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1.
Genome Biol ; 25(1): 89, 2024 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-38589921

RESUMO

Advancements in cytometry technologies have enabled quantification of up to 50 proteins across millions of cells at single cell resolution. Analysis of cytometry data routinely involves tasks such as data integration, clustering, and dimensionality reduction. While numerous tools exist, many require extensive run times when processing large cytometry data containing millions of cells. Existing solutions, such as random subsampling, are inadequate as they risk excluding rare cell subsets. To address this, we propose SuperCellCyto, an R package that builds on the SuperCell tool which groups highly similar cells into supercells. SuperCellCyto is available on GitHub ( https://github.com/phipsonlab/SuperCellCyto ) and Zenodo ( https://doi.org/10.5281/zenodo.10521294 ).


Assuntos
Pesquisa , Análise de Célula Única , Análise por Conglomerados , Software
2.
Genome Biol ; 25(1): 99, 2024 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-38637899

RESUMO

Spatial molecular data has transformed the study of disease microenvironments, though, larger datasets pose an analytics challenge prompting the direct adoption of single-cell RNA-sequencing tools including normalization methods. Here, we demonstrate that library size is associated with tissue structure and that normalizing these effects out using commonly applied scRNA-seq normalization methods will negatively affect spatial domain identification. Spatial data should not be specifically corrected for library size prior to analysis, and algorithms designed for scRNA-seq data should be adopted with caution.


Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Perfilação da Expressão Gênica/métodos , Algoritmos , Biologia
3.
Cytometry A ; 103(1): 54-70, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35758217

RESUMO

Mapping the dynamics of immune cell populations over time or disease-course is key to understanding immunopathogenesis and devising putative interventions. We present TrackSOM, a novel method for delineating cellular populations and tracking their development over a time- or disease-course cytometry datasets. We demonstrate TrackSOM-enabled elucidation of the immune response to West Nile Virus infection in mice, uncovering heterogeneous subpopulations of immune cells and relating their functional evolution to disease severity. TrackSOM is easy to use, encompasses few parameters, is quick to execute, and enables an integrative and dynamic overview of the immune system kinetics that underlie disease progression and/or resolution.


Assuntos
Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Camundongos , Animais , Vírus do Nilo Ocidental/fisiologia , Febre do Nilo Ocidental/patologia , Imunidade , Análise por Conglomerados
4.
Bioinformatics ; 38(10): 2943-2945, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35561197

RESUMO

SUMMARY: HTSeq 2.0 provides a more extensive application programming interface including a new representation for sparse genomic data, enhancements for htseq-count to suit single-cell omics, a new script for data using cell and molecular barcodes, improved documentation, testing and deployment, bug fixes and Python 3 support. AVAILABILITY AND IMPLEMENTATION: HTSeq 2.0 is released as an open-source software under the GNU General Public License and is available from the Python Package Index at https://pypi.python.org/pypi/HTSeq. The source code is available on Github at https://github.com/htseq/htseq. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Documentação , Genômica , Licenciamento
5.
Immunol Cell Biol ; 100(6): 453-467, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35416319

RESUMO

B cells play a major role in multiple sclerosis (MS), with many successful therapeutics capable of removing them from circulation. One such therapy, alemtuzumab, is thought to reset the immune system without the need for ongoing therapy in a proportion of patients. The exact cells contributing to disease pathogenesis and quiescence remain to be identified. We utilized mass cytometry to analyze B cells from the blood of patients with relapse-remitting MS (RRMS) before and after alemtuzumab treatment, and during relapse. A complementary RRMS cohort was analyzed by single-cell RNA sequencing. The R package "Spectre" was used to analyze these data, incorporating FlowSOM clustering, sparse partial least squares-discriminant analysis and permutational multivariate analysis of variance. Immunoglobulin (Ig)A+ and IgG1 + B-cell numbers were altered, including higher IgG1 + B cells during relapse. B-cell linker protein (BLNK), CD40 and CD210 expression by B cells was lower in patients with RRMS compared with non-MS controls, with similar results at the transcriptomic level. Finally, alemtuzumab restored BLNK, CD40 and CD210 expression by IgA+ and IgG1 + B cells, which was altered again during relapse. These data suggest that impairment of IgA+ and IgG1 + B cells may contribute to MS pathogenesis, which can be restored by alemtuzumab.


Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Alemtuzumab/uso terapêutico , Doença Crônica , Humanos , Imunoglobulina A , Imunoglobulina G , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Recidiva
6.
Cytometry A ; 101(3): 237-253, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33840138

RESUMO

As the size and complexity of high-dimensional (HD) cytometry data continue to expand, comprehensive, scalable, and methodical computational analysis approaches are essential. Yet, contemporary clustering and dimensionality reduction tools alone are insufficient to analyze or reproduce analyses across large numbers of samples, batches, or experiments. Moreover, approaches that allow for the integration of data across batches or experiments are not well incorporated into computational toolkits to allow for streamlined workflows. Here we present Spectre, an R package that enables comprehensive end-to-end integration and analysis of HD cytometry data from different batches or experiments. Spectre streamlines the analytical stages of raw data pre-processing, batch alignment, data integration, clustering, dimensionality reduction, visualization, and population labelling, as well as quantitative and statistical analysis. Critically, the fundamental data structures used within Spectre, along with the implementation of machine learning classifiers, allow for the scalable analysis of very large HD datasets, generated by flow cytometry, mass cytometry, or spectral cytometry. Using open and flexible data structures, Spectre can also be used to analyze data generated by single-cell RNA sequencing or HD imaging technologies, such as Imaging Mass Cytometry. The simple, clear, and modular design of analysis workflows allow these tools to be used by bioinformaticians and laboratory scientists alike. Spectre is available as an R package or Docker container. R code is available on Github (https://github.com/immunedynamics/spectre).


Assuntos
Algoritmos , Análise de Célula Única , Análise por Conglomerados , Citometria de Fluxo/métodos , Software
7.
Cell Rep Med ; 2(3): 100208, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33564749

RESUMO

SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3+cTFH1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies.


Assuntos
Formação de Anticorpos , COVID-19/imunologia , Imunidade Adaptativa , Adulto , Idoso , Anticorpos Antivirais/sangue , Linfócitos B/citologia , Linfócitos B/metabolismo , COVID-19/patologia , COVID-19/virologia , Feminino , Humanos , Imunidade Inata , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores CXCR3/metabolismo , Receptores de Interleucina-6/metabolismo , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Células Th1/citologia , Células Th1/metabolismo , Adulto Jovem
8.
Bioinformatics ; 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33508103

RESUMO

MOTIVATION: Many 'automated gating' algorithms now exist to cluster cytometry and single cell sequencing data into discrete populations. Comparative algorithm evaluations on benchmark datasets rely either on a single performance metric, or a few metrics considered independently of one another. However, single metrics emphasise different aspects of clustering performance and do not rank clustering solutions in the same order. This underlies the lack of consensus between comparative studies regarding optimal clustering algorithms and undermines the translatability of results onto other non-benchmark datasets. RESULTS: We propose the Pareto fronts framework as an integrative evaluation protocol, wherein individual metrics are instead leveraged as complementary perspectives. Judged superior are algorithms that provide the best trade-off between the multiple metrics considered simultaneously. This yields a more comprehensive and complete view of clustering performance. Moreover, by broadly and systematically sampling algorithm parameter values using the Latin Hypercube sampling method, our evaluation protocol minimises (un)fortunate parameter value selections as confounding factors. Furthermore, it reveals how meticulously each algorithm must be tuned in order to obtain good results, vital knowledge for users with novel data. We exemplify the protocol by conducting a comparative study between three clustering algorithms (ChronoClust, FlowSOM and Phenograph) using four common performance metrics applied across four cytometry benchmark datasets. To our knowledge, this is the first time Pareto fronts have been used to evaluate the performance of clustering algorithms in any application domain. AVAILABILITY: Implementation of our Pareto front methodology and all scripts to reproduce this article are available at https://github.com/ghar1821/ParetoBench.

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