally exchangeable form. CONCLUSIONS: Reporting standards and open file formats are essential for scientific collaboration and independent validation. The recently developed FCM data standards are now being incorporated into third party software tools and data repositories, which will ultimately facilitate understanding and data reuse.
| S-EPMC3060130 | biostudies-literature
Project description:IntroductionThe information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training.MethodsWe aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self-generated data sets.ResultsWe developed Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs "healthy", "old" vs "young", and facilitates the identification of cell populations with potential biologic relevance to states of disease and health.ConclusionsWe provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets.
| S-EPMC6688088 | biostudies-literature
Project description:Cell cycle progression can be studied with computational models that allow to describe and predict its perturbation by agents as ionizing radiation or drugs. Such models can then be integrated in tools for pre-clinical/clinical use, e.g. to optimize kinetically-based administration protocols of radiation therapy and chemotherapy. We present a deterministic compartmental model, specifically reproducing how cells that survive radiation exposure are distributed in the cell cycle as a function of dose and time after exposure. Model compartments represent the four cell-cycle phases, as a function of DNA content and time. A system of differential equations, whose parameters represent transition rates, division rate and DNA synthesis rate, describes the temporal evolution. Initial model inputs are data from unexposed cells in exponential growth. Perturbation is implemented as an alteration of model parameters that allows to best reproduce cell-cycle profiles post-irradiation. The model is validated with dedicated in vitro measurements on human lung fibroblasts (IMR90). Cells were irradiated with 2 and 5 Gy with a Varian 6 MV Clinac at IRCCS Maugeri. Flow cytometry analysis was performed at the RadBioPhys Laboratory (University of Pavia), obtaining cell percentages in each of the four phases in all studied conditions up to 72 h post-irradiation. Cells show early [Formula: see text]-phase block (increasing in duration as dose increases) and later [Formula: see text]-phase accumulation. For each condition, we identified the best sets of model parameters that lead to a good agreement between model and experimental data, varying transition rates from [Formula: see text]- to S- and from [Formula: see text]- to M-phase. This work offers a proof-of-concept validation of the new computational tool, opening to its future development and, in perspective, to its integration in a wider framework for clinical use.
| S-EPMC7806866 | biostudies-literature
Project description:BackgroundFlow cytometry, with its high throughput nature, combined with the ability to measure an increasing number of cell parameters at once can surpass the throughput of prevalent genomic and metagenomic approaches in the study of microbiomes. Novel computational approaches to analyze flow cytometry data will result in greater insights and actionability as compared to traditional tools used in the analysis of microbiomes. This paper is a demonstration of the fruitfulness of machine learning in analyzing microbial flow cytometry data generated in anaerobic microbiome perturbation experiments.ResultsAutoencoders were found to be powerful in detecting anomalies in flow cytometry data from nanoparticles and carbon sources perturbed anaerobic microbiomes but was marginal in predicting perturbations due to antibiotics. A comparison between different algorithms based on predictive capabilities suggested that gradient boosting (GB) and deep learning, i.e. feed forward artificial neural network with three hidden layers (DL) were marginally better under tested conditions at predicting overall community structure while distributed random forests (DRF) worked better for predicting the most important putative microbial group(s) in the anaerobic digesters viz. methanogens, and it can be optimized with better parameter tuning. Predictive classification patterns with DL (feed forward artificial neural network with three hidden layers) were found to be comparable to previously demonstrated multivariate analysis. The potential applications of this approach have been demonstrated for monitoring the syntrophic resilience of the anaerobic microbiomes perturbed by synthetic nanoparticles as well as antibiotics.ConclusionMachine learning can benefit the microbial flow cytometry research community by providing rapid screening and characterization tools to discover patterns in the dynamic response of microbiomes to several stimuli.
