Last updated: 2020-12-19

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Knit directory: meta-liver/

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Rmd 961f67d christianholland 2020-12-19 added acute ccl4 study

Introduction

Here we analysis a mouse model of CCl4 induced acute liver damage. The transcriptomic profiles were measured at 9 different time points ranging from 2 hours to 16 days.

Libraries and sources

These libraries and sources are used for this analysis.

library(mouse4302.db)

library(tidyverse)
library(tidylog)
library(here)

library(oligo)
library(annotate)
library(limma)
library(biobroom)

library(janitor)

library(AachenColorPalette)
library(cowplot)
library(lemon)
library(patchwork)

options("tidylog.display" = list(print))
source(here("code/utils-microarray.R"))
source(here("code/utils-utils.R"))
source(here("code/utils-plots.R"))

Definition of global variables that are used throughout this analysis.

# i/o
data_path <- "data/mouse-acute-ccl4"
output_path <- "output/mouse-acute-ccl4"

# graphical parameters
# fontsize
fz <- 9

Data processing

Load .CEL files and quality control

The array quality is controlled based on the relative log expression values (RLE) and the normalized unscaled standard errors (NUSE).

# load cel files and check quality
platforms <- readRDS(here("data/annotation/platforms.rds"))
raw_eset <- list.celfiles(here(data_path), listGzipped = T, full.names = T) %>%
  read.celfiles() %>%
  ma_qc() # Discarding in total 1 arrays: 6h_m3_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_2hm1_16_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_2hm2_17_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_2hm3_18_(Mouse430_2)_2.CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_2hm4_19_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_2hm5_20_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_8hm1_21_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_8hm2_22_(Mouse430_2)_2.CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_8hm3_23_(Mouse430_2)_2.CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_8hm4_24_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_8hm5_25_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D16M1_51_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D16M2_52_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D16M3_53_(Mouse430_2)_2.CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D16M4_54_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D16M5_55_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D16M6_56_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d1m1_26_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d1m2_27_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d1m3_28_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d1m4_29_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d1m5_30_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d2m1_31_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d2m2_32_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d2m3_33_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d2m4_34_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d2m5_35_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d4m1_36_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d4m2_37_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d4m3_38_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d4m4_39_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d4m5_40_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d6m1_41_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d6m2_42_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d6m3_43_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d6m4_44_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_d6m5_45_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D8M1_46_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D8M2_47_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D8M3_48_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D8M4_49_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_D8M5_50_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_KM1_1_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_KM2_2_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_KM3_3_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_KM4_4_(Mouse430_2).CEL
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-ccl4/Patricio_KM5_5_(Mouse430_2).CEL

Normalization and probe annotation

Probe intensities are normalized with the rma() function. Probes are annotated with MGI symbols.

eset <- rma(raw_eset)
#> Background correcting
#> Normalizing
#> Calculating Expression

# annotate microarray probes with mgi symbols
expr <- ma_annotate(eset, platforms)
pattern <- str_c(c(
  "Patricio_",
  "_\\(Mouse430_2\\).CEL",
  "_\\(Mouse430_2\\)_2.CEL"
), collapse = "|")

colnames(expr) <- str_c(
  "sample", str_remove_all(colnames(expr), pattern),
  sep = "_"
) %>%
  str_to_lower()

# save normalized expression
saveRDS(expr, here(output_path, "normalized_expression.rds"))

Build meta data

Meta information are parsed from the sample names.

# build meta data
meta <- colnames(expr) %>%
  enframe(name = NULL, value = "sample") %>%
  separate(sample, into = c("tmp", "key", "run"), remove = F) %>%
  dplyr::select(-tmp) %>%
  separate(key, into = c("group", "rep"), sep = "m", remove = T) %>%
  mutate(time = case_when(
    group == "k" ~ 0,
    str_detect(group, "h") ~ parse_number(group),
    str_detect(group, "d") ~ parse_number(group) * 24
  )) %>%
  mutate(time = ordered(time)) %>%
  mutate(group = case_when(
    group == "k" ~ "control",
    str_detect(group, "h") ~ str_c("h", parse_number(group)),
    TRUE ~ group
  )) %>%
  mutate(group = factor(group, levels = c(
    "control", "h2", "h8", "d1", "d2",
    "d4", "d6", "d8", "d16"
  ))) %>%
  arrange(sample)
#> mutate: new variable 'time' (double) with 9 unique values and 0% NA
#> mutate: converted 'time' from double to ordered factor (0 new NA)
#> mutate: changed 15 values (33%) of 'group' (0 new NA)
#> mutate: converted 'group' from character to factor (0 new NA)

