Last updated: 2021-02-10

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Knit directory: melanoma_publication_old_data/

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Introduction

This script generates plots for Figure 5.

Preparations

knitr::opts_chunk$set(echo = TRUE, message= FALSE)
knitr::opts_knit$set(root.dir = rprojroot::find_rstudio_root_file())

Load libraries

First, we will load the libraries needed for this part of the analysis.

sapply(list.files("code/helper_functions", full.names = TRUE), source)
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        code/helper_functions/sceChecks.R
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library(SingleCellExperiment)
library(reshape2)
library(tidyverse)
library(dplyr)
library(data.table) 
library(ggplot2)
library(cba)
library(ComplexHeatmap)
library(colorRamps)
library(circlize)
library(RColorBrewer)
library(ggpubr)
library(ggbeeswarm)
library(gridExtra)
library(tidyr)
library(ggpmisc)
library(circlize)
library(coxme)
library(dittoSeq)
library(scater)
library(cowplot)
library(survminer)
library(ggalluvial)
library(cytomapper)
library(corrplot)
library(ggridges)
library(rstatix)

Read the data

sce_rna = readRDS(file = "data/sce_RNA.rds")
sce_prot = readRDS(file = "data/sce_protein.rds")

# meta data
dat_relation = fread(file = "data/protein/Object relationships.csv",stringsAsFactors = FALSE)
dat_relation_rna = fread(file = "data/RNA/Object relationships.csv",stringsAsFactors = FALSE)

# image
image_mat_rna <- read.csv("data/rna/Image.csv")

# surv_dat
dat_survival_prot <- fread(file = "data/protein/clinical_data_protein.csv")
time_data = fread(file = "data/survdat_for_modelling.csv",stringsAsFactors = FALSE)
time_data$BlockID <- time_data$Block.ID

Prepare Relation Data Protein

# prepare data and add cellID
dat_relation$cellID_first <- paste("protein", paste(dat_relation$`First Image Number`, dat_relation$`First Object Number`, sep = "_"), sep = "_")
dat_relation$cellID_second <- paste("protein", paste(dat_relation$`Second Image Number`, dat_relation$`Second Object Number`, sep = "_"), sep = "_")

# add celltype status to first and second label
celltype_first <- data.frame(colData(sce_prot))[,c("cellID", "celltype", "celltype_clustered")]
colnames(celltype_first) <- c("cellID_first", "celltype_first", "celltype_clust_first")
celltype_second <- data.frame(colData(sce_prot))[,c("cellID", "celltype", "celltype_clustered")]
colnames(celltype_second) <- c("cellID_second", "celltype_second", "celltype_clust_second")

dat_relation <- left_join(dat_relation, celltype_first, by = "cellID_first")
dat_relation <- left_join(dat_relation, celltype_second, by = "cellID_second")

colnames(dat_relation)[5] <- "FirstImageNumber"

Prepare Relation Data RNA

# prepare data and add cellID
dat_relation_rna$cellID_first <- paste("RNA", paste(dat_relation_rna$`First Image Number`, dat_relation_rna$`First Object Number`, sep = "_"), sep = "_")
dat_relation_rna$cellID_second <- paste("RNA", paste(dat_relation_rna$`Second Image Number`, dat_relation_rna$`Second Object Number`, sep = "_"), sep = "_")

# add celltype status to first and second label
celltype_first <- data.frame(colData(sce_rna))[,c("cellID", "celltype_rf", "celltype_clustered")]
colnames(celltype_first) <- c("cellID_first", "celltype_first", "celltype_clust_first")
celltype_second <- data.frame(colData(sce_rna))[,c("cellID", "celltype_rf", "celltype_clustered")]
colnames(celltype_second) <- c("cellID_second", "celltype_second", "celltype_clust_second")

dat_relation_rna <- left_join(dat_relation_rna, celltype_first, by = "cellID_first")
dat_relation_rna <- left_join(dat_relation_rna, celltype_second, by = "cellID_second")

colnames(dat_relation_rna)[5] <- "FirstImageNumber"

