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Knit directory: ATAC_learning/

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package loading
library(tidyverse)
library(kableExtra)
library(broom)
library(RColorBrewer)
library("TxDb.Hsapiens.UCSC.hg38.knownGene")
library("org.Hs.eg.db")
library(rtracklayer)
library(ggfortify)
library(readr)
library(BiocGenerics)
library(gridExtra)
library(VennDiagram)
library(scales)
library(ggVennDiagram)
library(BiocParallel)
library(ggpubr)
library(edgeR)
library(genomation)
library(ggsignif)
library(plyranges)
library(ggrepel)
library(ComplexHeatmap)
library(cowplot)
library(smplot2)
library(readxl)
drug_pal <- c("#8B006D","#DF707E","#F1B72B", "#3386DD","#707031","#41B333")

prop_var_percent <- function(pca_result){ 
  # Ensure the input is a PCA result object
  if (!inherits(pca_result, "prcomp")) {
    stop("Input must be a result from prcomp()")
  }
  
  # Get the standard deviations from the PCA result
  sdev <- pca_result$sdev
  
  # Calculate the proportion of variance
  proportion_variance <- (sdev^2) / sum(sdev^2)*100
  
  return(proportion_variance)
}

Figure 1.A, B and C: Anthracycline treatment of iPSC-CMs results in DNA damage and chromatin changes.

knitr::include_graphics("assets/Figure\ 1.png", error=FALSE)

Version Author Date
d64bc29 E. Renee Matthews 2025-02-21
knitr::include_graphics("docs/assets/Figure\ 1.png",error = FALSE)

Figure 1.D: Quantification of the proportion of DNA damage-associated nuclei following treatment with DOX, EPI,DNR, MTX, TRZ, and VEH for three and 24 hours.

drug_pal <- c("#8B006D","#DF707E","#F1B72B", "#3386DD","#707031","#41B333")
Nuclei_gamma_count_87<- read_delim("data/Final_four_data/re_analysis/IF_data/Nuclei_gamma_count_87.txt",delim="\t")
Nuclei_gamma_count_77<- read_delim("data/Final_four_data/re_analysis/IF_data/Nuclei_gamma_count_77.txt",delim="\t")
Nuclei_gamma_count_71<- read_delim("data/Final_four_data/re_analysis/IF_data/Nuclei_gamma_count_71.txt",delim="\t")

Nuclei_gamma_count_75<- read_delim("data/Final_four_data/re_analysis/IF_data/Nuclei_gamma_count_75.txt",delim="\t")

Ind_A_table <- Nuclei_gamma_count_87 %>% 
  dplyr::select(Sample,percentage) %>% 
  separate_wider_delim(Sample,delim = "_",names = c("time","ind","trt","focus"),too_many = "merge") %>% 
  group_by(trt,time) %>%
  mutate(group_number = rep(c("A","B"), length.out = dplyr::n())) %>%
  mutate(trt=if_else(trt=="MTZ","MTX",trt)) %>% 
  ungroup() %>% 
  pivot_wider(., id_cols=c(time, ind,trt), names_from = group_number, values_from = percentage) %>% 
  rowwise() %>% 
  mutate(total = round(mean(c_across(c(A, B)), na.rm = TRUE), digits = 1)) %>% 
  ungroup() %>% 
  mutate(ind="A")

Ind_B_table <- Nuclei_gamma_count_77 %>% 
  dplyr::select(Sample,percentage) %>% 
  separate_wider_delim(Sample,delim = "_",names = c("time","ind","trt","focus"),too_many = "merge") %>% 
  group_by(trt,time) %>%
  mutate(group_number = rep(c("A","B"), length.out =  dplyr::n())) %>%
   mutate(trt=if_else(trt=="MTZ","MTX",trt)) %>% 
  ungroup() %>% 
  pivot_wider(., id_cols=c(time, ind,trt), names_from = group_number, values_from = percentage) %>% 
  rowwise() %>% 
  mutate(total = round(mean(c_across(c(A, B)), na.rm = TRUE), digits = 1)) %>% 
  ungroup() %>% 
  mutate(ind="B")

