Last updated: 2021-04-12

Checks: 6 1

Knit directory: melanoma_publication_old_data/

This reproducible R Markdown analysis was created with workflowr (version 1.6.2). The Checks tab describes the reproducibility checks that were applied when the results were created. The Past versions tab lists the development history.


The R Markdown file has unstaged changes. To know which version of the R Markdown file created these results, you’ll want to first commit it to the Git repo. If you’re still working on the analysis, you can ignore this warning. When you’re finished, you can run wflow_publish to commit the R Markdown file and build the HTML.

Great job! The global environment was empty. Objects defined in the global environment can affect the analysis in your R Markdown file in unknown ways. For reproduciblity it’s best to always run the code in an empty environment.

The command set.seed(20200728) was run prior to running the code in the R Markdown file. Setting a seed ensures that any results that rely on randomness, e.g. subsampling or permutations, are reproducible.

Great job! Recording the operating system, R version, and package versions is critical for reproducibility.

Nice! There were no cached chunks for this analysis, so you can be confident that you successfully produced the results during this run.

Great job! Using relative paths to the files within your workflowr project makes it easier to run your code on other machines.

Great! You are using Git for version control. Tracking code development and connecting the code version to the results is critical for reproducibility.

The results in this page were generated with repository version 3203891. See the Past versions tab to see a history of the changes made to the R Markdown and HTML files.

Note that you need to be careful to ensure that all relevant files for the analysis have been committed to Git prior to generating the results (you can use wflow_publish or wflow_git_commit). workflowr only checks the R Markdown file, but you know if there are other scripts or data files that it depends on. Below is the status of the Git repository when the results were generated:


Ignored files:
    Ignored:    .DS_Store
    Ignored:    .Rproj.user/
    Ignored:    ._.DS_Store
    Ignored:    analysis/._clinical metadata preparation.Rmd
    Ignored:    code/.DS_Store
    Ignored:    code/._.DS_Store
    Ignored:    data/.DS_Store
    Ignored:    data/._.DS_Store
    Ignored:    data/data_for_analysis/
    Ignored:    data/full_data/
    Ignored:    output/.DS_Store
    Ignored:    output/._.DS_Store
    Ignored:    output/._protein_neutrophil.png
    Ignored:    output/._rna_neutrophil.png
    Ignored:    output/PSOCKclusterOut/
    Ignored:    output/bcell_grouping.png
    Ignored:    output/dysfunction_correlation.pdf

Unstaged changes:
    Modified:   .gitignore
    Modified:   analysis/10_Dysfunction_Score.rmd
    Modified:   analysis/11_Bcell_Score.Rmd
    Modified:   analysis/Figure_1.rmd
    Modified:   analysis/Figure_2.rmd
    Modified:   analysis/Figure_4.rmd
    Modified:   analysis/Figure_5.rmd
    Modified:   analysis/Summary_Statistics.rmd
    Modified:   analysis/Supp-Figure_1.rmd
    Modified:   analysis/Supp-Figure_2.rmd
    Modified:   analysis/Supp-Figure_4.rmd
    Modified:   analysis/Supp-Figure_5.rmd
    Modified:   analysis/XX_hazard_ratio.rmd

Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.


These are the previous versions of the repository in which changes were made to the R Markdown (analysis/Figure_2.rmd) and HTML (docs/Figure_2.html) files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view the files as they were in that past version.

File Version Author Date Message
Rmd 3203891 toobiwankenobi 2021-02-19 change celltype names
html 3203891 toobiwankenobi 2021-02-19 change celltype names
Rmd ee1595d toobiwankenobi 2021-02-12 clean repo and adapt files
html ee1595d toobiwankenobi 2021-02-12 clean repo and adapt files
html 3f5af3f toobiwankenobi 2021-02-09 add .html files
Rmd afa7957 toobiwankenobi 2021-02-08 minor changes on figures and figure order
Rmd 20a1458 toobiwankenobi 2021-02-04 adapt figure order
Rmd f9bb33a toobiwankenobi 2021-02-04 new Figure 5 and minor changes in figure order
Rmd 2ac1833 toobiwankenobi 2021-01-08 changes to Figures
Rmd 9442cb9 toobiwankenobi 2020-12-22 add all new files
Rmd 1af3353 toobiwankenobi 2020-10-16 add stuff
Rmd a6b51cd toobiwankenobi 2020-10-14 clean scripts, add new subfigures
Rmd d8819f2 toobiwankenobi 2020-10-08 read new data (nuclei expansion) and adapt scripts
Rmd a21c858 toobiwankenobi 2020-08-06 adapt pipeline
Rmd 2c11d5c toobiwankenobi 2020-08-05 add new scripts

Introduction

This script generates plots for Figure 2.