Figure 5A

Generate adjacency matrix for all images

# subset sce for inflamed/exhausted in high samples
sce_protein_sub <- sce_prot[, sce_prot$dysfunction_density %in% c("high - high dysfunction", "high - low dysfunction")]

# sample 9 images each
images <- data.frame(colData(sce_protein_sub)) %>%
  distinct(ImageNumber, .keep_all = T) %>%
  group_by(dysfunction_density) %>%
  #filter(ImageNumber %in% sample(ImageNumber, 9)) %>%
  select(ImageNumber, dysfunction_density)

return <- list()

for (i in c("high - high dysfunction", "high - low dysfunction")){
  # title name
  title_name <- i

  # count interactions in 20 sample images
  cur_dat_relation <- data.frame(dat_relation) %>%
    filter(FirstImageNumber %in% images[images$dysfunction_density == i,]$ImageNumber) %>%
    select("celltype_first" ,"celltype_second") %>%
    dplyr::count(celltype_first,celltype_second) %>%
    data.frame()
  
  # remove tumor-tumor interactions
  cur_dat_relation <- cur_dat_relation[cur_dat_relation$celltype_first != "Tumor",]
  cur_dat_relation <- cur_dat_relation[cur_dat_relation$celltype_first != "unknown",]
  cur_dat_relation <- cur_dat_relation[cur_dat_relation$celltype_second != "unknown",]
  
    # count interactions
  cur_dat_relation_subcluster <- data.frame(dat_relation) %>%
    filter(FirstImageNumber %in% images[images$dysfunction_density == i,]$ImageNumber) %>%
    select("celltype_first" , "celltype_clust_second") %>%
    dplyr::count(celltype_first, celltype_clust_second) %>%
    data.frame()
  
  # remove tumor-tumor interactions
  cur_dat_relation_subcluster <- cur_dat_relation_subcluster[cur_dat_relation_subcluster$celltype_first == "Tcytotoxic",]
  cur_dat_relation_subcluster <- cur_dat_relation_subcluster[cur_dat_relation_subcluster$celltype_clust_second %in% 
                                                               unique(sce_prot[,sce_prot$celltype == "Tumor"]$celltype_clustered),]
  # return subcluster adj df
  return[[i]] <- cur_dat_relation_subcluster
  
  # make coord diagram
  chordDiagramFromDataFrame(cur_dat_relation,
                            grid.col = metadata(sce_prot)$colour_vectors$celltype,
                            reduce = 0.05, 
                            transparency = ifelse(cur_dat_relation[[1]] %in% c("Tcytotoxic"),0,0.6),
                            annotationTrack = c("grid"))
  
  # add percentages
  circos.track(track.index = 1, panel.fun = function(x, y) {
    xlim = get.cell.meta.data("xlim")
    ylim = get.cell.meta.data("ylim")
    sector.name = get.cell.meta.data("sector.index")
    xplot = get.cell.meta.data("xplot")
    
    circos.lines(xlim, c(mean(ylim), mean(ylim)), lty = 3) # dotted line
    by = ifelse(abs(xplot[2] - xplot[1]) > 30, 0.2, 0.5)
    for(p in seq(by, 1, by = by)) {
        circos.text(p*(xlim[2] - xlim[1]) + xlim[1], mean(ylim) + 0.1, 
            paste0(p*100, "%"), cex = 0.5, adj = c(0.5, 0), niceFacing = TRUE)
    }
}, bg.border = NA)
  
}
# create legend for tumor subclusters
#lgd1 = Legend(labels = names(col_vec[grep("Tumor", names(col_vec))]), title = "Tumor\nSubclusters", legend_gp = gpar(fill = unname(col_vec[grep("Tumor", names(col_vec))])))
lgd2 = Legend(labels = names(metadata(sce_prot)$colour_vector$celltype), title = "Cell Type", legend_gp = gpar(fill = unname(metadata(sce_prot)$colour_vector$celltype)))
draw(packLegend(lgd2, 
                #lgd1, 
                gap = unit(2, "cm")))