# save meta data
saveRDS(meta, here(output_path, "meta_data.rds"))

Exploratory analysis

PCA of normalized data

PCA plot of normalized expression data contextualized based on the time point. Only the top 1000 most variable genes are used as features.

expr <- readRDS(here(output_path, "normalized_expression.rds"))
meta <- readRDS(here(output_path, "meta_data.rds"))

pca_result <- do_pca(expr, meta, top_n_var_genes = 1000)
#> left_join: added 4 columns (group, rep, run, time)
#>            > rows only in x    0
#>            > rows only in y  ( 0)
#>            > matched rows     46
#>            >                 ====
#>            > rows total       46

saveRDS(pca_result, here(output_path, "pca_result.rds"))

plot_pca(pca_result, feature = "time") +
  my_theme()

Differential gene expression analysis

Running limma

Differential gene expression analysis via limma with the aim to identify the effect of APAP intoxication for the different time points.

# load expression and meta data
expr <- readRDS(here(output_path, "normalized_expression.rds"))
meta <- readRDS(here(output_path, "meta_data.rds"))

stopifnot(colnames(expr) == meta$sample)

# build design matrix
design <- model.matrix(~ 0 + group, data = meta)
rownames(design) <- meta$sample
colnames(design) <- levels(meta$group)

# define contrasts
contrasts <- makeContrasts(
  # effect of ccl4 treatment
  ccl_2h_vs_0h = h2 - control,
  ccl_8h_vs_0h = h8 - control,
  ccl_24h_vs_0h = d1 - control,
  ccl_48h_vs_0h = d2 - control,
  ccl_96h_vs_0h = d4 - control,
  ccl_144h_vs_0h = d6 - control,
  ccl_192h_vs_0h = d8 - control,
  ccl_384h_vs_0h = d16 - control,

  # consecutive time point comparison
  consec_2h_vs_0h = h2 - control,
  consec_8h_vs_2h = h8 - h2,
  consec_24h_vs_8h = d1 - h8,
  consec_48h_vs_24h = d2 - d1,
  consec_96h_vs_48h = d4 - d2,
  consec_144h_vs_96h = d6 - d4,
  consec_192h_vs_144h = d8 - d6,
  consec_384h_vs_192h = d16 - d8,
  levels = design
)

limma_result <- run_limma(expr, design, contrasts) %>%
  assign_deg()
#> select: renamed 3 variables (contrast, logFC, pval) and dropped one variable
#> group_by: one grouping variable (contrast)
#> mutate (grouped): new variable 'fdr' (double) with 151,077 unique values and 0% NA
#> ungroup: no grouping variables
#> mutate: new variable 'regulation' (character) with 3 unique values and 0% NA
#> mutate: converted 'regulation' from character to factor (0 new NA)

deg_df <- limma_result %>%
  mutate(contrast = fct_inorder(contrast)) %>%
  mutate(contrast_reference = case_when(
    str_detect(contrast, "ccl") ~ "ccl4", 
    str_detect(contrast, "consec") ~ "consec"
  ))
#> mutate: changed 0 values (0%) of 'contrast' (0 new NA)
#> mutate: new variable 'contrast_reference' (character) with 2 unique values and 0% NA

saveRDS(deg_df, here(output_path, "limma_result.rds"))

Volcano plots

Volcano plots visualizing the effect of APAP on gene expression.

df <- readRDS(here(output_path, "limma_result.rds"))

df %>%
  filter(contrast_reference == "ccl4") %>%
  plot_volcano() +
  my_theme(grid = "y", fsize = fz)
#> filter: removed 163,792 rows (50%), 163,792 rows remaining
#> rename: renamed one variable (p)

Time series clustering

Gene expression trajectories are clustered using the STEM software. The cluster algorithm is described here.

