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Weisel, F. & Shlomchik, M. Memory B cells of mice and humans. Note that @timoast from the Seurat team recommended otherwise, although I never seen an explanation why would this not best way to go. contributed to patient recruitment and data collection. Austin, J. W. et al. In e, two-sided Wilcoxon test was used with Holm multiple comparison correction. Andrews, S. F. et al. SARS-CoV-2-nave healthy controls (n=11) were sampled before their SARS-CoV-2 mRNA vaccination, at week 2 post-second dose, month 6 post-second dose and at week 2 post-third dose. ## [130] mnormt_2.1.1 sctransform_0.3.5 multcomp_1.4-22 But reading a few posts and issues here, it's not the way to go and I would like to understand why and to know how to do it properly. ## [13] htmltools_0.5.4 fansi_1.0.4 magrittr_2.0.3 6g and Extended Data Fig. 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). Similar to @amayer21 I am wondering what the best way to approach this is, and why treating a subsetted data set as new is not the correct way to run an integrated analysis pipeline? subsetting seurat object with multiple samples. We probed the Bm cell response to antigen reexposure in 35 of the 65 patients with COVID-19 who had received mRNA vaccination between month 6 and month 12 post-infection (Extended Data Fig. Just to demonstrate, a more complicated logical subset would be: And as Chase points out, %in% would be more efficient in your example: As Chase also points out, make sure you understand the difference between | and ||. The flow cytometry dataset is available upon request from the corresponding authors. e and f, UMAP represents Monocle 3 analysis on all Bm cells in scRNA-seq dataset, colored by clusters identified (e) or pseudotime annotation (f). Bioinformatics 31, 33563358 (2015). 2 Flow cytometry gating strategies and frequencies of SARS-CoV-2 spike-specific B, Extended Data Fig. 3j,k). First, we focused on samples from nonvaccinated individuals at acute infection (n=59, day 14 on average after symptom onset), month 6 (n=61, day 202 after symptom onset) and month 12 (n=17, day 374) (Fig. 3d). Shown are 30 most frequently used VH segments, sorted by hierarchical clustering, with colors indicating frequencies. ## [82] stringr_1.5.0 fastmap_1.1.1 yaml_2.3.7 This function performs differential gene expression testing for each dataset/group and combines the p-values using meta-analysis methods from the MetaDE R package. # One of these Assay objects is called the "default assay", meaning it's used for all analyses and visualization. Functional groups of genes were ordered by hierarchical clustering. Adding EV Charger (100A) in secondary panel (100A) fed off main (200A). 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. 8g). I hope it is useful. BCR diversity was slightly reduced in S+ CD21CD27FcRL5+ compared with S+ CD21+ resting Bm cells (Extended Data Fig. B cell clonality analysis was performed mainly with the changeo-10x pipeline from the Immcantation suite65 using the singularity image provided by Immcantation developers. Included were only pre-vaccination samples. Lines connect paired samples. 2e, as are preVac and nonVac SHM counts. ## [73] later_1.3.0 munsell_0.5.0 tools_4.2.0 1e,f). ## [106] lattice_0.20-45 Matrix_1.5-3 multtest_2.54.0 https://doi.org/10.1038/s41590-023-01497-y, DOI: https://doi.org/10.1038/s41590-023-01497-y. that a certain variable was either 1, 2 or 3. Does anyone has found a better solution to re-project a cluster of the dataset? Thank you for the wonderful package. Purtha, W. E., Tedder, T. F., Johnson, S., Bhattacharya, D. & Diamond, M. S. Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants. Elsner, R. A. Nat. Immunol. b, Hill numbers diversity curves show clonal diversities over a range of diversity orders for indicated S+ Bm cell subsets and nave B cells. Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine. Parabolic, suborbital and ballistic trajectories all follow elliptic paths. 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. Sci. Asking for help, clarification, or responding to other answers. ## [70] labeling_0.4.2 rlang_1.0.6 reshape2_1.4.4 VH/VL were clustered hierarchically, with colors indicating frequencies. