6f), the results are similar to AT2 cells with subject having the highest areas under the ROC and PR curves (0.88 and 0.15, respectively), followed by mixed (0.86 and 0.05, respectively) and wilcox (0.83 and 0.01, respectively). "poisson" : Likelihood ratio test assuming an . Our study highlights user-friendly approaches for analysis of scRNA-seq data from multiple biological replicates. RNA-Seq Data Heatmap: Is it necessary to do a log2 . Each panel shows results for 100 simulated datasets in one simulation setting. Analysis of AT2 cells and AMs from healthy and IPF lungs. S14f), wilcox produces better ranked gene lists of known markers than both subject and wilcox and again, the mixed method has the worst performance. Step 4: Customise it! I keep receiving an error that says: "data must be a , or an object coercible by fortify(), not an S4 object with class . The analyses presented here have illustrated how different results could be obtained when data were analysed using different units of analysis. ## [43] miniUI_0.1.1.1 Rcpp_1.0.10 viridisLite_0.4.1 For macrophages (Supplementary Fig. Single-cell RNA-sequencing (scRNA-seq) enables analysis of the effects of different conditions or perturbations on specific cell types or cellular states. One such subtype, defined by expression of CD66, was further processed by sorting basal cells according to detection of CD66 and profiling by bulk RNA-seq. ## [49] htmlwidgets_1.6.2 httr_1.4.5 RColorBrewer_1.1-3 The marginal distribution of Kij is approximately negative binomial with mean ij=sjqij and variance ij+iij2. (a) t-SNE plot shows AT2 cells (red) and AM (green) from single-cell RNA-seq profiling of human lung from healthy subjects and subjects with IPF. In addition to the inference reports and the associated Volcano plot views that allow users to visualize the distribution of fold change of all genes from say, one cluster to another, or one cluster to all cells, users can also visualize the normalized read . Marker detection methods were found to have unacceptable FDR due to pseudoreplication bias, in which cells from the same individual are correlated but treated as independent replicates, and pseudobulk methods were found to be too conservative, in the sense that too many differentially expressed genes were undiscovered. Rows correspond to different proportions of differentially expressed genes, pDE and columns correspond to different SDs of (natural) log fold change, . ## [73] fastmap_1.1.1 yaml_2.3.7 ragg_1.2.5 For the T cells, (Supplementary Fig. (Crowell et al., 2020) provides a thorough comparison of a variety of DGE methods for scRNA-seq with biological replicates including: (i) marker detection methods, (ii) pseudobulk methods, where gene counts are aggregated between cells from different biological samples and (iii) mixed models, where models for gene expression are adjusted for sample-specific or batch effects. ## [13] SeuratData_0.2.2 SeuratObject_4.1.3 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. (a) AUPR, (b) PPV with adjusted P-value cutoff 0.05 and (c) NPV with adjusted P-value cutoff 0.05 for 7 DS analysis methods. Applying themes to plots. We proceed as follows. Simply add the splitting variable to object, # metadata and pass it to the split.by argument, # SplitDotPlotGG has been replaced with the `split.by` parameter for DotPlot, # DimPlot replaces TSNEPlot, PCAPlot, etc. The implemented methods are subject (red), wilcox (blue), NB (green), MAST (purple), DESeq2 (orange), monocle (gold) and mixed (brown). Search for other works by this author on: Iowa Institute of Human Genetics, Roy J. and Lucille A. Published by Oxford University Press. The other six methods involved DS testing with cells as the units of analysis. This model implicitly assumes that the only systematic variation in expression is due to subject-level covariates, and for a fixed level of covariates, any additional variation between subjects or cells is due to chance. DGE methods to address this additional complexity, which have been referred to as differential state (DS) analysis are just being explored in the scRNA-seq field (Crowell et al., 2020; Lun et al., 2016; McCarthy et al., 2017; Van den Berge et al., 2019; Zimmerman et al., 2021). To consider characteristics of a real dataset, we matched fixed quantities and parameters of the model to empirical values from a small airway secretory cell subset from the newborn pig data we present again in Section 3.2. 