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library(LSD)
library(SingleCellExperiment)
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library(ggplot2)
library(scater)
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library(CATALYST)
library(workflowr)
sce = readRDS(file = "data/data_for_analysis/sce_protein.rds")
for(i in 1:10){
scatter_x_y(sce,x = "CD8",y = "pERK",imagenumber =i, xlim = c(0,5), ylim = c(0,5))
abline(h = 1.25)
abline(v=1.5)
abline(a = 0, b= 1)
}
# Aggregate the counts. calculateSummary is a function written from Nils
mean_sce <- calculateSummary(sce, split_by = "celltype",
exprs_values = "counts")
# Exclude DNA and Histone
mean_sce <- mean_sce[!grepl("DNA|Histone", rownames(mean_sce)),]
# Transform and scale
assay(mean_sce, "arcsinh") <- asinh(assay(mean_sce, "meanCounts"))
assay(mean_sce, "arcsinh_scaled") <- t(scale(t(asinh(assay(mean_sce, "meanCounts")))))
# Linear scale
plotHeatmap(mean_sce, exprs_values = "arcsinh",
features = c(rownames(mean_sce)),
colour_columns_by = "celltype",
color = viridis(100), cluster_rows = FALSE, gaps_row = 10)
# Linear scale
plotHeatmap(mean_sce, exprs_values = "arcsinh_scaled",
features = c(rownames(mean_sce)),
colour_columns_by = "celltype",
color = colorRampPalette(c("dark blue", "white", "dark red"))(100),
zlim = c(-3,3), cluster_rows = TRUE, gaps_row = 10)
# Select markers
marker_list <- list()
marker_list$Tumor <- c("Ki67", "PDL1", "PARP", "H3K27me3","CXCR2","HLADR","S100","Sox9","pERK","CD36",
"p75","MiTF","bCatenin","Collagen1","pS6","IDO1","SOX10")
marker_list$Neutrophil <- c("CD15", "PDL1", "MPO", "CD11b")
marker_list$`FOXP3+ T cell` <- c("IDO1", "PD1", "FOXP3", "ICOS", "CD4", "Ki67", "GrzB",
"CD45RA", "CD45RO")
marker_list$`CD8+ T cell` <- c("IDO1", "TOX1", "PD1", "TCF7", "ICOS", "CD8","Ki67", "GrzB",
"CD45RA", "CD45RO")
marker_list$`CD4+ T cell` <- c("IDO1", "TOX1", "PD1", "TCF7",
"FOXP3", "ICOS", "CD4", "Ki67", "GrzB",
"CD45RA", "CD45RO")
marker_list$`BnT cell` <- c("IDO1", "TOX1", "PD1", "TCF7",
"FOXP3", "ICOS", "CD8", "CD4", "Ki67", "GrzB",
"CD45RA", "CD45RO", "CD20","HLADR","H3K27me3","pS6","pERK", "CD7")
marker_list$pDC <- c("CD303","IDO1","GrzB","CXCR2","CD11b")
marker_list$`B cell` <- c("Ki67","CD45RA", "CD45RO", "CD20","HLADR","H3K27me3","pS6","pERK")
marker_list$Macrophage <- c("Caveolin1", "PDL1", "PARP", "H3K27me3","CXCR2","HLADR","CD45RO","CD45RA","CD68","pERK","CD36", "Collagen1","CD11c","pS6","IDO1","CD206")
marker_list$Stroma <- c("Caveolin1", "CD68","CD36", "Collagen1","SMA")
marker_list$unknown <- rownames(sce[rowData(sce)$good_marker,])
FlowSOM first because it is faster
## the FlowSOM function from CATALYST needs an another column in the rowData of the sce to work properly:
rowData(sce)$marker_class <- "state"
# vector for clustering
fs_clustering <- vector(length = ncol(sce))
# create the "exprs" slot in the assay data (needed for CATALYST)
assay(sce, "exprs") <- assay(sce,"asinh")
# Macrophage, B cells, CD4+ T cell, CD8+ T cell, Tother and BnT cells will be clustered for a total of 6 clustes each
set.seed(12345)
for(i in c("Macrophage","CD4+ T cell","CD8+ T cell")){
cur_sce <- sce[,sce$celltype == i]
cur_sce <- CATALYST::cluster(cur_sce,features = marker_list[i][[1]], ydim = 2,xdim = 3,maxK = 4)
fs_clustering[sce$celltype == i] <- cur_sce$cluster_id
}
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
# pDCs and Neutrophils will be clustered to 4 clusteres
for(i in c("pDC","Neutrophil","BnT cell","B cell","FOXP3+ T cell","Stroma", "unknown")){
cur_sce <- sce[,sce$celltype == i]
cur_sce <- CATALYST::cluster(cur_sce,features = marker_list[i][[1]],ydim = 2,xdim = 2,maxK = 3)
fs_clustering[sce$celltype == i] <- cur_sce$cluster_id
}
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
# Tumor will be clustered into 9 clusters
cur_sce <- sce[,sce$celltype == "Tumor"]
cur_sce <- CATALYST::cluster(cur_sce,features = marker_list["Tumor"][[1]],ydim = 3,xdim = 3,maxK = 7)
o running FlowSOM clustering...