Preparation

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

Load libraries

sapply(list.files("code/helper_functions", full.names = TRUE), source)
        code/helper_functions/calculateSummary.R
value   ?                                       
visible FALSE                                   
        code/helper_functions/censor_dat.R
value   ?                                 
visible FALSE                             
        code/helper_functions/detect_mRNA_expression.R
value   ?                                             
visible FALSE                                         
        code/helper_functions/DistanceToClusterCenter.R
value   ?                                              
visible FALSE                                          
        code/helper_functions/findMilieu.R code/helper_functions/findPatch.R
value   ?                                  ?                                
visible FALSE                              FALSE                            
        code/helper_functions/getInfoFromString.R
value   ?                                        
visible FALSE                                    
        code/helper_functions/getSpotnumber.R
value   ?                                    
visible FALSE                                
        code/helper_functions/plotCellCounts.R
value   ?                                     
visible FALSE                                 
        code/helper_functions/plotCellFractions.R
value   ?                                        
visible FALSE                                    
        code/helper_functions/plotDist.R
value   ?                               
visible FALSE                           
        code/helper_functions/scatter_function.R
value   ?                                       
visible FALSE                                   
        code/helper_functions/sceChecks.R
value   ?                                
visible FALSE                            
        code/helper_functions/validityChecks.R
value   ?                                     
visible FALSE                                 
library(SingleCellExperiment)
library(ComplexHeatmap)
library(data.table)
library(dplyr)
library(janitor)
library(tidyr)
library(ggpmisc)
library(cowplot)
library(corrplot)
library(gridExtra)
library(ggalluvial)
library(ggbeeswarm)
library(ggpubr)
library(RColorBrewer)
library(colorRamps)
library(circlize)
library(forcats)
library(ggpmisc)

Load data

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

Figure 2B

UpSet plot with ComplexHeatmap - including CellType Annotation and Met Location (with controls)

cur_dt <- as.data.table(colData(sce_rna))

# combinations with more than 600 occurrences
targets <- metadata(sce_rna)$chemokines_morethan600_withcontrol
targets_no_control <- replace(targets, match(c("CXCL8", "CCL18"),targets), c("CCL18", "CXCL8"))

# remove control samples
cur_dt <- cur_dt[MM_location_simplified != "control",]

# extract chemokine columns
chemokines <- cbind(cur_dt[,c("ImageNumber", "MM_location_simplified", "celltype")], cur_dt[,grepl(glob2rx("C*L*"),names(cur_dt)), with = F])

# create combination matrix
m <- make_comb_mat(chemokines, top_n_sets = 11)

# filter based on combination size and combination degree
m <- m[comb_size(m) >= 600 & comb_degree(m) > 0]

# sort according to abundance
m <- m[order(-comb_size(m))]

# extract comb names
comb_names <- comb_name(m)

# count celltypes for each combination
celltypes <- data.table()
location <- data.table()

# summarize statistics for each combination (celltype fractions, location)
for (i in comb_names){
  # subset
  set <- chemokines[extract_comb(m, i)]
  
  # chemokines celltypes
  set1 <- set %>%
    group_by(celltype) %>%
    summarise(n=n()) %>%
    reshape2::dcast(.,i ~ celltype, value.var = "n")
  
  # chemokines by location
  set2 <- set %>%
    group_by(ImageNumber, MM_location_simplified) %>%
    summarise(n=n())
  
  # add images with no combinations to not distort median
  set2_add <- distinct(cur_dt[,c("ImageNumber", "MM_location_simplified")], ImageNumber, .keep_all = T)
  set2_add$n <- 0
  
