Sci. In humans, resting Bm cells are typically CD21hi, and express the tumor necrosis factor (TNF) receptor superfamily member CD27. Victora, G. D. & Nussenzweig, M. C. Germinal centers. and reading this issue I only got more confused. The FCRL4hiENTPD1hiTNFRSF13Bhi cluster (cluster 6) probably represented the FcRL4+ B cell subset, and contained very few SWT+ Bm cells (Fig. SARS-CoV-2 infection generates tissue-localized immunological memory in humans. 5c). 25,26,27,28,29). Samples in a and cf were compared using a Kruskal-Wallis test with Dunns multiple comparison correction. b, Distribution of S+ Bm cell subsets is provided at month 6 preVac, month 12 nonVac and month 12 postVac. Academic theme for We associated this with an incident during sample preparation in one of our experiments and decided to exclude most cells of this dataset from the analysis. We included a total of 65 patients of the full cohort51,52 on the basis of a power calculation from pre-experiments and according to sample availability of at least paired samples from two timepoints. The text was updated successfully, but these errors were encountered: @attal-kush I hope its okay to piggyback of your question. 3d). Browse other questions tagged, Where developers & technologists share private knowledge with coworkers, Reach developers & technologists worldwide. Nature 602, 148155 (2021). To see help pages for operators, use ? 13, 446 (2022). Gene expression data and TotalSeq surface proteome data were integrated separately. Nowicka, M. et al. # To see all keys for all objects, use the Key function. dg, Stacked bar graphs display tissue (d) and isotype distribution (e) in Bm cell clusters, and isotype (f) and cluster distribution (g) in SWT+ Bm cells in tonsils and blood. Immunol. Default is INF. CD21CD27 Bm cells depend on the transcription factor T-bet for their development30, are CD11chi and express inhibitory coreceptors, such as Fc receptor-like protein 5 (FcRL5) (refs. The num_dim parameter of Monocles preprocess_cds() function was set to 20. I want to know: Weighted-nearest neighbor (WNN) clustering identified nave B cells (IgMhiIgDhiFCER2hi), nave/activated B cells (IgMhiIgDhiFCER2hiFCRL5hi), GC B cells (CD27hiCD38hiAICDAhi) and Bm cells (IgMloIgDloCD27int) (Extended Data Fig. Google Scholar. Very few S+ tonsillar Bm cells expressed FcRL4 in both vaccinated and recovered individuals (Extended Data Fig. Samples in f were compared using a Kruskal-Wallis test with Dunns multiple comparison correction, with adjusted P values shown. ident.remove = NULL, a, CD21 and CD27 expression on S+ Bm cells during acute infection (top) and month 6 post-infection (bottom) of patient CoV-P2 was determined by flow cytometry. c, Stacked bar plots (mean + SD) show isotypes of S+ Bm cells at week 2 (n=10) and month 6 (n=11) post-second dose and at week 2 post-third dose (n=10). Branch lengths represent mutation numbers per site between each node. F1000Res. e, Violin plots of geometric mean fluorescence intensities (gMFI) or percentages of indicated markers in S+ Bm cells at indicated time points. Thank you for the wonderful package. IFI6 and ISG15, on the other hand, are core interferon response genes and are upregulated accordingly in all cell types. The authors declare no competing interests. After subsetting clusters of interest (subsetting by ident) I have a Seurat object with RNA, SCT and integrated assay, and dimensional reduction (pca, tsne, umap) coming from the original Seurat object. What are the differences between "=" and "<-" assignment operators? Is it necessary to run FindVariableFeatures on the RNA assay of the subset and get new variables to use in PCA in order to properly cluster the subset? Cervia, C. et al. Extended Data Fig. Nature 604, 141145 (2022). In the scRNA-seq dataset, CD21+CD27+ resting Bm cells were the main S+ Bm cell subset at months 6 and 12 post-infection in nonvaccinated individuals, whereas CD21CD27+CD71+ activated and CD21CD27FcRL5+ Bm cells became predominant post-vaccination at month 12 post-infection (Fig. SCT_integrated <- FindClusters(SCT_integrated), control_subset <- subset(SCT_integrated, orig.ident = 'Chow') Did the Golden Gate Bridge 