molkart.QC_spots

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Last updated: 2023-06-12

Checks: 7 0

Knit directory: mi_spatialomics/

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File	Version	Author	Date	Message
Rmd	3b5ca40	FloWuenne	2023-06-12	Added code for supplementary Figures.
html	3b5ca40	FloWuenne	2023-06-12	Added code for supplementary Figures.

---

Introduction

Correlation between technical replicates

Read data

## This script will take the Molecular Cartography spot count tables transform them from tsv
data_dir <- here("../data/nf_MolCart_results/dedup_spots")

all_samples <- list()

all_files <- list.files(data_dir)
for(this_file in all_files){
  if(grepl("txt",this_file)){
    print(this_file)
    sample_tx <- vroom(paste(data_dir,this_file,sep="/"),col_names = c("x","y","z","gene"),col_select = c(x,y,z,gene))
    sample_tx$sample <- this_file
    
    sample_tx_sums <- sample_tx %>%
      subset(gene != "Duplicated") %>%
      count(gene) %>%
      mutate("sample" = this_file) %>%
      separate(sample, into = c("x","time","replicate","slide","spots"),
               remove = FALSE) %>%
      select(-c(x,spots)) %>%
      mutate("sample_ID" = paste("sample",time,replicate,sep="_"),
             "total_tx" = n) %>%
      select(-n) %>%
      arrange(desc(total_tx))
    
    all_samples[[this_file]] <- sample_tx_sums
  }
}

[1] "sample_2d_r1_s1.spots_markedDups.txt"

Rows: 940788 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 99 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_2d_r1_s2.spots_markedDups.txt"

Rows: 2242464 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 99 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_2d_r2_s1.spots_markedDups.txt"

Rows: 1055509 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_2d_r2_s2.spots_markedDups.txt"

Rows: 1855385 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 98 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4d_r1_s1.spots_markedDups.txt"

Rows: 4988178 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4d_r1_s2.spots_markedDups.txt"

Rows: 1225229 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4d_r2_s1.spots_markedDups.txt"

Rows: 1231209 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 99 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4d_r2_s2.spots_markedDups.txt"

Rows: 758844 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 99 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4h_r1_s1.spots_markedDups.txt"

Rows: 3037103 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4h_r1_s2.spots_markedDups.txt"

Rows: 2200936 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4h_r2_s1.spots_markedDups.txt"

Rows: 417879 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 98 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_4h_r2_s2.spots_markedDups.txt"

Rows: 2132153 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 98 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_control_r1_s1.spots_markedDups.txt"

Rows: 5558060 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 99 rows [1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_control_r1_s2.spots_markedDups.txt"

Rows: 5188851 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_control_r2_s1.spots_markedDups.txt"

Rows: 4657621 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

[1] "sample_control_r2_s2.spots_markedDups.txt"

Rows: 4854327 Columns: 4
── Column specification ────────────────────────────────────────────────────────
Delimiter: "\t"
chr (1): gene
dbl (3): x, y, z

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Warning: Expected 5 pieces. Additional pieces discarded in 100 rows [1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ...].

Merge data per biological replicate by slide

all_samples_df <- bind_rows(all_samples, .id = "column_label")
all_samples_df <- all_samples_df %>%
  select(total_tx, gene, sample_ID, slide,time)

slide1 <- subset(all_samples_df,slide == "s1") %>% select(-slide)
slide2 <- subset(all_samples_df,slide == "s2") %>% select(-slide)
merge_tx_sums <- full_join(slide1,slide2, by = c("gene","sample_ID","time"), suffix = c("_rep1","_rep2"))

Write data for plotting

vroom_write(merge_tx_sums,
            file = here("./output/tx_abundances_per_slide.tsv"))

Principal component analysis of spot counts

all_samples_df <- bind_rows(all_samples, .id = "column_label")
all_samples_df <- all_samples_df %>%mutate("sample_ID" = paste("sample",time,replicate,slide,sep="_"))

metadata <- all_samples_df %>%
  select(sample_ID,replicate,slide,time)

exp_mat <- all_samples_df %>%
  select(sample_ID,total_tx,gene) %>%
  pivot_wider(names_from = "gene",
             values_from = "total_tx")

samples <- exp_mat$sample_ID
exp_mat <- exp_mat %>% select(-sample_ID)

exp_mat <- as.matrix(exp_mat)
#colnames(exp_mat) <- samples
rownames(samples)

NULL

exp_mat[is.na(exp_mat)] <- 0

## Perform PCA
library(factoextra)

Welcome! Want to learn more? See two factoextra-related books at https://goo.gl/ve3WBa

res.pca <- prcomp(exp_mat, scale = TRUE)

fviz_eig(res.pca)