  # subset to only contain images which are not already part of set2
  set2_add <- set2_add[!(ImageNumber %in% set2$ImageNumber),]
  
  set2 <- set2 %>%
    rbind(., set2_add) %>%
    group_by(MM_location_simplified) %>%
    mutate(median = median(n)) %>%
    distinct(MM_location_simplified, median) %>%
    reshape2::dcast(.,i ~ MM_location_simplified, value.var = "median")
  
  # add to data.frame
  celltypes <- rbind(celltypes, set1, fill = TRUE)
  location <- rbind(location, set2, fill = TRUE)
}

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

# properties of combination matrix
ss = set_size(m)
cs = comb_size(m)

# create plot
ht = UpSet(m, 
    set_order = order(ss),
    comb_order = order(cs, decreasing = T),
    top_annotation = HeatmapAnnotation(
        "Number of\nExpressing Cells" = anno_barplot(celltypes[,-1], 
            ylim = c(0, max(cs)*1.1),
            border = FALSE, 
            gp = gpar(fill = metadata(sce_rna)$colour_vectors$celltype[colnames(celltypes[,-1])]), 
            axis_param = list(gp = gpar(fontsize=20)),
            height = unit(12, "cm")), 
        annotation_name_side = "left", 
        annotation_name_rot = 0,
        annotation_name_gp = gpar(fontsize=20)),
    left_annotation = rowAnnotation(
        "Total Number of\nExpressing Cells" = anno_barplot(-ss, 
            baseline = 0,
            axis_param = list(
                at = c(0, -5000, -10000, -15000),
                labels = c(0, 5000, 10000, 15000),
                labels_rot = 0,
                gp = gpar(fontsize = 15)),
            border = FALSE, 
            gp = gpar(fill = "black"), 
            width = unit(5, "cm"),
        ),
        set_name = anno_text(set_name(m), 
            location = 0.5, 
            gp = gpar(fontsize=15),
            just = "center",
            width = max_text_width(set_name(m)) + unit(4, "mm")),
        annotation_name_gp = gpar(fontsize=20)),
    right_annotation = NULL,
    show_row_names = FALSE,
    pt_size = unit(5, "mm"),
    lwd = 2,
    width = unit(14, "cm"),
    height = unit(14, "cm")
    )

# draw heatmap
ht = draw(ht)

# add absolute numbers on top of barplot
od = column_order(ht)
row_od = row_order(ht)
decorate_annotation("Number of\nExpressing Cells", {
  grid.text(cs[od], x = seq_along(cs), y = unit(cs[od], "native") + unit(2, "pt"), 
            default.units = "native", just = c("left", "bottom"), 
            gp = gpar(fontsize = 15, col = "#404040"), rot = 45)
  })
decorate_annotation("Total Number of\nExpressing Cells", {
    grid.text(ss[row_od], 
        x = unit(-ss[row_od], "native") + unit(-0.75, "cm"), 
        y = rev(seq_len(length(-ss))), 
        default.units = "native", rot = 0,
        gp = gpar(fontsize = 13))
})

Legend for Figure

# legend for celltypes
lgd1 = Legend(labels = colnames(celltypes[,-1]), 
             title = "Celltypes", 
             legend_gp = gpar(fill = metadata(sce_rna)$colour_vectors$celltype[colnames(celltypes[,-1])],
                              fontsize = 18),
             nrow = 5)

draw(packLegend(lgd1,column_gap = unit(0.5, "cm"),
                max_height = unit(7, "cm")))

Figure 2C

Heatmap Marker Expression

# add marker expression to cells
marker_expression <- data.frame(t(assay(sce_rna[rowData(sce_rna)$good_marker,], "asinh")))
marker_expression$cellID <- rownames(marker_expression)

# chemokine info
chemo <- data.frame(colData(sce_rna))[,c("cellID", "expressor", "celltype")] 

dat <- left_join(chemo, marker_expression, by = "cellID")
dat$cellID <- NULL

# aggregate data
dat_aggr <- dat %>%
  filter(expressor %in% colnames(colData(sce_rna))[grepl("CXCL|CCL", colnames(colData(sce_rna)))]) %>%
  group_by(celltype, expressor) %>%
  summarise_all(funs(mean))
Warning: `funs()` is deprecated as of dplyr 0.8.0.
Please use a list of either functions or lambdas: 