Ind_C_table <- Nuclei_gamma_count_71 %>% 
  dplyr::select(Sample,percentage) %>% 
  separate_wider_delim(Sample,delim = "_",names = c("time","ind","trt","focus"),too_many = "merge") %>% 
  group_by(trt,time) %>%
  mutate(group_number = rep(c("A","B"), length.out =  dplyr::n())) %>%
   mutate(trt=if_else(trt=="MTZ","MTX",trt)) %>% 
  ungroup() %>% 
  pivot_wider(., id_cols=c(time, ind,trt), names_from = group_number, values_from = percentage) %>% 
  rowwise() %>% 
  mutate(total = round(mean(c_across(c(A, B)), na.rm = TRUE), digits = 1)) %>% 
  ungroup() %>% 
  mutate(ind="C")



Ind_D_table <- Nuclei_gamma_count_75 %>% 
  dplyr::select(Sample,percentage) %>% 
  separate_wider_delim(Sample,delim = "_",names = c("time","ind","trt","focus"),too_many = "merge") %>% 
  group_by(trt,time) %>%
  mutate(group_number = rep(c("A","B"), length.out =  dplyr::n())) %>%
   mutate(trt=if_else(trt=="MTZ","MTX",trt)) %>% 
  ungroup() %>% 
  pivot_wider(., id_cols=c(time, ind,trt), names_from = group_number, values_from = percentage) %>% 
  rowwise() %>% 
  mutate(total = round(mean(c_across(c(A, B)), na.rm = TRUE), digits = 1)) %>% 
  ungroup() %>% 
  mutate(ind="D")

bind_rows(Ind_A_table,Ind_B_table) %>%
  bind_rows(., Ind_C_table) %>% 
  bind_rows(., Ind_D_table) %>% 
  mutate(time=factor(time, levels =c("3h","24h")),
         trt=factor(trt, levels = c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(.,aes(x=trt, y=total))+
  geom_boxplot(aes(fill=trt))+
  geom_point(aes(colour = ind))+
  ggsignif:: geom_signif(comparisons = list(
      c("VEH", "DOX"),
      c("VEH", "EPI"),
      c("VEH", "DNR"),
      c("VEH", "MTX"),
      c("VEH", "TRZ")),
      step_increase = 0.1,
      map_signif_level = FALSE, 
      test = "t.test")+
      facet_wrap(~time)+
  theme_bw()+
  scale_fill_manual(values=drug_pal)

Version Author Date
67a1456 reneeisnowhere 2025-08-07

Figure 1.E: Principal component analysis of ATAC-seq-derived accessibility measurements across 48 samples

filt_counts_raw <- readRDS("data/Final_four_data/ATAC_filtered_raw_counts_allsamples.RDS")
filt_raw_counts_noY <- filt_counts_raw[!grepl("chrY",rownames(filt_counts_raw)),]

filt4_matrix_lcpm <- filt_raw_counts_noY  %>%
  as.data.frame() %>% 
  rename_with(.,~gsub(pattern = "Ind1_75", replacement = "D_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind2_87", replacement = "A_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind3_77", replacement = "B_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind6_71", replacement = "C_",.)) %>% 
  rename_with(.,~gsub( "DX" ,'DOX',.)) %>% 
  rename_with(.,~gsub( "DA" ,'DNR',.)) %>% 
  rename_with(.,~gsub( "E" ,'EPI',.)) %>% 
  rename_with(.,~gsub( "T" ,'TRZ',.)) %>%
  rename_with(.,~gsub( "M" ,'MTX',.)) %>% 
  rename_with(.,~gsub( "V" ,'VEH',.)) %>% 
  rename_with(.,~gsub("24h","_24h",.)) %>% 
  rename_with(.,~gsub("3h","_3h",.)) %>% 
  as.matrix() %>% 
  cpm(., log = TRUE) 

annotation_mat <- data.frame(timeset=colnames(filt_raw_counts_noY)) %>% 
  mutate(sample = timeset) %>% 
  mutate(timeset=gsub("Ind1_75","D_",timeset)) %>% 
  mutate(timeset=gsub("Ind2_87","A_",timeset)) %>% 
  mutate(timeset=gsub("Ind3_77","B_",timeset)) %>% 
  mutate(timeset=gsub("Ind6_71","C_",timeset)) %>% 
  mutate(timeset = gsub("24h","_24h",timeset), 
       timeset = gsub("3h","_3h",timeset)) %>%
  separate(timeset, into = c("indv","trt","time"), sep= "_") %>% 
  mutate(trt= case_match(trt, 'DX' ~'DOX', 'E'~'EPI', 'DA'~'DNR', 'M'~'MTX', 'T'~'TRZ', 'V'~'VEH',.default = trt)) %>% 
  mutate(time = factor(time, levels = c("3h", "24h"), labels= c("3 hours","24 hours"))) %>% 
  mutate(trt = factor(trt, levels = c("DOX","EPI", "DNR", "MTX", "TRZ", "VEH"))) 