Figure 5B

Celltypes

celltypes <- data.frame(colData(sce_prot)) %>%
  filter(celltype != "Tumor") %>%
  group_by(ImageNumber, bcell_patch_score, dysfunction_score, celltype) %>%
  summarise(n=n()) %>%
  mutate(fraction = n/sum(n)) %>%
  filter(is.na(dysfunction_score) == FALSE)

celltypes$bcell_patch_score <- as.character(celltypes$bcell_patch_score)
celltypes$bcell_patch_score <- factor(celltypes$bcell_patch_score, levels = c("no B cells", "no B cell patches", "small B cell patches", "TLS"))

stat.test <- celltypes %>%
  group_by(celltype) %>%
  wilcox_test(data = ., fraction ~ dysfunction_score) %>%
  adjust_pvalue(method = "BH") %>%
  add_significance("p.adj") %>%
  add_x_position(x = "dysfunction_score", dodge = 0.8)


ggplot(celltypes, aes(x=celltype, y=fraction)) +
  geom_boxplot(alpha=.5, lwd = 1, outlier.shape = NA, aes(fill=dysfunction_score)) +
  #geom_jitter(alpha=1, position = position_jitterdodge(dodge.width = 0.75,jitter.width = 0.1), size = 4, 
              #aes(x = celltype, y = fraction, #col=ifelse(celltype %in% c("Bcell", "BnTcell"), bcell_patch_score, " "),
                  #group = dysfunction_score)) + 
  geom_quasirandom(dodge.width=0.75, alpha=1, size=1) + 
  stat_pvalue_manual(stat.test, x = "celltype", label = "p.adj.signif", size = 7, y.position = -0.05) + 
  theme_bw() +
  theme(text = element_text(size = 16),
        axis.text.x = element_text(angle = 45, vjust = 1, hjust=1)) + 
  guides(fill=guide_legend(title="Dysfunction Score", override.aes = c(lwd=0.5, alpha=1)), col=guide_legend(title="Bcell Score")) +
  xlab("") + 
  ylab("Cell Type Fractions") +
  scale_color_manual(values = c("black", "lightblue", "darkblue", "red", "red4"),
                    labels = c(" ", "no Bcells", "few Bcells", "small patches", "large patches"),
                    guide = TRUE)
# check images above median in low dysfunction group for B cells (all have large patches)
celltypes %>%
  filter(dysfunction_score == "low dysfunction") %>%
  filter(celltype == "Bcell") %>%
  group_by(dysfunction_score) %>%
  mutate(median_frac = median(fraction)) %>%
  filter(fraction > median_frac)
# A tibble: 7 x 7
# Groups:   dysfunction_score [1]
  ImageNumber bcell_patch_sco… dysfunction_sco… celltype     n fraction
        <int> <fct>            <chr>            <chr>    <int>    <dbl>
1          29 TLS              low dysfunction  Bcell      885    0.134
2          33 TLS              low dysfunction  Bcell     4831    0.365
3          86 TLS              low dysfunction  Bcell     2400    0.252
4          95 TLS              low dysfunction  Bcell     1603    0.116
5         109 TLS              low dysfunction  Bcell     1333    0.254
6         114 TLS              low dysfunction  Bcell     2881    0.360
7         118 TLS              low dysfunction  Bcell      787    0.206
# … with 1 more variable: median_frac <dbl>

Composition of Dysfunction Groups (Met Location and Punch Location)

c <- data.frame(colData(sce_rna)) %>%
  distinct(Description, .keep_all = T) %>%
  filter(dysfunction_score %in% c("low dysfunction", "high dysfunction")) %>%
  dplyr::count(MM_location, Location, dysfunction_score) %>%
  ggplot(aes(axis1 = MM_location, axis2 = Location, axis3 = dysfunction_score,y=n))+
  geom_alluvium(aes(fill=as.factor(dysfunction_score)))+
  geom_stratum()+
  geom_text(stat = "stratum", aes(label = after_stat(stratum))) +
  theme_minimal()+
  scale_x_discrete(limits = c("Met Location", "Punch Location", "Dysfunction Score"), expand = c(.2, .05)) +
  theme(text = element_text(size=20)) +
  guides(fill=guide_legend(title="Dysfunction Score")) +
  ylab("Number of Samples")
plot(c)