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. When samples correspond to different experimental subjects, the first stage characterizes biological variation in gene expression between subjects. Data for the analysis of human trachea were obtained from GEO accessions GSE143705 (bulk RNA-seq) and GSE143706 (scRNA-seq). For example, a simple definition of sjc is the number of unique molecular identifiers (UMIs) collected from cell c of subject j. As increases, the width of the distribution of effect sizes increases, so that the signal-to-noise ratio for differentially expressed genes is larger. 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. 5c). 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. Results for alternative performance measures, including receiver operating characteristic (ROC) curves, TPRs and false positive rates (FPRs) can be found in Supplementary Figures S7 and S8. The null and alternative hypotheses for the i-th gene are H0i:i2=0 and H0i:i20, respectively. Second, there may be imbalances in the numbers of cells collected from different subjects. Red and blue dots represent genes with a log 2 FC (fold . The data from pig airway epithelia underlying this article are available in GEO and can be accessed with GEO accession GSE150211. ## loaded via a namespace (and not attached): ## [1] systemfonts_1.0.4 plyr_1.8.8 igraph_1.4.1, ## [4] lazyeval_0.2.2 sp_1.6-0 splines_4.2.0, ## [7] crosstalk_1.2.0 listenv_0.9.0 scattermore_0.8, ## [10] digest_0.6.31 htmltools_0.5.5 fansi_1.0.4, ## [13] magrittr_2.0.3 memoise_2.0.1 tensor_1.5, ## [16] cluster_2.1.3 ROCR_1.0-11 limma_3.54.1, ## [19] globals_0.16.2 matrixStats_0.63.0 pkgdown_2.0.7, ## [22] spatstat.sparse_3.0-1 colorspace_2.1-0 rappdirs_0.3.3, ## [25] ggrepel_0.9.3 textshaping_0.3.6 xfun_0.38, ## [28] dplyr_1.1.1 crayon_1.5.2 jsonlite_1.8.4, ## [31] progressr_0.13.0 spatstat.data_3.0-1 survival_3.3-1, ## [34] zoo_1.8-11 glue_1.6.2 polyclip_1.10-4, ## [37] gtable_0.3.3 leiden_0.4.3 future.apply_1.10.0, ## [40] abind_1.4-5 scales_1.2.1 spatstat.random_3.1-4, ## [43] miniUI_0.1.1.1 Rcpp_1.0.10 viridisLite_0.4.1, ## [46] xtable_1.8-4 reticulate_1.28 ggmin_0.0.0.9000, ## [49] htmlwidgets_1.6.2 httr_1.4.5 RColorBrewer_1.1-3, ## [52] ellipsis_0.3.2 ica_1.0-3 farver_2.1.1, ## [55] pkgconfig_2.0.3 sass_0.4.5 uwot_0.1.14, ## [58] deldir_1.0-6 utf8_1.2.3 tidyselect_1.2.0, ## [61] labeling_0.4.2 rlang_1.1.0 reshape2_1.4.4, ## [64] later_1.3.0 munsell_0.5.0 tools_4.2.0, ## [67] cachem_1.0.7 cli_3.6.1 generics_0.1.3, ## [70] ggridges_0.5.4 evaluate_0.20 stringr_1.5.0, ## [73] fastmap_1.1.1 yaml_2.3.7 ragg_1.2.5, ## [76] goftest_1.2-3 knitr_1.42 fs_1.6.1, ## [79] fitdistrplus_1.1-8 purrr_1.0.1 RANN_2.6.1, ## [82] pbapply_1.7-0 future_1.32.0 nlme_3.1-157, ## [85] mime_0.12 formatR_1.14 compiler_4.2.0, ## [88] plotly_4.10.1 png_0.1-8 spatstat.utils_3.0-2, ## [91] tibble_3.2.1 bslib_0.4.2 stringi_1.7.12, ## [94] highr_0.10 desc_1.4.2 lattice_0.20-45, ## [97] Matrix_1.5-3 vctrs_0.6.1 pillar_1.9.0, ## [100] lifecycle_1.0.3 spatstat.geom_3.1-0 lmtest_0.9-40, ## [103] jquerylib_0.1.4 RcppAnnoy_0.0.20 data.table_1.14.8, ## [106] cowplot_1.1.1 irlba_2.3.5.1 httpuv_1.6.9, ## [109] R6_2.5.1 promises_1.2.0.1 KernSmooth_2.23-20, ## [112] gridExtra_2.3 parallelly_1.35.0 codetools_0.2-18, ## [115] MASS_7.3-56 rprojroot_2.0.3 withr_2.5.0, ## [118] sctransform_0.3.5 parallel_4.2.0 grid_4.2.0, ## [121] tidyr_1.3.0 rmarkdown_2.21 Rtsne_0.16, ## [124] spatstat.explore_3.1-0 shiny_1.7.4, Fast integration using reciprocal PCA (RPCA), Integrating scRNA-seq and scATAC-seq data, Demultiplexing with hashtag oligos (HTOs), Interoperability between single-cell object formats. However, in studies with biological replication, gene expression is influenced by both cell-specific and subject-specific effects. ## [19] globals_0.16.2 matrixStats_0.63.0 pkgdown_2.0.7 In (b), rows correspond to different genes, and columns correspond to different pigs. Default is 0.25. 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. We considered three values for pDE{0.01,0.3,0.6}, giving 1%, 30% and 60% of genes as differentially expressed, respectively, and we considered three values for {0.5,1.0,1.5}, representing low, medium and high signal-to-noise ratios, respectively.
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