  # Simple named list: 
  list(mean = mean, median = median)

  # Auto named with `tibble::lst()`: 
  tibble::lst(mean, median)

  # Using lambdas
  list(~ mean(., trim = .2), ~ median(., na.rm = TRUE))
This warning is displayed once every 8 hours.
Call `lifecycle::last_warnings()` to see where this warning was generated.
# prepare matrix for heatmap
dat_aggr <- dat_aggr %>%
  arrange(celltype, expressor)

stats <- dat %>%
  filter(expressor %in% colnames(colData(sce_rna))[grepl("CXCL|CCL", colnames(colData(sce_rna)))]) %>%
  group_by(celltype, expressor) %>%
  summarise(n=n()) %>%
  filter(n>1000) %>%
  arrange(celltype, expressor)

dat_aggr <- dat_aggr %>%
  filter(paste0(celltype,expressor) %in% paste0(stats$celltype,stats$expressor))

# factorize expressor for column sorting in heatmap
dat_aggr$expressor <- factor(dat_aggr$expressor, levels = c("CCL4", "CCL18", "CCL22", "CXCL8", 
                                                            "CCL8", "CXCL9", "CXCL10", "CXCL13", "CCL2", "CXCL12", "CCL19"))
stats$expressor <- factor(stats$expressor, levels = c("CCL4", "CCL18", "CCL22", "CXCL8", 
                                                            "CCL8", "CXCL9", "CXCL10", "CXCL13", "CCL2", "CXCL12", "CCL19"))

dat_aggr <- dat_aggr %>%
  arrange(celltype, expressor)

stats <- stats %>%
  arrange(celltype, expressor)

# create and scale scale matrix
m <- as.matrix(t(dat_aggr[,-c(1:2)]))
m <- t(scale(t(m)))
colnames(m) <- dat_aggr$celltype

# create top annotations
ha <- HeatmapAnnotation("Chemokine" = dat_aggr$expressor,
                        "Cells" = anno_barplot(stats[,3],
                                               height = unit(1.5,"cm"),
                                               axis_param = list(gp = gpar(fontsize=14))),
                        "Cell Numbers" = anno_text(t(stats[,3]), 
                                                   which = "column", 
                                                   rot = 90, 
                                                   just = "center", 
                                                   location = 0.5,
                                                   gp = gpar(fontsize=10)),
                        col = list("Chemokine" = metadata(sce_rna)$colour_vectors$chemokine_single),
                        show_legend = FALSE,
                        annotation_name_gp = gpar(fontsize = 16))


# row_split for markers
rowData(sce_rna)$heatmap_relevance <- ""
rowData(sce_rna[rowData(sce_rna)$good_marker,])$heatmap_relevance <- "Lineage"
rowData(sce_rna[grepl("CXCL|CCL|DapB", rownames(sce_rna)),])$heatmap_relevance <- "Chemokine"
rowData(sce_rna[grepl("B2M|GLUT1|CD134|Lag3|CD163|cleavedPARP|pRB", rownames(sce_rna)),])$heatmap_relevance <- "Other"
             
# create heatmap
h <- Heatmap(m, name = "Scaled\nExpression",
             row_split = rowData(sce_rna[rowData(sce_rna)$good_marker,])$heatmap_relevance,
             cluster_columns = FALSE,
             show_column_names = FALSE,
             top_annotation = ha,
             show_heatmap_legend = FALSE,
             column_split = colnames(m),
             column_title_rot = 90,
             cluster_column_slices = TRUE,
             row_names_gp = gpar(fontsize = 16),
             column_title_gp = gpar(fontsize = 23),
             row_title_gp = gpar(fontsize = 23),
             col = colorRamp2(c(-3, 0, 3), c("blue", "white", "red")),
             height = unit(20, "cm"),
             width = unit(25,"cm"))

# draw heatmap
draw(h)

Legend heatmap

lgd1 = color_mapping_legend(h@matrix_color_mapping, plot = FALSE, legend_direction = "horizontal", legend_width=unit(3,"cm"), at = c(-3:3))
lgd2 = color_mapping_legend(ha@anno_list$Chemokine@color_mapping, plot = FALSE, legend_direction = "horizontal", nrow = 4)

lgd_list = packLegend(lgd1,lgd2,direction = "horizontal", gap = unit(1,"cm"))
draw(lgd_list)