| S-EPMC6134764 | biostudies-literature
Project description:Flow cytometry is commonly used to evaluate the performance of engineered bacteria. With increasing use of high-throughput experimental methods, there is a need for automated analysis methods for flow cytometry data. Here, we describe FlowGateNIST, a Python package for automated analysis of bacterial flow cytometry data. The main components of FlowGateNIST perform automatic gating to differentiate between cells and background events and then between singlet and multiplet events. FlowGateNIST also includes a method for automatic calibration of fluorescence signals using fluorescence calibration beads. FlowGateNIST is open source and freely available with tutorials and example data to facilitate adoption by users with minimal programming experience.
| S-EPMC8372958 | biostudies-literature
Project description:Flow cytometry (FCM) software packages from R/Bioconductor, such as flowCore and flowViz, serve as an open platform for development of new analysis tools and methods. We created plateCore, a new package that extends the functionality in these core packages to enable automated negative control-based gating and make the processing and analysis of plate-based data sets from high-throughput FCM screening experiments easier. plateCore was used to analyze data from a BD FACS CAP screening experiment where five Peripheral Blood Mononucleocyte Cell (PBMC) samples were assayed for 189 different human cell surface markers. This same data set was also manually analyzed by a cytometry expert using the FlowJo data analysis software package (TreeStar, USA). We show that the expression values for markers characterized using the automated approach in plateCore are in good agreement with those from FlowJo, and that using plateCore allows for more reproducible analyses of FCM screening data.
| S-EPMC2777006 | biostudies-literature
Project description:Flow cytometry is one of the fundamental research tools available to the life scientist. The ability to observe multidimensional changes in protein expression and activity at single-cell resolution for a large number of cells provides a unique perspective on the behavior of cell populations. However, the analysis of complex multidimensional data is one of the obstacles for wider use of polychromatic flow cytometry. Recent enhancements to an open-source platform-R/Bioconductor-enable the graphical and data analysis of flow cytometry data. Prior examples have focused on high-throughput applications. To facilitate wider use of this platform for flow cytometry, the analysis of a dataset, obtained following isolation of CD4(+)CD62L(+) T cells from Balb/c splenocytes using magnetic microbeads, is presented as a form of tutorial. A common workflow for analyzing flow cytometry data was presented using R/Bioconductor. In addition, density function estimation and principal component analysis are provided as examples of more complex analyses. The compendium presented here is intended to help illuminate a path for inquisitive readers to explore their own data using R/Bioconductor (available as Supporting Information).
| S-EPMC2941976 | biostudies-literature
Project description:Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks: (i) mammalian cell population identification, to determine whether automated algorithms can reproduce expert manual gating and (ii) sample classification, to determine whether analysis pipelines can identify characteristics that correlate with external variables (such as clinical outcome). This analysis presents the results of the first FlowCAP challenges. Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis.
| S-EPMC3906045 | biostudies-literature
Project description:The arrival of mass cytometry (MC) and, more recently, spectral flow cytometry (SFC) has revolutionized the study of cellular, functional and phenotypic diversity, significantly increasing the number of characteristics measurable at the single-cell level. As a consequence, new computational techniques such as dimensionality reduction and/or clustering algorithms are necessary to analyze, clean, visualize, and interpret these high-dimensional data sets. In this small comparison study, we investigated splenocytes from the same sample by either MC or SFC and compared both high-dimensional data sets using expert gating, t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP) analysis and FlowSOM. When we downsampled each data set to their equivalent cell numbers and parameters, our analysis yielded highly comparable results. Differences between the data sets only became apparent when the maximum number of parameters in each data set were assessed, due to differences in the number of recorded events or the maximum number of assessed parameters. Overall, our small comparison study suggests that mass cytometry and spectral flow cytometry both yield comparable results when analyzed manually or by high-dimensional clustering or dimensionality reduction algorithms such as t-SNE, UMAP, or FlowSOM. However, large scale studies combined with an in-depth technical analysis will be needed to assess differences between these technologies in more detail. © 2020 International Society for Advancement of Cytometry.
| S-EPMC7682594 | biostudies-literature
Project description:BackgroundIn 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.ResultsWe compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts.ConclusionsOur results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available.
| S-EPMC3243046 | biostudies-literature