molkart.QC_spots
FloWuenne
2023-06-12
Last updated: 2023-06-12
Checks: 6 1
Knit directory: mi_spatialomics/
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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)NULLexp_mat[is.na(exp_mat)] <- 0## Perform PCA
library(factoextra)Welcome! Want to learn more? See two factoextra-related books at https://goo.gl/ve3WBares.pca <- prcomp(exp_mat, scale = TRUE)
fviz_eig(res.pca)
## 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
write.table(pcs,
file = here("./output/pca_spots.tsv"),
sep = "\t",
col.names = TRUE,
row.names = FALSE,
quote = FALSE)
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