'flatten' under the weight of 300,000 people in 1987? analyzed scRNA-seq data. . That way, one would avoid the pitfall described in @Zha0rong's first scenario because the sub-clustering would have been driven by the variable features recalculated in the data subset. ISSN 1529-2916 (online) Colors indicate Bm cell subsets. What are the advantages of running a power tool on 240 V vs 120 V? Nucleic Acids Res. Weiss, G. E. et al. a, Dot plots and medians of frequencies of S+ Bm cells are provided at baseline (n=10), week 2 post-second dose (n=10) and month 6 post-second dose (n=11). ), Filling the Gap Program of UZH (to M.E.R. 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. Replies here and in some other GitHub issues have slightly different approaches but they all make general sense. Human memory B cells show plasticity and adopt multiple fates upon recall response to SARS-CoV-2. Now that weve aligned the stimulated and control cells, we can start to do comparative analyses and look at the differences induced by stimulation. Most functions now take an assay parameter, but you can set a Default Assay to avoid repetitive statements. 9e). Transl. Primary Handling Editor: Ioana Visan in collaboration with the Nature Immunology team. If I decide that batch correction is not required for my samples, could I subset cells from my original Seurat Object (after running Quality Control and clustering on it), set the assay to "RNA", and and run the standard SCTransform pipeline. Frequencies of S+ Bm cells were comparable in patients with mild and severe COVID-19 (Fig. K.W. Rev. 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). Setliff, I. et al. Differential gene expression identified higher expression of CR2, CD44, CCR6 and CD69 in tonsillar SWT+ Bm cells compared with blood SWT+ Bm cells, whereas the activation-related genes FGR and CD52 were higher in blood SWT+ Bm cells compared with their tonsillar counterparts (Extended Data Fig. Each dot represents an individual (n=6). Sorted B cells were analyzed by scRNA-seq using the commercial 5 Single Cell GEX and VDJ v1.1 platform (10x Genomics). Immunol. Viral Hepat. d, Percentages of Ki-67+ S+ Bm cells are provided in paired blood and tonsil samples of SARS-CoV-2-vaccinated and recovered individuals (n=16). ## [19] ROCR_1.0-11 limma_3.54.1 globals_0.16.2 ## [67] deldir_1.0-6 utf8_1.2.3 tidyselect_1.2.0 J. Immunol. Use of this site constitutes acceptance of our User Agreement and Privacy P values are shown if significant (p<0.05). c, Dot plot shows expression of selected genes in main B cell populations. 1b. (palm-face-impact)@MariaKwhere were you 3 months ago?! Goel, R. R. et al. ## [64] pkgconfig_2.0.3 sass_0.4.5 uwot_0.1.14 IFN induces epigenetic programming of human T-bethi B cells and promotes TLR7/8 and IL-21 induced differentiation. f, Representative contour plots of CD21 and CD27 expression on S+ Bm cells are shown at preVac and day 9 and day 78 postVac. Heat maps were generated using the ComplexHeatmap package (v2.13.1) or pheatmap package (v1.0.12) (ref. By clicking Accept all cookies, you agree Stack Exchange can store cookies on your device and disclose information in accordance with our Cookie Policy. Making statements based on opinion; back them up with references or personal experience. Connect and share knowledge within a single location that is structured and easy to search. Browse other questions tagged, Start here for a quick overview of the site, Detailed answers to any questions you might have, Discuss the workings and policies of this site. e, Circos plots of all persistent S+ Bm cell clones (left) and those adopting multiple Bm cell fates (right) are shown, with arrows connecting cells of months 6 with 12 and colored according to Bm cell phenotype at month 12. f, SHM counts were calculated in indicated S+ Bm cell subsets (unswitched, n=53; CD27lo resting, n=122; CD27hi resting, n=535; activated, n=713; CD21CD27FcRL5+, n=531). ## [31] xfun_0.37 dplyr_1.1.0 crayon_1.5.2 Atypical B cells up-regulate costimulatory molecules during malaria and secrete antibodies with T follicular helper cell support. Unless a gene is not expressed (n-reads) at 1/p* try to forget about it just like a bad day (p* being the relative mean gene expression taking into account cDNA library construction efficiency, which in the case of 10x is 15%, or 1/p* = 1/0.15 7 reads/cell/gene). Sci. Since the data I am analyzing comes from different diets as well as different batches, will batch-correction make me unable to determine differences in gene expression of cells from different diets? 