PCA4_info_filter <- (prcomp(t(filt4_matrix_lcpm), scale. = TRUE))

pca_final_four_anno <- data.frame(annotation_mat, PCA4_info_filter$x)
plotting_var_names <- prop_var_percent(PCA4_info_filter)

pca_final_four_anno %>%
  ggplot(.,aes(x = PC1, y = PC2, col=trt, shape=time, group=indv))+
  geom_point(size= 5)+
  scale_color_manual(values=drug_pal)+
   ggrepel::geom_text_repel(aes(label = indv))+
   ggtitle(expression("PCA of log"[2]*"(cpm) filtered peak set"))+
  theme_bw()+
  guides(col="none", size =4)+
  labs(y = paste0("PC 2 (",round(plotting_var_names[2],2),"%)")
       , x =paste0("PC 1 (",round(plotting_var_names[1],2),"%)"))+
  theme(plot.title=element_text(size= 14,hjust = 0.5),
        axis.title = element_text(size = 12, color = "black"))

Version Author Date
67a1456 reneeisnowhere 2025-08-07

sessionInfo()
R version 4.4.2 (2024-10-31 ucrt)
Platform: x86_64-w64-mingw32/x64
Running under: Windows 11 x64 (build 26100)

Matrix products: default


locale:
[1] LC_COLLATE=English_United States.utf8 
[2] LC_CTYPE=English_United States.utf8   
[3] LC_MONETARY=English_United States.utf8
[4] LC_NUMERIC=C                          
[5] LC_TIME=English_United States.utf8    

time zone: America/Chicago
tzcode source: internal

attached base packages:
[1] grid      stats4    stats     graphics  grDevices utils     datasets 
[8] methods   base     

other attached packages:
 [1] readxl_1.4.5                            
 [2] smplot2_0.2.5                           
 [3] cowplot_1.1.3                           
 [4] ComplexHeatmap_2.22.0                   
 [5] ggrepel_0.9.6                           
 [6] plyranges_1.26.0                        
 [7] ggsignif_0.6.4                          
 [8] genomation_1.38.0                       
 [9] edgeR_4.4.2                             
[10] limma_3.62.2                            
[11] ggpubr_0.6.1                            
[12] BiocParallel_1.40.2                     
[13] ggVennDiagram_1.5.4                     
[14] scales_1.4.0                            
[15] VennDiagram_1.7.3                       
[16] futile.logger_1.4.3                     
[17] gridExtra_2.3                           
[18] ggfortify_0.4.18                        
[19] rtracklayer_1.66.0                      
[20] org.Hs.eg.db_3.20.0                     
[21] TxDb.Hsapiens.UCSC.hg38.knownGene_3.20.0
[22] GenomicFeatures_1.58.0                  
[23] AnnotationDbi_1.68.0                    
[24] Biobase_2.66.0                          
[25] GenomicRanges_1.58.0                    
[26] GenomeInfoDb_1.42.3                     
[27] IRanges_2.40.1                          
[28] S4Vectors_0.44.0                        
[29] BiocGenerics_0.52.0                     
[30] RColorBrewer_1.1-3                      
[31] broom_1.0.8                             
[32] kableExtra_1.4.0                        
[33] lubridate_1.9.4                         
[34] forcats_1.0.0                           
[35] stringr_1.5.1                           
[36] dplyr_1.1.4                             
[37] purrr_1.0.4                             
[38] readr_2.1.5                             
[39] tidyr_1.3.1                             
[40] tibble_3.3.0                            
[41] ggplot2_3.5.2                           
[42] tidyverse_2.0.0                         
[43] workflowr_1.7.1                         