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. low.threshold = -Inf, Reyes, R. A. et al. Long-lived plasma cells can continuously secrete high-affinity antibodies that are protective against a homologous pathogen7, whereas Bm cells encode a broader repertoire which allows protection against variants of the initial pathogen after restimulation8. Creates a Seurat object containing only a subset of the cells in the BCR and IFN- signaling appears to be a defining feature of CD21CD27 Bm cells, and probably induces and governs the T-bet-dependent transcriptional program in these cells32. ## 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. Convergent antibody responses to SARS-CoV-2 in convalescent individuals. The point is that you need a series of single comparisons, not a comparison of a series of options. Cell 162, 184197 (2015). By default, this is set to the VariableFeatures. | NoLegend | Remove all legend elements | I followed a similar approach to @attal-kush. Flow cytometry using the multimer probe approach (Extended Data Fig. Annu. ## [64] pkgconfig_2.0.3 sass_0.4.5 uwot_0.1.14 From reading the other issues posted regarding the topic it appears that any kind of re-analysis prior to integration is not recommended, and that once subsetted a integrated data set should just be re-scaled and the pipeline followed on from this point on. To extend our analyses to SARS-CoV-2-specific Bm cells in the peripheral lymphoid organs, we analyzed paired tonsil and blood samples from a cohort of 16 patients (9 females and 7 males) undergoing tonsillectomy who were exposed to SARS-CoV-2 by infection, vaccination or both. ## [31] xfun_0.37 dplyr_1.1.0 crayon_1.5.2 Sci. between condition A cluster 1 vs. condition B cluster 1 cells). 4 Unsupervised analysis of circulating S, Extended Data Fig. Semilog line was fitted to data (R2=0.2695). 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). This study was approved by the Cantonal Ethics Committee of Zurich (BASEC #2016-01440). Kim, W. et al. F1000Res. ## [79] mathjaxr_1.6-0 ggridges_0.5.4 evaluate_0.20 Hao, Y. et al. Cells are colored by timepoint (left) and by clusters identified by PhenoGraph algorithm (right). Longitudinal tracking of S+ Bm cell clones between month 6 and month 12 post-infection identified 30 persistent clones in individuals vaccinated during that period (Fig. For example, In FeaturePlot, one can specify multiple genes and also split.by to further split to multiple the conditions in the meta.data. The transcription factors ZEB2 and T-bet cooperate to program cytotoxic T cell terminal differentiation in response to LCMV viral infection. The markers were ordered by hierarchical clustering. 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. 2d). Creates a Seurat object containing only a subset of the cells in the original object. Our data showing expression of ZEB2 in CD21CD27 Bm cells suggest unidirectional plasticity, as ZEB2 acts together with T-bet to commit CD8+ effector T cells to a terminal differentiation state and has been proposed to act similarly in B cells16,40. Black lines indicate trajectory. Google Scholar. Thank you. GSEA was performed on this preranked list using the R package fgsea (v.1.2). 3g,h and Extended Data Fig. Seurat has a vast, ggplot2-based plotting library. Med. ## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 Have a question about this project? Here, we take the average expression of both the stimulated and control naive T cells and CD14 monocyte populations and generate the scatter plots, highlighting genes that exhibit dramatic responses to interferon stimulation. f, Violin plots of percentages of Ki-67+ S+ Bm cells are shown at indicated timepoints. Statistical analysis was performed with GraphPad Prism (version 9.4.1, GraphPad Software, USA) and R (version 4.1.0). (I ask because in the new integration vignette, it explicitly mentions not to run ScaleData after running the IntegrateData function)? e, Shown are gating strategy (left) and stacked bar plots (mean+standard deviation; right) of IgG+, IgM+ and IgA+ S+ Bm cells at indicated timepoints (acute, n=23; month 6, n=52; month 12, n=16). Open access funding provided by University of Zurich. Bm cells are colored by cluster (f, left), tissue origin (f, right) or SWT binding (g). For this, a count matrix was created with HC/LC segments as rows and samples as columns. ## Sci. ## other attached packages: Sakharkar, M. et al. ## [127] MASS_7.3-56 rprojroot_2.0.3 withr_2.5.0 Whereas S+ Bm cells were predominantly resting CD21+ Bm cells