Prepare input

# prepare input for stem analysis
df <- readRDS(here(output_path, "limma_result.rds"))

stem_inputs <- df %>%
  mutate(class = str_c("Hour ", parse_number(as.character(contrast)))) %>%
  mutate(class = factor(class, levels = unique(.$class))) %>%
  select(gene, class, logFC, contrast_reference)
#> mutate: new variable 'class' (character) with 8 unique values and 0% NA
#> mutate: converted 'class' from character to factor (0 new NA)
#> select: dropped 5 variables (contrast, statistic, pval, fdr, regulation)

stem_inputs %>%
  filter(contrast_reference == "ccl4") %>%
  select(-contrast_reference) %>%
  pivot_wider(names_from = class, values_from = logFC) %>%
  write_delim(here(output_path, "stem/input/ccl4.txt"), delim = "\t")
#> filter: removed 163,792 rows (50%), 163,792 rows remaining
#> select: dropped one variable (contrast_reference)
#> pivot_wider: reorganized (class, logFC) into (Hour 2, Hour 8, Hour 24, Hour 48, Hour 96, …) [was 163792x3, now 20474x9]

Run STEM

STEM is implemented in Java. The .jar file is called from R. Only significant time series clusters are visualized.

# execute stem
stem_res <- run_stem(here(output_path, "stem"), clear_output = T)
#> distinct: no rows removed
#> mutate: new variable 'gene' (character) with 20,474 unique values and 0% NA
#> distinct: no rows removed
#> mutate: new variable 'key' (character) with one unique value and 0% NA
#> select: dropped one variable (spot)
#> gather: reorganized (x0, hour_2, hour_8, hour_24, hour_48, …) into (time, value) [was 2084x12, now 18756x5]
#> mutate: converted 'time' from character to double (0 new NA)
#> mutate: new variable 'key' (character) with one unique value and 0% NA
#> select: renamed 4 variables (profile, y_coords, size, p) and dropped 2 variables
#> inner_join: added 3 columns (y_coords, size, p)
#>             > rows only in x  (     0)
#>             > rows only in y  (     0)
#>             > matched rows     18,756
#>             >                 ========
#>             > rows total       18,756
#> inner_join: added one column (symbol)
#>             > rows only in x  (     0)
#>             > rows only in y  (18,390)
#>             > matched rows     18,756
#>             >                 ========
#>             > rows total       18,756
#> transmute: dropped one variable (symbol)
#>            changed 18,630 values (99%) of 'gene' (0 new NA)

saveRDS(stem_res, here(output_path, "stem_result.rds"))

stem_res %>%
  filter(p <= 0.05) %>%
  filter(key == "ccl4") %>%
  distinct() %>%
  plot_stem_profiles(model_profile = F, ncol = 2) +
  labs(x = "Time in Hours", y = "logFC") +
  my_theme(grid = "y", fsize = fz)
#> filter: removed 2,781 rows (15%), 15,975 rows remaining
#> filter: no rows removed
#> distinct: no rows removed
#> group_by: 4 grouping variables (key, profile, p, time)
#> ungroup: no grouping variables


