3d). d, Frequency of S+ Bm cells was measured by flow cytometry and separated by mild (acute, n=40; month 6, n=39; month 12, n=11) and severe COVID-19 (acute, n=19; month 6, n=22; month 12, n=6). Lung-resident memory B cells established after pulmonary influenza infection display distinct transcriptional and phenotypic profiles. arguments. I wanted to base an analysis on data that that was matching one of a few criteria, e.g. PhenoGraph clustering identified an IgG+CD21CD27 cluster (cluster 2), which was TbethiCD11c+FcRL5+, and CD21CD27+ clusters characterized by high expression of CD71, Blimp-1 and Ki-67 (clusters 1, 7 and 8) (Extended Data Fig. https://doi.org/10.1038/s41590-023-01497-y, DOI: https://doi.org/10.1038/s41590-023-01497-y. 3c). Sci. e, Stacked bar graphs (mean + SD) display isotype distribution in S+ Bm cell subsets in samples of SARS-CoV-2-recovered individuals postVac at months 6 and 12 post-infection from flow cytometry dataset (n=37). Asking for help, clarification, or responding to other answers. How about saving the world? I hope it is useful. Moreover, expression of inhibitory receptors, including FCRL2, FCRL3, FCRL5, SIGLEC6, SIGLEC10, LAIR1, LILRB1 and LILRB2, and proteins involved in antigen presentation and processing, such as HLA-DPA1, HLA-DPB1, HLA-DRB1, HLA-DRB5, CD74 and CD86, was particularly high in CD21CD27FcRL5+ Bm cells (Fig. Serum and blood was obtained, and peripheral blood mononuclear cells were isolated by density centrifugation, washed and frozen in fetal bovine serum (FBS) with 10% dimethyl sulfoxide and stored in liquid nitrogen until use. ## [43] future.apply_1.10.0 BiocGenerics_0.44.0 abind_1.4-5 subsetting seurat object with multiple samples, Traffic: 1812 users visited in the last hour, User Agreement and Privacy Hi Team Seurat, These results suggest that CD21CD27 Bm cells partake in the normal immune response to pathogens37. 3j,k). SCT_integrated <- RunPCA(SCT_integrated) Fourteen cycles (in one case 17) of initial cDNA amplification were used for all sample batches, and single-cell sequencing libraries for whole-transcriptome analysis (GEX), BCR profiling (VDJ) and TotalSeq (BioLegend) barcode detection (ADT) were generated. Immunol. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. a, Uniform manifold approximation and projection (UMAP) plots of S+ Bm cells are provided during acute SARS-CoV-2 infection and at months 6 and 12, showing samples of nonvaccinated individuals from the SARS-CoV-2 Infection Cohort, subsampled to maximally 25 cells per sample (Acute, n=44; month 6, n=59; month 12, n=17). 15, 149159 (2015). Victora, G. D. & Nussenzweig, M. C. Germinal centers. F1000Res. Immunol. 3i). 1b and Extended Data Fig. ## This study was approved by the Cantonal Ethics Committee of Zurich (BASEC #2016-01440). Accessing data in Seurat is simple, using clearly defined accessors and setters to quickly find the data needed. Choose a subset of cells, and then split by samples and then re-run the integration steps (select integration features, find anchors and integrate data). Gene sets were obtained from the Molecular Signatures Database (v7.5.1, collections H and C5) and loaded in R by the package msigdbr (v.7.5.1). As one can see in the pic below, the quality is quite different in each of the duplicated conditions. Learn more about Stack Overflow the company, and our products. I'm also interested in understanding better how to do this. Prolonged evolution of the human B cell response to SARS-CoV-2 infection. PubMedGoogle Scholar. Rev. Immunol. Zurbuchen, Y., Michler, J., Taeschler, P. et al. c, Frequency of S+ Bm cells in total B cells was measured by flow cytometry at acute infection (n=59) and months 6 (n=61) and 12 post-infection (n=17). e, Heatmap of log2-fold change of indicated markers is shown in blood and tonsillar S+ Bm cells of vaccinated and recovered individuals (top; n=16) and N+ Bm cells of recovered individuals (bottom; n=8), with red indicating higher expression in tonsils and blue in blood. Immunity 55, 945964 (2022). Sign up for a free GitHub account to open an issue and contact its maintainers and the community. For UMAP generation in the SARS-CoV-2 Infection Cohort datasets, the embedding parameters were manually set to a=1.4 and b=0.75. Asking for help, clarification, or responding to other answers. 