at month 6, vaccination strongly induced the appearance of S+ CD21CD27+ and CD21CD27 Bm cells in blood (Fig. Immunity 53, 11361150 (2020). Note, that tested this on one data set only so far. | RotatedAxis | Rotates x-axis labels |. Looking for job perks? I have a Seurat object that I have run through doubletFinder. assay = NULL, 2b,c). it makes no sense to me the not to use the integrated assay on every downstream analysis. Immunity 52, 842855.e6 (2020). max per cell ident. The DotPlot() function with the split.by parameter can be useful for viewing conserved cell type markers across conditions, showing both the expression level and the percentage of cells in a cluster expressing any given gene. Immunol. A. et al. After discussing with colleagues and reading other articles I decided to go for option b). A longitudinal cohort (Extended Data Fig. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. 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. RNA, ADT, etc.) Extended Data Fig. All the best, and O.B. Kurosaki, T., Kometani, K. & Ise, W. Memory B cells. | ----------- | ----------- | By clicking Accept all cookies, you agree Stack Exchange can store cookies on your device and disclose information in accordance with our Cookie Policy. Hopp, C. S. et al. Correspondence to 5a and Extended Data Fig. | object@dr$pca | object[["pca"]] | SCT_integrated <- FindClusters(SCT_integrated), control_subset <- subset(SCT_integrated, orig.ident = 'Chow') 9b). Eight patients were vaccinated against SARS-CoV-2 (analyzed on average at day 144 after last vaccination), whereas the other eight patients were considered SARS-CoV-2-recovered based on a history of SARS-CoV-2 infection or positive anti-nucleocapsid (N) serum antibody measurement, with six of them additionally vaccinated against SARS-CoV-2 (assessed on average at day 118 post-last vaccination) (Extended Data Fig. contributed to patient recruitment. 5f,g). Here we plot 2-3 strong marker genes for each of our 14 clusters. | object@cell.names | colnames(x = object) | 5 Flow cytometry analysis of tonsillar and circulating SARS-CoV-2-specific B. Can I general this code to draw a regular polyhedron? | Seurat v2.X | Seurat v3.X | Yang, R. et al. By clicking Accept all cookies, you agree Stack Exchange can store cookies on your device and disclose information in accordance with our Cookie Policy. Thanks for contributing an answer to Stack Overflow! All plotting functions will return a ggplot2 plot by default, allowing easy customization with ggplot2. Dan, J. M. et al. It only takes a minute to sign up. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. a, Sorting strategy for SARS-CoV-2 S+ Bm cells and S B cells, gated on CD19+ non-PB, for scRNA-seq is provided. Conversely, CD21+CD27+ and CD21+CD27 Bm cells were prominent at months 6 and 12, amounting to 60.5% and 29.1% of S+ Bm cells at month 12, respectively (Fig. Lines connect paired samples. Immunol. ## [37] survival_3.3-1 zoo_1.8-11 glue_1.6.2 Stack Exchange network consists of 181 Q&A communities including Stack Overflow, the largest, most trusted online community for developers to learn, share their knowledge, and build their careers. Following 20min staining with fixable viability dye eFluor 780 (eBioscience) and TruStain FcX and subsequently 1h antigen-specific staining mix, cells were incubated at 4C for 30min with a surface staining mix containing fluorescently labeled and barcoded antibodies, and each sample was marked with a hashtag antibody that allowed multiplexing (Supplementary Table 6). J. Is there a way to do that? For f and g, statistical analysis of the gene set enrichment and variation analyses was performed as outlined in Methods, and all adjusted P values are shown. Samples in bd were compared using KruskalWallis test with Dunns multiple comparison correction, showing adjusted P values if significant. I tried. 23, 10081020 (2022). Analysis of V heavy and light chain frequencies identified several chains enriched in RBD+ Bm cells compared with RBD Bm cells described to encode RBD-binding antibodies, including IGHV3-30, IGHV3-53, IGHV3-66, IGKV1-9 and IGKV1-33 (refs. Invest. It did always just select values that matched the first of the criteria, here 1. The clonality distance threshold was set to 0.20 for the longitudinal analysis of the SARS-CoV-2 Infection Cohort dataset and to 0.05 for the SARS-CoV-2 Tonsil Cohort dataset. Rev. Which of course included re-calculating the variable genes (on the "RNA" Slot) and re-integration. Btw, regarding DE analysis in your question 1, according to #1836 (comment), it says that both RNA and SCT assay could be used for DE analysis if my understanding is correct. 