Figure 5C

B cell patch grouping for dysfunction groups

a <- data.frame(colData(sce_rna)) %>%
  distinct(Description, .keep_all = T) %>%
  group_by(bcell_patch_score, dysfunction_score) %>%
  summarise(n=n()) %>%
  filter(is.na(dysfunction_score) == F) %>%
  group_by(dysfunction_score) %>%
  mutate(fraction = n / sum(n)) %>%
  ungroup()

ggplot(a) + 
  geom_col(aes(y=dysfunction_score, x=fraction, fill=bcell_patch_score)) +
  theme_minimal() + 
  theme(text = element_text(size = 22)) +
  xlab("Fraction of Samples") +
  ylab("") +
  guides(fill=guide_legend(title="Bcell Score"))

Figure 5D

Analysis

# im_size
im_size <- as.data.frame(cbind(image_mat_rna$Metadata_Description, (image_mat_rna$Height_cellmask * image_mat_rna$Width_cellmask)/1000000))
names(im_size) <- c("Description", "mm2")
im_size$mm2 <- as.numeric(im_size$mm2)

cxcl13 <- data.frame(colData(sce_rna)) %>%
  mutate(mmLocationPunch = paste(MM_location, Location, sep = "_")) %>%
  filter(mmLocationPunch != "LN_M") %>% # remove LN margin samples 
  filter(CXCL13 == 1) %>%
  group_by(Description, MM_location, celltype) %>%
  summarise(n=n()) %>%
  reshape2::dcast(Description + MM_location ~ celltype, value.var = "n", fill = 0)

all_images <- data.frame(colData(sce_rna)) %>%
  mutate(mmLocationPunch = paste(MM_location, Location, sep = "_")) %>%
  filter(mmLocationPunch != "LN_M") %>%
  distinct(Description, .keep_all = T) %>%
  select(Description)

# left_join to have all images
all_images <- left_join(all_images, cxcl13)

# replace NA
all_images[is.na(all_images)] <- 0

all_images <- all_images %>%
  reshape2::melt(id.vars=c("Description", "MM_location"), variable.name = "celltype", value.name = "n")

# add mm2 and calculate density
all_images <- left_join(all_images, im_size)
all_images$density <- all_images$n / all_images$mm2

#
Bcell <- data.frame(colData(sce_rna)) %>%
  distinct(Description, .keep_all = T) %>%
  group_by(Description) %>%
  select(Description, bcell_patch_score)

# add grouping
all_images <- left_join(all_images, Bcell[,c("Description","bcell_patch_score")]) %>%
  filter(celltype %in% c("Tcytotoxic", "Tcell", "HLA-DR")) 

#all_images$interaction <- interaction(all_images$celltype, all_images$bcell_patch_score)

ggplot(all_images,aes(x=bcell_patch_score, y = log10(as.numeric(density+1)), fill=celltype)) + 
  geom_boxplot(alpha=1, lwd=1, outlier.shape = NA) + 
  #scale_x_discrete(labels = rep(unique(all_images$bcell_patch_score), each = 3)) +
  geom_quasirandom(dodge.width=0.75, alpha=1, size=2) +
  #geom_line(aes(group = Description), alpha = ifelse(all_images$celltype != "HLA-DR", 0.6, 0), colour = "black") +
  #geom_point(aes(group= interaction), position = position_dodge(0.75)) +
  ylab("CXCL13 Expressing Cells per mm2\n(log10 + 1)") +
  xlab("") + 
  theme_bw() +
  theme(text = element_text(size=18),
        axis.text.x = element_text(angle=45, vjust=1, hjust=1)) +
  scale_fill_manual(values = unname(metadata(sce_rna)$colour_vectors$celltype[c("Tcytotoxic", "Tcell", "HLA-DR")]),
                    breaks = names(metadata(sce_rna)$colour_vectors$celltype[c("Tcytotoxic", "Tcell", "HLA-DR")])) +
  #scale_color_manual(values = unname(metadata(sce_rna)$colour_vectors$celltype[c("Tcytotoxic", "Tcell", "HLA-DR")]),
                    #breaks = names(metadata(sce_rna)$colour_vectors$celltype[c("Tcytotoxic", "Tcell", "HLA-DR")]),
                    #guide = FALSE) +
  guides(fill=guide_legend(title="Cell Type", override.aes = c(lwd=0.5)))