6, eabh0891 (2021). Natl Acad. If split.by is not NULL, the ncol is ignored so you can not arrange the grid. In c and g, all P values are shown, in the other graphs adjusted P values are shown if significant (p<0.05). You signed in with another tab or window. 4ac). 4d). FindAllMarkers and FindMarkers functions were executed with logfc.thresholds set to 0.25 (0.1 for comparing resting Bm cells at month 6 versus month 12) and a min.pct cutoff at 0.1. Is there a generic term for these trajectories? 5c). e, Representative CD69 histograms in S+ Bm cells of patient CoV-T2 (left) and percentages of CD69+ S+ Bm cells (right) in blood and tonsils. How a top-ranked engineering school reimagined CS curriculum (Ep. Markers were scaled with arcsinh transformation (cofactor 6,000), samples were subsetted to maximally 25 S+ Bm cells per sample. c, UMAP as in a was colored by normalized expression of indicated markers. Additionally, CD21CD27+ activated Bm cells11 might represent a GC-derived population prone to plasma cell differentiation12, and CD21CD27 Bm cells have been reported in chronic infection, immunodeficiency and autoimmune diseases and are thought to be of extrafollicular origin13,14,15,16,17,18. | object@hvg.info | HVFInfo(object = object) | rev2023.4.21.43403. Otherwise, will return an object consissting only of these cells, Parameter to subset on. Antibody affinity shapes the choice between memory and germinal center B cell fates. customize FeaturePlot in Seurat for multi-condition comparisons using B cells that differentiate in the GC undergo affinity maturation through somatic hypermutation (SHM) of the B cell receptor (BCR) following which B cells can become long-lived plasma cells or Bm cells4,5,6. All authors edited and approved the final paper. Collectively, these data identify a durable, IgG1-dominated S+ Bm cell response forming upon SARS-CoV-2 infection. ## [16] memoise_2.0.1 tensor_1.5 cluster_2.1.3 SCT_integrated <- FindClusters(SCT_integrated), control_subset <- subset(SCT_integrated, orig.ident = 'Chow') Look at what 1||2||3 evaluates to: and you'd get the same using | instead. 31,32). Activation dynamics and immunoglobulin evolution of pre-existing and newly generated human memory B cell responses to influenza hemagglutinin. The flow cytometry and scRNA-seq subcohort characteristics are presented in Supplementary Tables 1 and 2, respectively. How a top-ranked engineering school reimagined CS curriculum (Ep. Most functions now take an assay parameter, but you can set a Default Assay to avoid repetitive statements. Yang, R. et al. I have similar questions as @attal-kush with regards to reclustering of a subset of an integrated object. Sci. 6g and Extended Data Fig. Each dot represents an individual (n=6). Lau, D. et al. Content Discovery initiative April 13 update: Related questions using a Review our technical responses for the 2023 Developer Survey, R: subsetting data frame by both certain column names (as a variable) and field values. 1 Answer Sorted by: 1 There are a few ways to address this. Ritchie, M. E. et al. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in k, Venn diagram shows clonal overlap of SWT+ and SWT Bm cells in tonsils and blood from scRNA-seq dataset. Severe deficiency of switched memory B cells (CD27+IgMIgD) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. conceived the project, designed experiments and interpreted data. In e, two-sided Wilcoxon rank sum test was used and P values corrected by Bonferroni correction. Default is INF. The flow cytometry dataset is available upon request from the corresponding authors. We would all appreciate it if @timoast or others from the @satijalab can chime in. i, SHM counts are provided for nave B cells (n=1,607), blood (n=170) and tonsillar SWT+ Bm cells (n=1,128). 60). Dimensionality reduction and clustering analysis of flow cytometry data were performed in R using the CATALYST workflow (CATALYST package, version 1.18.1) (ref. One way to look broadly at these changes is to plot the average expression of both the stimulated and control cells and look for genes that are visual outliers on a scatter plot. P values for different comparisons are given below. Bioinformatics Stack Exchange is a question and answer site for researchers, developers, students, teachers, and end users interested in bioinformatics. 