3e and Extended Data Fig. 3j,k). Patients with COVID-19 and healthy individuals were recruited at one of four hospitals in the Canton of Zurich, Switzerland. I was able to achieve this in the following way: Would be interesting to know if Seurat provides such functionality out of the box. During acute infection S+ Bm cells were mainly immunoglobulin (Ig)M+ and IgG+, whereas IgG+ Bm cells predominated (8590%) at months 6 and 12 post-infection (Fig. But as you can see, %in% is far more useful and less verbose in such circumstances. Transcriptomes of individual cells were used as inputs for the gsva() function with default parameters. All samples were analyzed by flow cytometry, and paired week 2, month 6 post-second dose and week 2 post-third dose samples from three patients were additionally assessed by scRNA-seq. Yang, R. et al. | object@hvg.info | HVFInfo(object = object) | Jenks, S. A. et al. ## [112] lifecycle_1.0.3 Rdpack_2.4 spatstat.geom_3.0-6 Can I general this code to draw a regular polyhedron? batch effect correction), and to perform comparative scRNA-seq analysis of across experimental conditions. (default), then this list will be computed based on the next three 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). Rev. c, S+ Bm cell frequencies within B cells (n=41) are plotted against time post-last vaccination. The SWT+ Bm cells in the IgG+CD27hiCD45RBhi cluster (cluster 5) were mainly from blood, in the IgG+CD21hi cluster (cluster 2) predominantly tonsillar, while the IgG+CD27lo cluster (cluster 4) contained SWT+ Bm cells from both compartments. Why are these constructs using pre and post-increment undefined behavior? Open access funding provided by University of Zurich. X-axis shows log-fold change and y-axis the adjusted P values (p<0.05 was considered significant). | GetCellEmbeddings(object = object, reduction.type = "pca") | Embeddings(object = object, reduction = "pca") | 38 patients received SARS-CoV-2 mRNA vaccination during their recovery phase (three between acute infection and month 6, and 35 between month 6 and month 12). Nevertheless, I have seen that normalized RNA (log norm'd) is very reproducible in a PCR/bulk RNAseq/rnaFISH exp (if your DE gene FC is >1.5x and expressed in atleast 50% of cells). I have been subsetting a cluster from a Seurat object to find subclusters. The interrelatedness between these Bm cell subsets remains unknown. For example, to only cluster cells using a single sample group, control, we could run the following: . Below, we demonstrate methods for scRNA-seq integration as described in Stuart*, Butler* et al, 2019 to perform a comparative analysis of human immune cells (PBMC) in either a resting or interferon-stimulated state. ## [109] vctrs_0.5.2 mutoss_0.1-12 pillar_1.8.1 Therefore, I assume I cannot use Pearson residuals for DE analysis. 5a,b) identified S+ Bm cells in the blood and tonsils of both vaccinated and recovered individuals, whereas N+ Bm cells were enriched only in recovered individuals (Fig. ## [55] reticulate_1.28 stats4_4.2.0 htmlwidgets_1.6.1 Lines connect samples of same individual. 4f,g). Robbiani, D. F. et al. Genewise statistics were conducted using empirical Bayes quasi-likelihood F-tests. d, Exemplary dendrograms (IgPhyML B cell trees) display different persistent Bm cell clones at months 6 (triangles) and 12 (dots) post-infection. ), Innovation grant of University Hospital Zurich (to O.B. Genes such as CD3D and GNLY are canonical cell type markers (for T cells and NK/CD8 T cells) that are virtually unaffected by interferon stimulation and display similar gene expression patterns in the control and stimulated group. EDIT: RDocumentation. In the SARS-CoV-2 Infection Cohort, cells with fewer than 200 or more than 2,500 detected genes and cells with more than 