Help! ## [15] Seurat_4.2.1.9001 Although, in this work, we only consider the simple model presented above, the model could be extended to allow for systematic variation between cells by imposing a regression model in stage ii. The number of genes detected by wilcox, NB, MAST, DESeq2, Monocle and mixed were 6928, 7943, 7368, 4512, 5982 and 821, respectively. We will call genes significant here if they have FDR < 0.01 and a log2 fold change of 0.58 (equivalent to a fold-change of 1.5). Overall, the subject and mixed methods had the highest concordance between permutation and method P-values. Nine simulation settings were considered. We also assume that cell types or states have been identified, DS analysis will be performed within each cell type of interest and henceforth, the notation corresponds to one cell type. The value of pDE describes the relative number of differentially expressed genes in a simulated dataset, and the value of controls the signal-to-noise ratio. I prefer to apply a threshold when showing Volcano plots, displaying any points with extreme / impossible p-values (e.g. The color represents the average expression level, # Single cell heatmap of feature expression, # Plot a legend to map colors to expression levels. Introduction. Create volcano plot. Single-cell RNA-sequencing (scRNA-seq) provides more granular biological information than bulk RNA-sequencing; bulk RNA sequencing remains popular due to lower costs which allows processing more biological replicates and design more powerful studies. 1. healthy versus disease), an additional layer of variability is introduced. Increasing sequencing depth can reduce technical variation and achieve more precise expression estimates, and collecting samples from more subjects can increase power to detect differentially expressed genes. This interactive plotting feature works with any ggplot2-based scatter plots (requires a geom_point layer). ## [28] dplyr_1.1.1 crayon_1.5.2 jsonlite_1.8.4 #' @return Returns a volcano plot from the output of the FindMarkers function from the Seurat package, which is a ggplot object that can be modified or plotted. The recall, also known as the true positive rate (TPR), is the fraction of differentially expressed genes that are detected. ## BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3 With Seurat, all plotting functions return ggplot2-based plots by default, allowing one to easily capture and manipulate plots just like any other ggplot2-based plot. These approaches will likely yield better type I and type II error rate control, but as we saw for the mixed method in our simulation, the computation times can be substantially longer and the computational burden of these methods scale with the number of cells, whereas the pseudobulk method scales with the number of subjects. ## [115] MASS_7.3-56 rprojroot_2.0.3 withr_2.5.0 Rows correspond to different proportions of differentially expressed genes, pDE and columns correspond to different SDs of (natural) log fold change, . The marginal distribution of Kij is approximately negative binomial with mean ij=sjqij and variance ij+iij2. The volcano plot that is being produced after this analysis is wierd and seems not to be correct. Here, we introduce a mathematical framework for modeling different sources of biological variation introduced in scRNA-seq data, and we provide a mathematical justification for the use of pseudobulk methods for DS analysis. I have scoured the web but I still cannot figure out how to do this. ## [79] fitdistrplus_1.1-8 purrr_1.0.1 RANN_2.6.1 # search for positive markers monocyte.de.markers <- FindMarkers (pbmc, ident.1 = "CD14+ Mono", ident.2 = NULL, only.pos = TRUE) head (monocyte.de.markers) Figure 5 shows the results of the marker detection analysis. First, the CF and non-CF labels were permuted between subjects. Supplementary data are available at Bioinformatics online. In this case, Cj-1csjc=sj* and Cj-1csjc2=sj*2, and the theorem holds. The subject method has the strongest type I error rate control and highest PPVs, wilcox has the highest TPRs and mixed has intermediate performance with better TPRs than subject yet lower FPRs than wilcox (Supplementary Table S2). In a scRNA-seq study of human tracheal epithelial cells from healthy subjects and subjects with idiopathic pulmonary fibrosis (IPF), the authors found that the basal cell population contained specialized subtypes (Carraro et al., 2020). Whereas the pseudobulk method is a simple approach to DS analysis, it has limitations. Below is a brief demonstration but please see the patchwork package website here for more details and examples. In addition to returning a vector of cell names, CellSelector() can also take the selected cells and assign a new identity to them, returning a Seurat object with the identity classes already set. Multiple methods and bioinformatic tools exist for initial scRNA-seq data processing, including normalization, dimensionality