Figure 2D

Correlation of Chemokines with Celltypes

# top abundant chemokines
cur_rna <- data.frame(colData(sce_rna))

# sum
rna_sum <- cur_rna %>%
  group_by(Description, expressor) %>%
  summarise(n = n()) %>%
  reshape2::dcast(Description ~ expressor, value.var = "n", fill = 0) 

# only keep highly abundant chemokines
rna_sum <- rna_sum[,colnames(rna_sum) %in% targets]

# correlation
cor <- cor(rna_sum, rna_sum, method = "pearson")

corrplot(cor, 
         order = "FPC",
         addCoef.col = "white",
         method = "circle",
         tl.col="black",
         tl.cex = 1.5)

Figure 2E

Correlation of Chemokines with Celltypes (based on fractions)

# top abundant chemokines
cur_rna <- data.frame(colData(sce_rna))

# protein data
cur_prot <- data.frame(colData(sce_prot))

# sum
rna_sum <- cur_rna %>%
  group_by(Description) %>%
  mutate(total_cells=n()) %>%
  ungroup() %>%
  group_by(Description, total_cells, expressor) %>%
  summarise(n=n()) %>%
  mutate(fraction=n/total_cells) %>%
  reshape2::dcast(Description ~ expressor, value.var = "fraction", fill = 0) 

# only keep highly abundant chemokines
rna_sum <- rna_sum[,c("Description", targets)]

prot_sum <- cur_prot %>%
  group_by(Description, celltype) %>%
  summarise(n = n()) %>%
  group_by(Description) %>%
  mutate(fraction = n/sum(n)) %>%
  reshape2::dcast(Description ~ celltype, value.var = "fraction", fill = 0)

# equal images
all(rna_sum$Description == prot_sum$Description)
[1] TRUE
# correlation
cor <- cor(rna_sum[,-1], prot_sum[,-1], method = "pearson")

corrplot(cor, 
         addCoef.col = "white",
         method = "circle",
         tl.col="black",
         tl.cex = 1.5)

Scatter Plot for CD8+ and Chemokine Selection

# sum
chemokines <- data.frame(colData(sce_rna)) %>%
  group_by(Description) %>%
  mutate(total_cells=n()) %>%
  ungroup() %>%
  group_by(Description, total_cells, expressor) %>%
  summarise(n=n()) %>%
  mutate(fraction=n/total_cells) %>%
  reshape2::dcast(Description ~ expressor, value.var = "fraction", fill = 0) %>%
  select(Description, CXCL12, CXCL9, CCL22)

celltypes <- data.frame(colData(sce_prot)) %>%
  group_by(Description, celltype) %>%
  summarise(n=n()) %>%
  mutate(fraction=n/sum(n)) %>%
  reshape2::dcast(Description ~ celltype, value.var = "fraction", fill = 0) %>%
  select(Description, `CD8+ T cell`, pDC, `FOXP3+ T cell`) %>%
  reshape2::melt(id.vars = "Description", variable.name = "celltype", value.name = "cell_fraction")

sum <- left_join(celltypes, chemokines, by = "Description") %>%
  reshape2::melt(id.vars = c("Description", "celltype", "cell_fraction"), variable.name = "expressor", value.name = "chemo_fraction")

sum <- sum %>%
  mutate(celltype_chemo = paste(celltype, expressor, sep = " ~ ")) %>%
  filter(celltype_chemo %in% c("`CD8+ T cell` ~ CXCL9", "pDC ~ CXCL12", "`FOXP3+ T cell` ~ CCL22"))

sum$celltype_chemo <- factor(sum$celltype_chemo, levels = c("pDC ~ CXCL12", "`FOXP3+ T cell` ~ CCL22","`CD8+ T cell` ~ CXCL9"))