10% detected mitochondrial genes were excluded from the analysis. The beginning of pseudotime was manually set inside the partition with mostly unswitched B cells. Kurosaki, T., Kometani, K. & Ise, W. Memory B cells. Raw counts obtained from the cellranger gene expression matrix were used to create cell datasets, which were preprocessed using the Monocle 3 pipeline. and M.B.S. 1e). In short: I found that the first and second approaches lead to a nice integration while the third and fourth lead to an uncorrected batch effect (see the image below). Site design / logo 2023 Stack Exchange Inc; user contributions licensed under CC BY-SA. HolmBonferroni method was used for P value adjustment of multiple comparisons. I was wondering, if it make more sense to find subsetting parameters which will comply with all the samples, or one can do it one sample (or one condition) at a time by itself. Immunol. But I would like to be able to select data via logical operators, so: why did the first approach not work? CXCL10 shows a distinct upregulation in monocytes and B cells after interferon stimulation but not in other cell types. 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. & Warnatz, K. Naive- and memory-like CD21 low B cell subsets share core phenotypic and signaling characteristics in systemic autoimmune disorders. Thank you! Wang, Z. et al. It only takes a minute to sign up. Naradikian, M. S., Hao, Y. CD21CD27 Bm cells have also been identified during acute SARS-CoV-2 infection and post-SARS-CoV-2 vaccination22,25,26,27,28,29. g, Comparison of somatic hypermutation (SHM) counts are provided in SWT+ Bm cells at indicated timepoints (week 2 post-second dose, n=174 cells; month 6 post-second dose, n=271 cells; week 2 post-third dose, n=698 cells). Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. DefaultAssay(control_subset) <- "RNA" b, Shown is weighted-nearest neighbor (WNN) UMAP analysis from scRNA-seq analysis of fluorescence-activated cell-sorted B cells from paired tonsil and blood samples (SARS-CoV-2-recovered, n=2; SARS-CoV-2-vaccinated, n=2). Differential gene expression analyses were done using assay RNA of the integrated datasets. ## [13] bmcite.SeuratData_0.3.0 SeuratData_0.2.2 3c). However, antibody responses to several previously applied vaccines were normal in T-bet-deficient patients30. USA 104, 97709775 (2007). I used the first way as @Zha0rong described for re-clustering of subset cells, choosing a subset and then use the integration assay to Run PCA, umap, findneighbors and findclusters to do subclustering. Med. J.N. high.threshold = Inf, Each set of modal data (eg. This study was approved by the Cantonal Ethics Committee of Zurich (BASEC #2016-01440). 4 Unsupervised analysis of circulating S, Extended Data Fig. Viant, C. et al. c, Stacked bar graphs show single patient contribution to the WNN clusters. d, Violin plots of frequencies of Bm cell subsets of S+ Bm cells at the indicated time points. ## Matrix products: default 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). | AddMetaData(object = object, metadata = vector, col.name = "name") | object$name <- vector | 2d). Gupta, N. T. et al. d, Sorting strategy for S+ and S Bm cells, gated on CD19+ non-plasmablasts (non-PB, PB identified as CD38++CD27+) that were IgD and/or CD27+ and decoy, and for nave B cells, gated on CD19+ non-PB that were IgD+CD27 and S decoy. Choose a subset of cells, and use the integration assay to Run PCA, umap, findneighbors and findclusters to do subclustering. English version of Russian proverb "The hedgehogs got pricked, cried, but continued to eat the cactus", Effect of a "bad grade" in grad school applications. 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). Signature of long-lived memory CD8+ T cells in acute SARS-CoV-2 infection. Accessing data in Seurat is simple, using clearly defined accessors and setters to quickly find the data needed. Koutsakos, M. et al. You signed in with another tab or window. Qi, H., Liu, B., Wang, X. subsetting seurat object with multiple samples, Traffic: 1812 users visited in the last hour, User Agreement and Privacy Niessl, J. et al. 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).