o running ConsensusClusterPlus metaclustering...
fs_clustering[sce$celltype == "Tumor"] <- cur_sce$cluster_id
# Save in SCE object
colData(sce)$celltype_clustered <- as.factor(fs_clustering)
sce$celltype_clustered <- paste0(sce$celltype, "_", sce$celltype_clustered)
# Aggregate the counts
mean_sce <- calculateSummary(sce, split_by = c("celltype","celltype_clustered"),
exprs_values = "counts")
# Exclude DNA and Histone
mean_sce <- mean_sce[!grepl("DNA|Histone", rownames(mean_sce)),]
# Transform and scale
assay(mean_sce, "arcsinh") <- asinh(assay(mean_sce, "meanCounts"))
assay(mean_sce, "arcsinh_scaled") <- t(scale(t(asinh(assay(mean_sce, "meanCounts")))))
# Linear scale
plotHeatmap(mean_sce, exprs_values = "arcsinh",
features = rownames(sce)[rowData(sce)$good_marker],
colour_columns_by = c("celltype"),
color = viridis(100),
labels_col = mean_sce$celltype_clustered,
show_colnames = TRUE, annotation_legend = FALSE, borders_color = NA)
# Scaled
plotHeatmap(mean_sce, exprs_values = "arcsinh_scaled",
features = rownames(sce)[rowData(sce)$good_marker],
colour_columns_by = c("celltype"),
labels_col = mean_sce$celltype_clustered,
show_colnames = TRUE, annotation_legend = TRUE, borders_color = NA,
color = colorRampPalette(c("dark blue", "white", "dark red"))(100),
zlim = c(-4, 4),legend = TRUE)
table(sce$celltype_clustered)
B cell_1 B cell_2 B cell_3 B cell_4 BnT cell_1
4315 11731 12914 21486 7024
BnT cell_2 BnT cell_3 BnT cell_4 CD4+ T cell_1 CD4+ T cell_2
7961 6527 9082 11334 11205
CD4+ T cell_3 CD4+ T cell_4 CD4+ T cell_5 CD4+ T cell_6 CD8+ T cell_1
9717 11569 13073 2997 3671
CD8+ T cell_2 CD8+ T cell_3 CD8+ T cell_4 CD8+ T cell_5 CD8+ T cell_6
11058 11808 12549 9536 4671
FOXP3+ T cell_1 FOXP3+ T cell_2 FOXP3+ T cell_3 FOXP3+ T cell_4 Macrophage_1
2976 2375 2827 1293 12010
Macrophage_2 Macrophage_3 Macrophage_4 Macrophage_5 Macrophage_6
8126 10022 8439 14226 9699
Neutrophil_1 Neutrophil_2 Neutrophil_3 Neutrophil_4 pDC_1
3339 2473 1770 3612 2508
pDC_2 pDC_3 pDC_4 Stroma_1 Stroma_2
1810 891 1882 14333 17709
Stroma_3 Stroma_4 Tumor_1 Tumor_2 Tumor_3
10161 18751 84616 76055 82520
Tumor_4 Tumor_5 Tumor_6 Tumor_7 Tumor_8
51185 72087 86565 43957 51597
Tumor_9 unknown_1 unknown_2 unknown_3 unknown_4
75909 2795 10315 1678 4665
annotations <- sce$celltype_clustered
# annotations[annotations == "other_4"] <- "Other_CD206Low_pS6Low"
# annotations[annotations == "other_2"] <- "Tumor_SOX9Low_SOX10+_MITF+_S100+_bCatenin++"
# annotations[annotations == "other_3"] <- "Other_SOX10Low_MITFLow_bCatenin+"
# annotations[annotations == "other_1"] <- "Other_CaveolinLow"
# annotations[annotations == "other_5"] <- "Vasculature_SMA++_Collagen+_Caveolin+"
# annotations[annotations == "other_6"] <- "Stroma_SMA+_Collagen++_Caveolin++_CD36++"
#
# annotations[annotations == "Tumor_2"] <- "Tumor_SOX9++_p75+"
# annotations[annotations == "Tumor_3"] <- "Tumor_SOX10+_MITFLow_CD36Low"
# annotations[annotations == "Tumor_4"] <- "Tumor_SOX10Low_S100+"
# annotations[annotations == "Tumor_5"] <- "Tumor_SOX9++_SOX10+_MITF+_S100Low"
# annotations[annotations == "Tumor_8"] <- "Tumor_SOX9Low_SOX10Low_MITFLow_p75+"
# annotations[annotations == "Tumor_9"] <- "Tumor_SOX10+_MITF+_S100++"
# annotations[annotations == "Tumor_6"] <- "Tumor_S100+"
# annotations[annotations == "Tumor_1"] <- "Tumor_SOX10Low_MITFLow_HLADR+"
# annotations[annotations == "Tumor_7"] <- "Tumor_SOX10+_MITF++_S100+_pS6+"
#
# annotations[annotations == "B cells_1"] <- "B cells_pErkLow_H3K27meLow"
# annotations[annotations == "B cells_4"] <- "B cells_pErkLow_H3K27meLow"
# annotations[annotations == "B cells_2"] <- "B cells_H3K27me_LowpS6Low"
# annotations[annotations == "B cells_3"] <- "B cells_CD19++_pErkLow_H3K27meLow"
# annotations[annotations == "B cells_5"] <- "B cells_Ki67++"
# annotations[annotations == "B cells_6"] <- "B cells_SMALow"
#
# annotations[annotations == "BnT_1"] <- "B cells_CD3Low_CD4Low"
# annotations[annotations == "BnT_2"] <- "B cells_pErkLow_H3K27meLow"
# annotations[annotations == "BnT_6"] <- "BnT_CD8+"
# annotations[annotations == "BnT_4"] <- "BnT_CD8+"
# annotations[annotations == "BnT_5"] <- "BnT_CD4+_TCF7+"
# annotations[annotations == "BnT_3"] <- "BnT_CD4+_ICOS++_PD1++_Ki67+"
#
# annotations[annotations == "T_other_4"] <- "Tcells_undefined"
# annotations[annotations == "T_other_5"] <- "Tcells_undefined"
# annotations[annotations == "T_other_3"] <- "Tcells_CD15_LowGrzB+"
# annotations[annotations == "T_other_1"] <- "Tcells_Ki67++"
# annotations[annotations == "T_other_2"] <- "Tcells_CaveolinLow_SMALow"
# annotations[annotations == "T_other_6"] <- "Tcells_undefined"
#
# annotations[annotations == "T_helper_2"] <- "CD4+ T cell_CD11cLow_HLADRLow"
# annotations[annotations == "T_helper_5"] <- "CD4+ T cell_SMALow_CaveolinLow"
# annotations[annotations == "T_helper_1"] <- "CD4+ T cell_TCF7++"
# annotations[annotations == "T_helper_6"] <- "CD4+ T cell_FoxP3++_ICOS+_CD11cLow_CD206Low"
# annotations[annotations == "T_helper_3"] <- "CD4+ T cell_FoxP3Low_pERKLow"
# annotations[annotations == "T_helper_4"] <- "CD4+ T cell_FoxP3Low"
#
# annotations[annotations == "T_cytotoxic_3"] <- "CD8+ T cell_PD1+_TOX1+_GrzBLow"
# annotations[annotations == "T_cytotoxic_5"] <- "CD8+ T cell_TCF7+_H3K27meLow"
# annotations[annotations == "T_cytotoxic_6"] <- "CD8+ T cell_PD1+_TOX1+_GrzB+_ICOSLow_Ki67+"
# annotations[annotations == "T_cytotoxic_1"] <- "CD8+ T cell_GrzBLow_SMALow_CaveolinLow"
# annotations[annotations == "T_cytotoxic_2"] <- "CD8+ T cell_GrzBLow_pS6Low"
# annotations[annotations == "T_cytotoxic_4"] <- "CD8+ T cell_PD1+_TOX1+_GrzBLow_ICOSLow_CD11cLow_CD206Low"
#
# annotations[annotations == "Macrophage_4"] <- "Macrophage_CD68Low_CaveolinLow"
# annotations[annotations == "Macrophage_6"] <- "Tumor_CD68Low_S100Low_bCateninLow_MITFLow_SOX10Low_HLADR+"
# annotations[annotations == "Macrophage_2"] <- "Macrophage_CD68Low_CD206++_CD36Low"
# annotations[annotations == "Macrophage_1"] <- "Macrophage_CD68Low_pS6Low_S100Low"
# annotations[annotations == "Macrophage_3"] <- "Macrophage_CD68+_CD11c+_CD206++_CXCR2+_PDL1+"
# annotations[annotations == "Macrophage_5"] <- "Macrophage_CD68++_CD11c+_CD36+_Caveolin+_Collagen+"
#
# annotations[annotations == "pDC_1"] <- "pDC_IDO1+_GrzB+"
# annotations[annotations == "pDC_2"] <- "pDC_IDO1+_TOX1+_CD11b+"
# annotations[annotations == "pDC_3"] <- "pDC_IDO1++_GrzB++"
# annotations[annotations == "pDC_4"] <- "pDC_Ido1+_PARP++"
#
# annotations[annotations == "Neutrophil_2"] <- "Neutrophil_CD11b+_CD15++_MPO+_PDL1++"
# annotations[annotations == "Neutrophil_4"] <- "Neutrophil_CD11b+_CD15+_MPO+_PDL1Low"
# annotations[annotations == "Neutrophil_3"] <- "Neutrophil_CD11b++_CD15++_MPO++_CXCR2Low_PDL1+"
# annotations[annotations == "Neutrophil_1"] <- "Neutrophil_CD11b+_CD15+_MPO++"
sce$cellAnnotation <- annotations
mean_sce <- calculateSummary(sce, split_by = c("celltype","cellAnnotation"),
exprs_values = "counts")
# Exclude DNA and Histone
mean_sce <- mean_sce[!grepl("DNA|Histone", rownames(mean_sce)),]
# Transform and scale
assay(mean_sce, "arcsinh") <- asinh(assay(mean_sce, "meanCounts"))
assay(mean_sce, "arcsinh_scaled") <- t(scale(t(asinh(assay(mean_sce, "meanCounts")))))
plotHeatmap(mean_sce, exprs_values = "arcsinh_scaled",
features = rownames(sce)[rowData(sce)$good_marker],
colour_columns_by = c("celltype"),
labels_col = mean_sce$cellAnnotation,
show_colnames = TRUE, annotation_legend = TRUE, borders_color = NA,
color = colorRampPalette(c("dark blue", "white", "dark red"))(100),
zlim = c(-4, 4),legend = TRUE)
clustering does not really pick up PDL1 positive Tumor cells (which definitely exist). Potentially we have to investigate this with a manual cut off. arcsinh > 1.75 seems to be reasonable
Additionally, the clustering did not pick up proliferating and non prolifertating tumor clusters. this should also be investigated by manual cut offs. arcsinh > 1.25 seems reasonable.
celltype_counts <- sce$celltype
table(celltype_counts)
# delete the "exprs" slot from the single cell experiment again.
assay(sce,"exprs") <- NULL
saveRDS(sce,file = "data/data_for_analysis/sce_protein.rds")
sessionInfo()
R version 4.1.2 (2021-11-01)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.3 LTS
Matrix products: default
BLAS/LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.8.so
locale:
[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
attached base packages:
[1] stats4 stats graphics grDevices utils datasets methods
[8] base
other attached packages:
[1] CATALYST_1.18.1 igraph_1.2.11
[3] viridis_0.6.2 viridisLite_0.4.0
[5] scater_1.22.0 scuttle_1.4.0
[7] ggplot2_3.3.5 SingleCellExperiment_1.16.0