Figure 5E

Example of CXCL10 Cluster and corresponding Community

example <- findClusters(sce_prot[,sce_prot$Description == "C9"], sce_prot[,colData(sce_prot)$celltype %in% c("Bcell", "BnTcell")]$cellID, 
                    'cellID', 
                    'Center_X', 'Center_Y', 
                    'Description', 
                    distance = 15, 
                    min_clust_size = 20,
                    output_colname = "example_patch")
Time difference of 2.19296 secs
[1] "clusters successfully added to sce object"
example <- findCommunity(example, 
              'cellID', 
              'Center_X', 'Center_Y', 
              'Description', 
              'example_patch', 
              distance = 50,
              output_colname = "example_milieu",
              plot = TRUE)
Time difference of 3.302347 secs
[1] "communities successfully added to sce object"

Figure 5F

Fraction of Tcf7 Tcells cells in bcell patch groups

celltypes <- data.frame(colData(sce_prot)) %>%
  mutate(mmLocationPunch = paste(MM_location, Location, sep = "_")) %>%
  filter(mmLocationPunch != "LN_M") %>% # remove LN margin images 
  #filter(celltype != "Tumor") %>%
  mutate(TCF7_PD1 = paste(PD1, TCF7, sep = "_")) %>%
  group_by(PatientID,BlockID, Description, bcell_patch_score, celltype, TCF7_PD1) %>%
  summarise(n=n()) %>% 
  reshape2::dcast(PatientID + BlockID + Description + bcell_patch_score + celltype ~ TCF7_PD1, value.var = "n", fill=0) %>%
  reshape2::melt(id.vars = c("PatientID","BlockID", "Description", "bcell_patch_score", "celltype"), 
                 variable.name = "TCF7_PD1", value.name = "n") %>%
  group_by(Description, celltype) %>%
  mutate(fraction = n/sum(n)) %>%
  mutate(total_cells = sum(n)) %>%
  ungroup() %>%
  filter(celltype %in% c("Tcytotoxic", "Thelper")) 

celltypes$bcell_patch_score <- as.character(celltypes$bcell_patch_score)
celltypes$bcell_patch_score <- factor(celltypes$bcell_patch_score, levels = c("no B cells", "no B cell patches", "small B cell patches", "TLS"))

stat.test <- celltypes %>%
  group_by(celltype, TCF7_PD1) %>%
  kruskal_test(data = ., fraction ~ bcell_patch_score) %>%
  adjust_pvalue(method = "BH") %>%
  add_significance("p.adj") %>%
  mutate(group1 = celltype, group2 = TCF7_PD1) %>%
  add_xy_position()

ggplot(celltypes, aes(x=TCF7_PD1, y=fraction)) +
  geom_boxplot(alpha=1, lwd = 0.5, outlier.size = 0.5, aes(fill=bcell_patch_score)) +
  stat_pvalue_manual(x = "group2", stat.test, y.position = -0.1, size = 6) +
  theme_bw() +
  theme(text = element_text(size = 14),
        axis.text.x = element_text(angle = 45, vjust = 1, hjust=1)) + 
  guides(fill=guide_legend(title="Bcell Score", override.aes = c(lwd=0.5))) +
  xlab("") + 
  ylab("Fraction of Population") +
  facet_wrap(~celltype, scales = "free") +
  ylim(-0.1,1.1)

Figure 5G

Percentage of TCF7+PD1+CD8+ cells that are part of a Bcell milieu

# what fraction of each celltype is part of a milieu?
cur_dat <- data.frame(colData(sce_prot)) %>%
  filter(Location != "CTRL") %>%
  filter(celltype %in% c("Tcytotoxic", "Thelper")) %>%
  mutate(status = paste(TCF7, PD1, sep = "_")) %>%
  #filter(status == "TCF7+_PD1+") %>%
  mutate(milieu_binary = ifelse(bcell_milieu > 0, 1, 0)) %>%
  group_by(Description, milieu_binary, celltype, status) %>%
  summarise(n=n()) %>%
  group_by(Description, celltype, status) %>%
  mutate(fraction_in_milieu = n / sum(n)) %>%
  filter(milieu_binary == 1)