loaded via a namespace (and not attached):
  [1] later_1.4.2                 BiocIO_1.16.0              
  [3] bitops_1.0-9                cellranger_1.1.0           
  [5] rpart_4.1.24                XML_3.99-0.18              
  [7] lifecycle_1.0.4             rstatix_0.7.2              
  [9] doParallel_1.0.17           rprojroot_2.0.4            
 [11] vroom_1.6.5                 processx_3.8.6             
 [13] lattice_0.22-7              backports_1.5.0            
 [15] magrittr_2.0.3              Hmisc_5.2-3                
 [17] sass_0.4.10                 rmarkdown_2.29             
 [19] jquerylib_0.1.4             yaml_2.3.10                
 [21] plotrix_3.8-4               httpuv_1.6.16              
 [23] DBI_1.2.3                   abind_1.4-8                
 [25] zlibbioc_1.52.0             RCurl_1.98-1.17            
 [27] nnet_7.3-20                 git2r_0.36.2               
 [29] circlize_0.4.16             GenomeInfoDbData_1.2.13    
 [31] svglite_2.2.1               codetools_0.2-20           
 [33] DelayedArray_0.32.0         xml2_1.3.8                 
 [35] tidyselect_1.2.1            shape_1.4.6.1              
 [37] UCSC.utils_1.2.0            farver_2.1.2               
 [39] base64enc_0.1-3             matrixStats_1.5.0          
 [41] GenomicAlignments_1.42.0    jsonlite_2.0.0             
 [43] GetoptLong_1.0.5            Formula_1.2-5              
 [45] iterators_1.0.14            systemfonts_1.2.3          
 [47] foreach_1.5.2               tools_4.4.2                
 [49] Rcpp_1.1.0                  glue_1.8.0                 
 [51] SparseArray_1.6.2           xfun_0.52                  
 [53] MatrixGenerics_1.18.1       withr_3.0.2                
 [55] formatR_1.14                fastmap_1.2.0              
 [57] callr_3.7.6                 digest_0.6.37              
 [59] timechange_0.3.0            R6_2.6.1                   
 [61] seqPattern_1.38.0           textshaping_1.0.1          
 [63] colorspace_2.1-1            dichromat_2.0-0.1          
 [65] RSQLite_2.4.1               generics_0.1.4             
 [67] data.table_1.17.6           htmlwidgets_1.6.4          
 [69] httr_1.4.7                  S4Arrays_1.6.0             
 [71] whisker_0.4.1               pkgconfig_2.0.3            
 [73] gtable_0.3.6                blob_1.2.4                 
 [75] impute_1.80.0               XVector_0.46.0             
 [77] htmltools_0.5.8.1           carData_3.0-5              
 [79] pwr_1.3-0                   clue_0.3-66                
 [81] png_0.1-8                   knitr_1.50                 
 [83] lambda.r_1.2.4              rstudioapi_0.17.1          
 [85] tzdb_0.5.0                  reshape2_1.4.4             
 [87] rjson_0.2.23                checkmate_2.3.2            
 [89] curl_6.4.0                  zoo_1.8-14                 
 [91] cachem_1.1.0                GlobalOptions_0.1.2        
 [93] KernSmooth_2.23-26          parallel_4.4.2             
 [95] foreign_0.8-90              restfulr_0.0.16            
 [97] pillar_1.11.0               vctrs_0.6.5                
 [99] promises_1.3.3              car_3.1-3                  
[101] cluster_2.1.8.1             htmlTable_2.4.3            
[103] evaluate_1.0.4              cli_3.6.5                  
[105] locfit_1.5-9.12             compiler_4.4.2             
[107] futile.options_1.0.1        Rsamtools_2.22.0           
[109] rlang_1.1.6                 crayon_1.5.3               
[111] labeling_0.4.3              ps_1.9.1                   
[113] getPass_0.2-4               plyr_1.8.9                 
[115] fs_1.6.6                    stringi_1.8.7              
[117] viridisLite_0.4.2           gridBase_0.4-7             
[119] Biostrings_2.74.1           Matrix_1.7-3               
[121] BSgenome_1.74.0             patchwork_1.3.1            
[123] hms_1.1.3                   bit64_4.6.0-1              
[125] KEGGREST_1.46.0             statmod_1.5.0              
[127] SummarizedExperiment_1.36.0 memoise_2.0.1              
[129] bslib_0.9.0                 bit_4.6.0