Past versions of unnamed-chunk-5-1.png

Version	Author	Date
3b5ca40	FloWuenne	2023-06-12

## Plot PCAs
pcs <- as.data.frame(res.pca$x)
pcs$sample <- samples
pcs <- pcs %>%
  mutate("time" = if_else(grepl("control",sample),"control",
                          if_else(grepl("4h",sample),"4h",
                                  if_else(grepl("2d",sample),"2d","4d")))
         )

pcs$time <- factor(pcs$time,levels= c("control","4h","2d","4d"))
pcs$label <- gsub(".spots.txt","",pcs$sample)
pcs <- pcs %>%
  separate("sample", into = c("string","time","replicate","slide"))
pcs <- pcs %>%
  select(-c(string))
pcs$slide <- gsub("s1","Slide 1",pcs$slide)
pcs$slide <- gsub("s2","Slide 2",pcs$slide)
pcs$time <- factor(pcs$time,
                   levels = c("control","4h","2d","4d"))

pca_plot <- ggplot(pcs,aes(PC1,PC2,label = label)) +
  geom_point(size = 4, aes(color = time, shape = slide)) +
  scale_color_brewer(palette = "Dark2",labels = c("control","4 hours","2 days","4 days")) +
  labs(color = "Time",
       shape = "Slide") + 
  background_grid()
pca_plot

Past versions of unnamed-chunk-6-1.png

Version	Author	Date
3b5ca40	FloWuenne	2023-06-12

write.table(pcs,
            file = here("./output/pca_spots.tsv"),
            sep = "\t",
            col.names = TRUE,
            row.names = FALSE,
            quote = FALSE)

Session information

sessionInfo()

R version 4.2.3 (2023-03-15)
Platform: aarch64-apple-darwin20 (64-bit)
Running under: macOS Ventura 13.4

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.2-arm64/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.2-arm64/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats     graphics  grDevices datasets  utils     methods   base     

other attached packages:
 [1] factoextra_1.0.7 ggsci_3.0.0      cowplot_1.1.1    ggrepel_0.9.3   
 [5] patchwork_1.1.2  ggpubr_0.6.0     lubridate_1.9.2  forcats_1.0.0   
 [9] stringr_1.5.0    dplyr_1.1.2      purrr_1.0.1      readr_2.1.4     
[13] tidyr_1.3.0      tibble_3.2.1     ggplot2_3.4.2    tidyverse_2.0.0 
[17] vroom_1.6.3      here_1.0.1       workflowr_1.7.0 

loaded via a namespace (and not attached):
 [1] httr_1.4.6         sass_0.4.6         bit64_4.0.5        jsonlite_1.8.4    
 [5] carData_3.0-5      bslib_0.4.2        getPass_0.2-2      highr_0.10        
 [9] renv_0.17.3        yaml_2.3.7         pillar_1.9.0       backports_1.4.1   
[13] glue_1.6.2         digest_0.6.31      RColorBrewer_1.1-3 promises_1.2.0.1  
[17] ggsignif_0.6.4     colorspace_2.1-0   htmltools_0.5.5    httpuv_1.6.11     
[21] pkgconfig_2.0.3    broom_1.0.5        scales_1.2.1       processx_3.8.0    
[25] whisker_0.4.1      later_1.3.1        tzdb_0.4.0         timechange_0.2.0  
[29] git2r_0.32.0       generics_0.1.3     farver_2.1.1       car_3.1-2         
[33] cachem_1.0.8       withr_2.5.0        cli_3.6.1          magrittr_2.0.3    
[37] crayon_1.5.2       evaluate_0.21      ps_1.7.4           fs_1.6.2          
[41] fansi_1.0.4        rstatix_0.7.2      tools_4.2.3        hms_1.1.3         
[45] lifecycle_1.0.3    munsell_0.5.0      callr_3.7.3        compiler_4.2.3    
[49] jquerylib_0.1.4    rlang_1.1.1        grid_4.2.3         rstudioapi_0.14   
[53] labeling_0.4.2     rmarkdown_2.21     gtable_0.3.3       abind_1.4-5       
[57] R6_2.5.1           knitr_1.42         fastmap_1.1.1      bit_4.0.5         
[61] utf8_1.2.3         rprojroot_2.0.3    stringi_1.7.12     parallel_4.2.3    
[65] Rcpp_1.0.10        vctrs_0.6.2        tidyselect_1.2.0   xfun_0.39