1e). Unexpected uint64 behaviour 0xFFFF'FFFF'FFFF'FFFF - 1 = 0? Each of the cells in cells.1 exhibit a higher level than each of the cells in cells.2). You signed in with another tab or window. 4ac). Single-cell RNA-seq: Pseudobulk differential expression analysis I'm writing here to be sure to receive an email when somebody will post an explanation here :-). I can figure out what it is by doing the following: Where meta_data = 'DF.classifications_0.25_0.03_252' and is a character class. Frequencies were compared in c using two-tailed Mann Whitney test, in d and e with a two-tailed Wilcoxon matched-pairs signed rank test and in g with a Kruskal-Wallis test with a Dunns multiple comparison correction, showing adjusted P values. Department of Immunology, University Hospital Zurich, Zurich, Switzerland, Yves Zurbuchen,Patrick Taeschler,Sarah Adamo,Carlo Cervia,Miro E. Raeber,Jakob Nilsson,Klaus Warnatz&Onur Boyman, Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland, Jan Michler,Ilhan E. Acar&Andreas E. Moor, Department of Rheumatology and Clinical Immunology, Faculty of Medicine, University of Freiburg, Freiburg, Germany, Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, Freiburg, Germany, Department of Otorhinolaryngology, Head and Neck Surgery, University and University Hospital Zurich, Zurich, Switzerland, Faculty of Medicine and Faculty of Science, University of Zurich, Zurich, Switzerland, You can also search for this author in Compared with their circulating counterparts, tonsillar S+ and N+ Bm cells expressed, on average, more CD69, less Ki-67, reduced T-bet and several chemokine receptors differently (Fig. contributed reagents and interpreted data. 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). Studies in patients with SLE or HIV infection have suggested that CD21CD27 Bm cells differentiate through an extrafollicular pathway16,17. Because we are confident in having identified common cell types across condition, we can ask what genes change in different conditions for cells of the same type. b, Representative flow cytometry plots show gating strategy for RBD+ Bm cells in patient CoV-P1, as in Fig. M.E.R. Thank you @satijalab for this amazing tool and the amazing tutorials !!!! I have been following the SCTransform integration tutorial and it doesn't mention how to FindClusters or identify cluster specific markers. The S+ CD21CD27 Bm cells identified here were transcriptionally very similar to their atypical counterparts in SLE. ## Platform: x86_64-pc-linux-gnu (64-bit) 31,32). DefaultAssay(control_subset) <- "RNA" Sci. c, Venn diagram shows clonal overlap of SARS-CoV-2-specific clones in different Bm cell subsets. Y.Z. 6, eabk0894 (2021). Learn more about Stack Overflow the company, and our products. They donated blood before vaccination, at days 813 (week 2) post-second dose, 6months after the second dose and days 1114 post-third dose. Then find the DEGs between 2 clusters with FindMarkers(ident.1=, ident.2=). f,g, WNN UMAP of Bm cells was derived from scRNA-seq analysis of blood and tonsillar B cells (n=4). J. Exp. | rownames(x = object@data) | rownames(x = object) | | WhichCells(object = object, max.cells.per.ident = 500) | WhichCells(object = object, downsample = 500) | Hi @vertesy , PubMed Niessl, J. et al. 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. Not the answer you're looking for? These circulating resting Bm cells might be able to rapidly respond to antigen rechallenge with the acquisition of different Bm cell fates or they might home to secondary lymphoid and peripheral organs to form a CD69+ tissue-resident Bm cells. JCI Insight 2, e92943 (2017). Qi, H., Liu, B., Wang, X. Seurat (version 3.1.4) Cervia, C. et al. Thank you @satijalab !!!! This process consists of data normalization and variable feature selection, data scaling, a PCA on variable features, construction of a shared-nearest-neighbors graph, and clustering using a modularity optimizer. CD21CD27 Bm cells have also been identified during acute SARS-CoV-2 infection and post-SARS-CoV-2 vaccination22,25,26,27,28,29. 8a). ## [58] httr_1.4.5 RColorBrewer_1.1-3 TFisher_0.2.0