ggplot(sum, aes(x=log10(chemo_fraction), y=log10(cell_fraction))) + 
  geom_point() + 
  geom_smooth(method="lm") +
  ylab("Celltype Fraction (log10)\n") +
  xlab("Chemokine Fraction (log10)") +
  scale_y_continuous(position = "right") +
  theme_bw() + 
  theme(text=element_text(size=16),
        plot.margin = margin(0.5,0.5,0.5,0.5, "cm"),
        axis.title.y = element_text(hjust=1)) +
  guides(col=guide_legend(title="Chemokines")) +
  stat_poly_eq(formula = y ~ x, size=7,
               aes(label = ..rr.label..), 
               parse = TRUE) + 
  facet_wrap(~celltype_chemo, ncol = 2, scales = "free", as.table = FALSE)
Warning: Removed 48 rows containing non-finite values (stat_smooth).
Warning: Removed 48 rows containing non-finite values (stat_poly_eq).

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] forcats_0.5.0               circlize_0.4.12            
 [3] colorRamps_2.3              RColorBrewer_1.1-2         
 [5] ggpubr_0.4.0                ggbeeswarm_0.6.0           
 [7] ggalluvial_0.12.3           gridExtra_2.3              
 [9] corrplot_0.84               cowplot_1.1.1              
[11] ggpmisc_0.3.7               ggplot2_3.3.3              
[13] tidyr_1.1.2                 janitor_2.1.0              
[15] dplyr_1.0.2                 data.table_1.13.6          
[17] ComplexHeatmap_2.4.3        SingleCellExperiment_1.12.0
[19] SummarizedExperiment_1.20.0 Biobase_2.50.0             
[21] GenomicRanges_1.42.0        GenomeInfoDb_1.26.2        
[23] IRanges_2.24.1              S4Vectors_0.28.1           
[25] BiocGenerics_0.36.0         MatrixGenerics_1.2.0       
[27] matrixStats_0.57.0          workflowr_1.6.2            

loaded via a namespace (and not attached):
 [1] colorspace_2.0-0       ggsignif_0.6.0         rjson_0.2.20          
 [4] ellipsis_0.3.1         rio_0.5.16             rprojroot_2.0.2       
 [7] snakecase_0.11.0       XVector_0.30.0         GlobalOptions_0.1.2   
[10] fs_1.5.0               clue_0.3-58            rstudioapi_0.13       
[13] farver_2.0.3           lubridate_1.7.9.2      splines_4.0.3         
[16] knitr_1.30             polynom_1.4-0          broom_0.7.3           
[19] cluster_2.1.0          png_0.1-7              compiler_4.0.3        
[22] backports_1.2.1        Matrix_1.3-2           later_1.1.0.1         
[25] htmltools_0.5.0        tools_4.0.3            gtable_0.3.0          
[28] glue_1.4.2             GenomeInfoDbData_1.2.4 reshape2_1.4.4        
[31] Rcpp_1.0.5             carData_3.0-4          cellranger_1.1.0      
[34] vctrs_0.3.6            nlme_3.1-151           xfun_0.20             
[37] stringr_1.4.0          openxlsx_4.2.3         lifecycle_0.2.0       
[40] rstatix_0.6.0          zlibbioc_1.36.0        scales_1.1.1          
[43] hms_0.5.3              promises_1.1.1         yaml_2.2.1            
[46] curl_4.3               stringi_1.5.3          zip_2.1.1             
[49] shape_1.4.5            rlang_0.4.10           pkgconfig_2.0.3       
[52] bitops_1.0-6           evaluate_0.14          lattice_0.20-41       
[55] purrr_0.3.4            labeling_0.4.2         tidyselect_1.1.0      
[58] plyr_1.8.6             magrittr_2.0.1         R6_2.5.0              
[61] generics_0.1.0         DelayedArray_0.16.0    pillar_1.4.7          
[64] haven_2.3.1            whisker_0.4            foreign_0.8-81        
[67] withr_2.3.0            mgcv_1.8-33            abind_1.4-5           
[70] RCurl_1.98-1.2         tibble_3.0.4           crayon_1.3.4          
[73] car_3.0-10             rmarkdown_2.6          GetoptLong_1.0.5      
[76] readxl_1.3.1           git2r_0.28.0           digest_0.6.27         
[79] httpuv_1.5.4           munsell_0.5.0          beeswarm_0.2.3        
[82] vipor_0.4.5