[9] SummarizedExperiment_1.24.0 Biobase_2.54.0
[11] GenomicRanges_1.46.1 GenomeInfoDb_1.30.1
[13] IRanges_2.28.0 S4Vectors_0.32.3
[15] BiocGenerics_0.40.0 MatrixGenerics_1.6.0
[17] matrixStats_0.61.0 LSD_4.1-0
[19] dplyr_1.0.7 workflowr_1.7.0
loaded via a namespace (and not attached):
[1] utf8_1.2.2 tidyselect_1.1.1
[3] grid_4.1.2 BiocParallel_1.28.3
[5] Rtsne_0.15 aws.signature_0.6.0
[7] flowCore_2.6.0 munsell_0.5.0
[9] ScaledMatrix_1.2.0 codetools_0.2-18
[11] withr_2.4.3 colorspace_2.0-2
[13] highr_0.9 knitr_1.37
[15] rstudioapi_0.13 ggsignif_0.6.3
[17] git2r_0.29.0 GenomeInfoDbData_1.2.7
[19] polyclip_1.10-0 farver_2.1.0
[21] pheatmap_1.0.12 flowWorkspace_4.6.0
[23] rprojroot_2.0.2 vctrs_0.3.8
[25] generics_0.1.2 TH.data_1.1-0
[27] xfun_0.29 R6_2.5.1
[29] doParallel_1.0.16 ggbeeswarm_0.6.0
[31] clue_0.3-60 rsvd_1.0.5
[33] bitops_1.0-7 DelayedArray_0.20.0
[35] assertthat_0.2.1 promises_1.2.0.1
[37] scales_1.1.1 multcomp_1.4-18
[39] beeswarm_0.4.0 gtable_0.3.0
[41] beachmat_2.10.0 processx_3.5.2
[43] RProtoBufLib_2.6.0 sandwich_3.0-1
[45] rlang_1.0.0 GlobalOptions_0.1.2
[47] splines_4.1.2 rstatix_0.7.0
[49] hexbin_1.28.2 broom_0.7.12
[51] reshape2_1.4.4 yaml_2.2.2
[53] abind_1.4-5 backports_1.4.1
[55] httpuv_1.6.5 RBGL_1.70.0
[57] tools_4.1.2 ellipsis_0.3.2
[59] jquerylib_0.1.4 RColorBrewer_1.1-2
[61] ggridges_0.5.3 Rcpp_1.0.8
[63] plyr_1.8.6 base64enc_0.1-3
[65] sparseMatrixStats_1.6.0 zlibbioc_1.40.0
[67] purrr_0.3.4 RCurl_1.98-1.5
[69] ps_1.6.0 FlowSOM_2.2.0
[71] ggpubr_0.4.0 GetoptLong_1.0.5
[73] cowplot_1.1.1 zoo_1.8-9
[75] ggrepel_0.9.1 cluster_2.1.2
[77] colorRamps_2.3 fs_1.5.2
[79] magrittr_2.0.2 ncdfFlow_2.40.0
[81] data.table_1.14.2 scattermore_0.7
[83] circlize_0.4.13 mvtnorm_1.1-3
[85] whisker_0.4 ggnewscale_0.4.5
[87] evaluate_0.14 XML_3.99-0.8
[89] jpeg_0.1-9 gridExtra_2.3
[91] shape_1.4.6 ggcyto_1.22.0
[93] compiler_4.1.2 tibble_3.1.6
[95] crayon_1.4.2 ggpointdensity_0.1.0
[97] htmltools_0.5.2 later_1.3.0
[99] tidyr_1.2.0 RcppParallel_5.1.5
[101] aws.s3_0.3.21 DBI_1.1.2
[103] tweenr_1.0.2 ComplexHeatmap_2.10.0
[105] MASS_7.3-55 Matrix_1.4-0
[107] car_3.0-12 cli_3.1.1
[109] parallel_4.1.2 pkgconfig_2.0.3
[111] getPass_0.2-2 xml2_1.3.3
[113] foreach_1.5.2 vipor_0.4.5
[115] bslib_0.3.1 XVector_0.34.0
[117] drc_3.0-1 stringr_1.4.0
[119] callr_3.7.0 digest_0.6.29
[121] ConsensusClusterPlus_1.58.0 graph_1.72.0
[123] rmarkdown_2.11 DelayedMatrixStats_1.16.0
[125] curl_4.3.2 gtools_3.9.2
[127] rjson_0.2.21 lifecycle_1.0.1
[129] jsonlite_1.7.3 carData_3.0-5
[131] BiocNeighbors_1.12.0 fansi_1.0.2
[133] pillar_1.7.0 lattice_0.20-45
[135] plotrix_3.8-2 fastmap_1.1.0
[137] httr_1.4.2 survival_3.2-13
[139] glue_1.6.1 png_0.1-7
[141] iterators_1.0.13 Rgraphviz_2.38.0
[143] nnls_1.4 ggforce_0.3.3
[145] stringi_1.7.6 sass_0.4.0
[147] BiocSingular_1.10.0 CytoML_2.6.0
[149] latticeExtra_0.6-29 cytolib_2.6.1
[151] irlba_2.3.5