reduction, visualization, cell type identification, lineage relationships and differential gene expression (DGE) analysis (Chen et al., 2019; Hwang et al., 2018; Luecken and Theis, 2019; Vieth et al., 2019; Zaragosi et al., 2020). It enables quick visual identification of genes with large fold changes that are also statistically significant. However, a better approach is to avoid using p-values as quantitative / rankable results in plots; they're not meant to be used in that way. Default is set to Inf. As scRNA-seq costs have decreased, collecting data from more than one biological replicate has become more feasible, but careful modeling of different layers of biological variation remains challenging for many users. If a gene was differentially expressed, i2 was simulated from a normal distribution with mean 0 and standard deviation (SD) . Applying the assumptions Cj-1csjck1 and Cj-1csjc2k2 completes the proof. d Volcano plots showing DE between T cells from random groups of unstimulated controls drawn . Data for the analysis of human trachea were obtained from GEO accessions GSE143705 (bulk RNA-seq) and GSE143706 (scRNA-seq). To illustrate scalability and performance of various methods in real-world conditions, we show results in a porcine model of cystic fibrosis and analyses of skin, trachea and lung tissues in human sample datasets. We can then change the identity of these cells to turn them into their own mini-cluster. ## [9] panc8.SeuratData_3.0.2 ifnb.SeuratData_3.1.0 For example, consider a hypothetical gene having heterogeneous expression in CF pigs, where cells were either low expressors or high expressors versus homogeneous expression in non-CF pigs, where cells were moderate expressors. The FindAllMarkers () function has three important arguments which provide thresholds for determining whether a gene is a marker: logfc.threshold: minimum log2 fold change for average expression of gene in cluster relative to the average expression in all other clusters combined. We have found this particularly useful for small clusters that do not always separate using unbiased clustering, but which look tantalizingly distinct. Comparisons of characteristics of the simulated and real data are shown in Supplementary Figures S1S6. PR curves for DS analysis methods. The lists of genes detected by the other six methods likely contain many false discoveries. ## [3] thp1.eccite.SeuratData_3.1.5 stxBrain.SeuratData_0.1.1 ## [70] ggridges_0.5.4 evaluate_0.20 stringr_1.5.0 I have been following the Satija lab tutorials and have found them intuitive and useful so far. The main idea of the theorem is that if gene counts are summed across cells and the number of cells grows large for each subject, the influence of cell-level variation on the summed counts is negligible. Define Kijc to be the count for gene i in cell ccollected from subject j, and a size factorsjc related to the amount of information collected from cell c in subject j (i=1,G; c=1,,Cj;j=1,,n). The use of the dotplot is only meaningful when the counts matrix contains zeros representing no gene counts. ## [16] cluster_2.1.3 ROCR_1.0-11 limma_3.54.1 We evaluated the performance of our tested approaches for human multi-subject DS analysis in health and disease. Aggregation technique accounting for subject-level variation in DS analysis. To measure heterogeneity in expression among different groups, we assume that mean expression for gene iin subject j is influenced by R subject-specific covariates xj1,,xjR. This can, # be changed with the `group.by` parameter, # Use community-created themes, overwriting the default Seurat-applied theme Install ggmin, # with remotes::install_github('sjessa/ggmin'), # Seurat also provides several built-in themes, such as DarkTheme; for more details see, # Include additional data to display alongside cell names by passing in a data frame of, # information Works well when using FetchData, ## [1] "AAGATTACCGCCTT" "AAGCCATGAACTGC" "AATTACGAATTCCT" "ACCCGTTGCTTCTA", # Now, we find markers that are specific to the new cells, and find clear DC markers, ## p_val avg_log2FC pct.1 pct.2 p_val_adj, ## FCER1A 3.239004e-69 3.7008561 0.800 0.017 4.441970e-65, ## SERPINF1 7.761413e-36 1.5737896 0.457 0.013 1.064400e-31, ## HLA-DQB2 1.721094e-34 0.9685974 0.429 0.010 2.360309e-30, ## CD1C 2.304106e-33 1.7785158 0.514 0.025 3.159851e-29, ## ENHO 5.099765e-32 1.3734708 0.400 0.010 6.993818e-28, ## ITM2C 4.299994e-29 1.5590007 0.371 0.010 5.897012e-25, ## [1] "selected" "Naive CD4 T" "Memory CD4 T" "CD14+ Mono" "B", ## [6] "CD8 T" "FCGR3A+ Mono" "NK" "Platelet", # LabelClusters and LabelPoints will label clusters (a coloring variable) or individual points, # Both functions support `repel`, which will intelligently stagger labels and draw connecting, # lines from the labels to the points or clusters, ## Platform: x86_64-pc-linux-gnu (64-bit), ## BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3, ## LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/liblapack.so.3, ## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C, ## [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8, ## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8, ## [7] LC_PAPER=en_US.UTF-8 LC_NAME=C, ## [9] LC_ADDRESS=C LC_TELEPHONE=C, ## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C, ## [1] stats graphics grDevices utils datasets methods base, ## [1] patchwork_1.1.2 ggplot2_3.4.1, ## [3] thp1.eccite.SeuratData_3.1.5 stxBrain.SeuratData_0.1.1, ## [5] ssHippo.SeuratData_3.1.4 pbmcsca.SeuratData_3.0.0, ## [7] pbmcMultiome.SeuratData_0.1.2 pbmc3k.SeuratData_3.1.4, ## [9] panc8.SeuratData_3.0.2 ifnb.SeuratData_3.1.0, ## [11] hcabm40k.SeuratData_3.0.0 bmcite.SeuratData_0.3.0, ## [13] SeuratData_0.2.2 SeuratObject_4.1.3. So, If I change the assay to "RNA", how we can trust that the DEGs are not due . RNA-seqR "Seurat" FindMarkers() FindMarkers() Volcano plotMA plot Among the other five methods, when the number of differentially expressed genes was small (pDE = 0.01), the mixed method had the highest PPV values, whereas for higher numbers of differentially expressed genes (pDE > 0.01), the DESeq2 method had the highest PPV values. Was this translation helpful? For each method, the computed P-values for all genes were adjusted to control the FDR using the BenjaminiHochberg procedure (Benjamini and Hochberg, 1995). This is the model used in DESeq2 (Love et al., 2014). Yes, you can use the second one for volcano plots, but it might help to understand what it's implying. Then, for each method, we defined the permutation test statistic to be the unadjusted P-value generated by the method. For each method, we compared the permutation P-values to the P-values directly computed by each method, which we define as the method P-values. I prefer to apply a threshold when showing Volcano plots, displaying any points with extreme / impossible p-values (e.g. FindMarkers: Finds markers (differentially expressed genes) for identified clusters. Here, we present the DS results comparing CF and non-CF pigs only in secretory cells from the small airways. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. 1). For a sequence of cutoff values between 0 and 1, precision, also known as positive predictive value (PPV), is the fraction of genes with adjusted P-values less than a cutoff (detected genes) that are differentially expressed. Returns a volcano plot from the output of the FindMarkers function from the Seurat package, which is a ggplot object that can be modified or plotted. In Supplementary Figure S14(ef), we quantify the ability of each method to correctly identify markers of T cells and macrophages from a database of known cell type markers (Franzen et al., 2019). It sounds like you want to compare within a cell cluster, between cells from before and after treatment. Carver College of Medicine, University of Iowa. If we omit DESeq2, which seems to be an outlier, the other six methods form two distinct clusters, with cluster 1 composed of wilcox, NB, MAST and Monocle, and cluster 2 composed of subject and mixed. I understand a little bit more now. (2019) used scRNA-seq to profile cells from the lungs of healthy subjects and those with pulmonary fibrosis disease subtypes, including hypersensitivity pneumonitis, systemic sclerosis-associated and myositis-associated interstitial lung diseases and IPF (Reyfman et al., 2019). Overall, these results suggest that the current marker detection analysis tools used in common practice, such as wilcox, will produce a reliable set of markers. As scRNA-seq studies grow in scope, due to technological advances making these studies both less labor-intensive and less expensive, biological replication will become the norm. Volcano plots in R: complete script. In extreme cases, where only a few cells have been collected for some subjects, interpretation of gene expression differences should be handled with caution. I have successfully installed ggplot, normalized my datasets, merged the datasets, etc., but what I do not understand is how to transfer the sequencing data to the ggplot function. Cons: Specifically, if Kijc is the count of gene i in cell c from pig j, we defined Eijc=Kijc/i'Ki'jc to be the normalized expression for cell c from subject j and Eij=cKijc/i'cKi'jc to be the normalized expression for subject j. Rows correspond to different proportions of differentially expressed genes, pDE and columns correspond to different SDs of (natural) log fold change, . For the AM cells (Fig. As you can see, there are four major groups of genes: - Genes that surpass our p-value and logFC cutoffs (blue). We propose an extension of the negative binomial model to scRNA-seq data by introducing an additional stage in the model hierarchy. "t" : Student's t-test. When only 1% of genes were differentially expressed (pDE = 0.01), all methods had NPV values near 1. does allah forgive if you hurt someone,
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