sessionInfo()
#> R version 4.0.2 (2020-06-22)
#> Platform: x86_64-apple-darwin17.0 (64-bit)
#> Running under: macOS Mojave 10.14.5
#> 
#> Matrix products: default
#> BLAS:   /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.dylib
#> LAPACK: /Library/Frameworks/R.framework/Versions/4.0/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] parallel  stats4    stats     graphics  grDevices datasets  utils    
#> [8] methods   base     
#> 
#> other attached packages:
#>  [1] pd.mouse430.2_3.12.0     DBI_1.1.0                RSQLite_2.2.1           
#>  [4] patchwork_1.1.1          lemon_0.4.5              cowplot_1.1.0           
#>  [7] AachenColorPalette_1.1.2 janitor_2.0.1            biobroom_1.22.0         
#> [10] broom_0.7.3              limma_3.46.0             annotate_1.68.0         
#> [13] XML_3.99-0.5             oligo_1.54.1             Biostrings_2.58.0       
#> [16] XVector_0.30.0           oligoClasses_1.52.0      here_1.0.1              
#> [19] tidylog_1.0.2            forcats_0.5.0            stringr_1.4.0           
#> [22] dplyr_1.0.2              purrr_0.3.4              readr_1.4.0             
#> [25] tidyr_1.1.2              tibble_3.0.4             ggplot2_3.3.2           
#> [28] tidyverse_1.3.0          mouse4302.db_3.2.3       org.Mm.eg.db_3.12.0     
#> [31] AnnotationDbi_1.52.0     IRanges_2.24.1           S4Vectors_0.28.1        
#> [34] Biobase_2.50.0           BiocGenerics_0.36.0      workflowr_1.6.2         
#> 
#> loaded via a namespace (and not attached):
#>  [1] colorspace_2.0-0            ellipsis_0.3.1             
#>  [3] rprojroot_2.0.2             snakecase_0.11.0           
#>  [5] GenomicRanges_1.42.0        fs_1.5.0                   
#>  [7] rstudioapi_0.13             farver_2.0.3               
#>  [9] affyio_1.60.0               bit64_4.0.5                
#> [11] fansi_0.4.1                 lubridate_1.7.9.2          
#> [13] xml2_1.3.2                  codetools_0.2-16           
#> [15] splines_4.0.2               knitr_1.30                 
#> [17] jsonlite_1.7.2              dbplyr_2.0.0               
#> [19] BiocManager_1.30.10         compiler_4.0.2             
#> [21] httr_1.4.2                  backports_1.2.1            
#> [23] assertthat_0.2.1            Matrix_1.2-18              
#> [25] cli_2.2.0                   later_1.1.0.1              
#> [27] htmltools_0.5.0             tools_4.0.2                
#> [29] gtable_0.3.0                glue_1.4.2                 
#> [31] GenomeInfoDbData_1.2.4      affxparser_1.62.0          
#> [33] Rcpp_1.0.5                  cellranger_1.1.0           
#> [35] vctrs_0.3.6                 preprocessCore_1.52.0      
#> [37] iterators_1.0.13            xfun_0.19                  
#> [39] rvest_0.3.6                 lifecycle_0.2.0            
#> [41] renv_0.12.3                 zlibbioc_1.36.0            
#> [43] scales_1.1.1                clisymbols_1.2.0           
#> [45] hms_0.5.3                   promises_1.1.1             
#> [47] MatrixGenerics_1.2.0        SummarizedExperiment_1.20.0
#> [49] yaml_2.2.1                  gridExtra_2.3              
#> [51] memoise_1.1.0               stringi_1.5.3              
#> [53] foreach_1.5.1               GenomeInfoDb_1.26.2        
#> [55] rlang_0.4.9                 pkgconfig_2.0.3            
#> [57] bitops_1.0-6                matrixStats_0.57.0         
#> [59] evaluate_0.14               lattice_0.20-41            
#> [61] labeling_0.4.2              bit_4.0.4                  
#> [63] tidyselect_1.1.0            plyr_1.8.6                 
#> [65] magrittr_2.0.1              R6_2.5.0                   
#> [67] generics_0.1.0              DelayedArray_0.16.0        
#> [69] pillar_1.4.7                haven_2.3.1                
#> [71] whisker_0.4                 withr_2.3.0                
#> [73] RCurl_1.98-1.2              modelr_0.1.8               
#> [75] crayon_1.3.4                rmarkdown_2.6              
#> [77] grid_4.0.2                  readxl_1.3.1               
#> [79] blob_1.2.1                  git2r_0.27.1               
#> [81] reprex_0.3.0                digest_0.6.27              
#> [83] xtable_1.8-4                ff_4.0.4                   
#> [85] httpuv_1.5.4                munsell_0.5.0              
#> [87] viridisLite_0.3.0