1 Overview of SARS-CoV-2 cohorts analyzed in this study. a, Gating strategy for SARS-CoV-2 spike (S)+ and receptor-binding domain (RBD)+ Bm cells. However, antibody responses to several previously applied vaccines were normal in T-bet-deficient patients30. So, my here is my workflow: seurat_object <- subset (seurat_object, subset = DF.classifications_0.25_0.03_252 == 'Singlet') #this approach works I would like to automate this process but the _0.25_0.03_252 of DF.classifications_0.25_0.03_252 is based on values that are calculated and will not be known in advance. ## [1] stats graphics grDevices utils datasets methods base ## [49] miniUI_0.1.1.1 Rcpp_1.0.10 plotrix_3.8-2 Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine. Nat. 65 patients were included and followed-up until month 12 post-infection. We identified 16 shared SWT+ Bm cell clones between these compartments (Fig. ## [58] httr_1.4.5 RColorBrewer_1.1-3 TFisher_0.2.0 Below, we demonstrate how to modify the Seurat integration workflow for datasets that have been normalized with the sctransform workflow. Generic Doubly-Linked-Lists C implementation. Of these, 35 received SARS-CoV-2 mRNA vaccination between month 6 and month 12, and 3 subjects between acute infection and month 6. f, Violin plots show percentages of IgG1+ (left) and IgG3+ (right) S+ Bm cells at indicated timepoints (acute, n=23; month 6, n=52; month 12, n=16). 4e). PubMed Central Out of all possible solutions, I feel like performing the analysis as @tilofreiwald's "option b" would be the best. Immunity 33, 451463 (2010). Thanks for contributing an answer to Stack Overflow! Bm cells can be subdivided into phenotypically and functionally distinct subsets10. Academic theme for This issue may help you address your question. I followed a similar approach to @attal-kush. h, Expression of selected genes (left) and surface protein markers (right) are shown in Bm cell clusters. Bm cells specific for RBD, wild-type spike (SWT) or spike variants B.1.351 (Sbeta) and B.1.617.2 (Sdelta) were identified by SAV multimers carrying specific oligonucleotide barcodes. Assa Yeroslaviz 1.8k. Nature 602, 148155 (2021). GSEA was performed on this preranked list using the R package fgsea (v.1.2). ## [109] vctrs_0.5.2 mutoss_0.1-12 pillar_1.8.1 # One of these Assay objects is called the "default assay", meaning it's used for all analyses and visualization. However, this brings the cost of flexibility. I would also like to know the recommended way of doing this. EDIT: The point is that you need a series of single comparisons, not a comparison of a series of options. ), Clinical Research Priority Program CYTIMM-Z of University of Zurich (UZH) (to O.B. | object@data | GetAssayData(object = object) | 13, 446 (2022). b, Representative flow cytometry plots show percentages of decoy-negative SARS-CoV-2 S+ Bm cells (gated as in Extended Data Fig. Cao, J. et al. To subscribe to this RSS feed, copy and paste this URL into your RSS reader. Source data are provided with this paper. Each set of modal data (eg. ## Robbiani, D. F. et al. Multi-Assay Features With Seurat, you can easily switch between different assays at the single cell level (such as ADT counts from CITE-seq, or integrated/batch-corrected data). Data-driven phenotypic dissection of AML reveals progenitor-like cells that correlate with prognosis. 37, 521546 (2019). In Hafemeister and Satija, 2019, we introduced an improved method for the normalization of scRNA-seq, based on regularized negative binomial regression. r - Conditional subsetting of Seurat object - Stack Overflow Proc. | WhichCells(object = object, ident.remove = "ident.remove") | WhichCells(object = object, idents = "ident.remove", invert = TRUE) | Slider with three articles shown per slide. P values are shown if significant (p<0.05). and JavaScript. We performed scRNA-seq combined with feature barcoding, which allowed us to assess surface phenotype and to perform BCR-seq in sorted S+ Bm cells and S B cells from paired blood and tonsil samples of four patients (two SARS-CoV-2-recovered and two SARS-CoV-2-vaccinated). Biol. Masopust, D. & Soerens, A. G. Tissue-resident T cells and other resident leukocytes. To visualize the two conditions side-by-side, we can use the split.by argument to show each condition colored by cluster. ## [106] lattice_0.20-45 Matrix_1.5-3 multtest_2.54.0 Samples were stained as described for spectral flow cytometry using biotinylated SWT, RBD, Sbeta and Sdelta (MiltenyiBiotec) and hemagglutinin (SinoBiological) that were multimerized at 4:1 molar ratios with fluorescently labeled and/or barcoded SAV (TotalSeqC, BioLegend).
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