# what fraction of the total cell area is made up by cells that are part of a milieu
milieu_area <- data.frame(colData(sce_prot)) %>%
  filter(Location != "CTRL") %>%
  mutate(milieu_binary = ifelse(bcell_milieu > 0, 1, 0)) %>%
  group_by(Description, milieu_binary) %>%
  summarise(area = sum(Area)) %>% 
  group_by(Description) %>%
  mutate(fraction_of_area = area / sum(area))  %>%
  filter(row_number() == n())

sum <- left_join(cur_dat, milieu_area, by="Description")

# calculate metric - milieu_fraction divided by milieu-area-fraction (this normalizes the first metric)
sum$metric <- sum$fraction_in_milieu / sum$fraction_of_area

# one-sample t test
stat.test <- sum %>%
  group_by(celltype, status) %>%
  mutate(log10_metric = log10(metric)) %>%
  t_test(log10_metric ~ 1, mu = 0, alternative = "greater", detailed = TRUE) %>%
  adjust_pvalue() %>%
  add_significance("p.adj")

ggplot(sum, aes(x=sum$status, y=log10(metric))) +
  geom_boxplot(outlier.shape = NA) + 
  geom_quasirandom(size=0.75) +
  geom_hline(yintercept = 0) +
  facet_wrap(~celltype) +
  theme_bw() + 
  theme(text=element_text(size=14),
        axis.text.x = element_text(angle=45, vjust=1, hjust=1)) +
  stat_pvalue_manual(
    stat.test, x = "status", y.position = 1.5,
    label = "p.adj.signif",
    position = position_dodge(0.8), 
    size=7) + 
  ylab("Area-Normalized Log10 Fold-Change") +
  xlab("") #+
  #coord_cartesian(ylim=c(-1,1.75))

sessionInfo()
R version 4.0.3 (2020-10-10)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04 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=C             
 [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] grid      parallel  stats4    stats     graphics  grDevices utils    
 [8] datasets  methods   base     

other attached packages:
 [1] rstatix_0.6.0               ggridges_0.5.3             
 [3] corrplot_0.84               cytomapper_1.3.1           
 [5] EBImage_4.32.0              ggalluvial_0.12.3          
 [7] survminer_0.4.8             cowplot_1.1.1              
 [9] scater_1.16.2               dittoSeq_1.0.2             
[11] coxme_2.2-16                bdsmatrix_1.3-4            
[13] survival_3.2-7              ggpmisc_0.3.7              
[15] gridExtra_2.3               ggbeeswarm_0.6.0           
[17] ggpubr_0.4.0                RColorBrewer_1.1-2         
[19] circlize_0.4.12             colorRamps_2.3             
[21] ComplexHeatmap_2.4.3        cba_0.2-21                 
[23] proxy_0.4-24                data.table_1.13.6          
[25] forcats_0.5.0               stringr_1.4.0              
[27] dplyr_1.0.2                 purrr_0.3.4                
[29] readr_1.4.0                 tidyr_1.1.2                
[31] tibble_3.0.4                ggplot2_3.3.3              
[33] tidyverse_1.3.0             reshape2_1.4.4             
[35] SingleCellExperiment_1.12.0 SummarizedExperiment_1.20.0
[37] Biobase_2.50.0              GenomicRanges_1.42.0       
[39] GenomeInfoDb_1.26.2         IRanges_2.24.1             
[41] S4Vectors_0.28.1            BiocGenerics_0.36.0        
[43] MatrixGenerics_1.2.0        matrixStats_0.57.0         
[45] workflowr_1.6.2            

loaded via a namespace (and not attached):
  [1] utf8_1.1.4                shinydashboard_0.7.1     
  [3] tidyselect_1.1.0          htmlwidgets_1.5.3        
  [5] BiocParallel_1.22.0       munsell_0.5.0            
  [7] units_0.6-7               codetools_0.2-18         
  [9] withr_2.3.0               colorspace_2.0-0         
 [11] knitr_1.30                rstudioapi_0.13          
 [13] ggsignif_0.6.0            labeling_0.4.2           
 [15] git2r_0.28.0              GenomeInfoDbData_1.2.4   
 [17] KMsurv_0.1-5              farver_2.0.3             
 [19] pheatmap_1.0.12           rprojroot_2.0.2          
 [21] vctrs_0.3.6               generics_0.1.0           
 [23] xfun_0.20                 R6_2.5.0                 
 [25] clue_0.3-58               rsvd_1.0.3               
 [27] locfit_1.5-9.4            concaveman_1.1.0         
 [29] bitops_1.0-6              DelayedArray_0.16.0      
 [31] assertthat_0.2.1          promises_1.1.1           
 [33] scales_1.1.1              beeswarm_0.2.3           
 [35] gtable_0.3.0              rlang_0.4.10             
 [37] systemfonts_0.3.2         GlobalOptions_0.1.2      
 [39] splines_4.0.3             broom_0.7.3              
 [41] yaml_2.2.1                abind_1.4-5              
 [43] modelr_0.1.8              backports_1.2.1          
 [45] httpuv_1.5.4              tools_4.0.3              
 [47] ellipsis_0.3.1            raster_3.4-5             
 [49] Rcpp_1.0.5                plyr_1.8.6               
 [51] zlibbioc_1.36.0           classInt_0.4-3           
 [53] RCurl_1.98-1.2            GetoptLong_1.0.5         
 [55] viridis_0.5.1             zoo_1.8-8                
 [57] haven_2.3.1               ggrepel_0.9.0            
 [59] cluster_2.1.0             fs_1.5.0                 
 [61] magrittr_2.0.1            openxlsx_4.2.3           
 [63] RANN_2.6.1                reprex_0.3.0             
 [65] whisker_0.4               hms_0.5.3                
 [67] mime_0.9                  evaluate_0.14            
 [69] fftwtools_0.9-9           xtable_1.8-4             
 [71] rio_0.5.16                jpeg_0.1-8.1             
 [73] readxl_1.3.1              shape_1.4.5              
 [75] compiler_4.0.3            V8_3.4.0                 
 [77] KernSmooth_2.23-18        crayon_1.3.4             
 [79] htmltools_0.5.0           later_1.1.0.1            
 [81] tiff_0.1-6                lubridate_1.7.9.2        
 [83] DBI_1.1.0                 dbplyr_2.0.0             
 [85] sf_0.9-7                  Matrix_1.3-2             
 [87] car_3.0-10                cli_2.2.0                
 [89] pkgconfig_2.0.3           km.ci_0.5-2              
 [91] foreign_0.8-81            sp_1.4-5                 
 [93] xml2_1.3.2                svglite_1.2.3.2          
 [95] vipor_0.4.5               XVector_0.30.0           
 [97] rvest_0.3.6               digest_0.6.27            
 [99] rmarkdown_2.6             cellranger_1.1.0         
[101] survMisc_0.5.5            edgeR_3.30.3             
[103] DelayedMatrixStats_1.10.1 gdtools_0.2.3            
[105] curl_4.3                  shiny_1.5.0              
[107] rjson_0.2.20              lifecycle_0.2.0          
[109] nlme_3.1-151              jsonlite_1.7.2           
[111] carData_3.0-4             BiocNeighbors_1.6.0      
[113] viridisLite_0.3.0         limma_3.44.3             
[115] fansi_0.4.1               pillar_1.4.7             
[117] lattice_0.20-41           fastmap_1.0.1            
[119] httr_1.4.2                glue_1.4.2               
[121] zip_2.1.1                 png_0.1-7                
[123] svgPanZoom_0.3.4          class_7.3-17             
[125] stringi_1.5.3             BiocSingular_1.4.0       
[127] e1071_1.7-4               irlba_2.3.3