Last updated: 2023-04-16

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Knit directory: Cardiotoxicity/

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This starts the documentation of the RNA-seq cardiotoxicity analysis for my manuscript

library(edgeR)#
library(limma)#
library(RColorBrewer)
library(mixOmics)
library(gridExtra)#
library(reshape2)#

library(AnnotationHub)
library(tidyverse)
library(scales)
library(biomaRt)#
library(Homo.sapiens)
library(cowplot)#
library(ggrepel)#
library(corrplot)
library(Hmisc)
library(ggpubr)
 ###now we add genenames to the geneid###
 
geneid <- rownames(mymatrix) ### pulls the names we have in the counts file
genes <- select(Homo.sapiens, keys=geneid, columns=c("SYMBOL"),
  keytype="ENTREZID")
genes <- genes[!duplicated(genes$ENTREZID),]
mymatrix$genes <- genes

Initial RNA-seq quality checks

Initial histograms from count matrix

[1] 28395    72
[1] 14084    72

PCA by treatment and as a whole



###  Vehicle 



###  Daunorubicin 



###  Doxorubicin 



###  Epirubicin 



###  Mitoxantrone 



###  Trastuzumab 

        samplenames indv         drug time RIN group       PC1      PC2
Da.1.3h MCW_RM_R_11    1 Daunorubicin   3h 9.3     1 -18.33154 61.71013
Do.1.3h MCW_RM_R_12    1  Doxorubicin   3h 9.8     2 -12.36280 73.97678
Ep.1.3h MCW_RM_R_13    1   Epirubicin   3h 9.8     3 -11.16205 66.48794
Mi.1.3h MCW_RM_R_14    1 Mitoxantrone   3h  10     4 -10.19948 73.48343
Tr.1.3h MCW_RM_R_15    1  Trastuzumab   3h 9.6     5 -12.17619 80.01454
Ve.1.3h MCW_RM_R_16    1      Vehicle   3h 9.9     6 -14.98226 76.62199
              PC3        PC4        PC5       PC6
Da.1.3h 44.039139  -4.547031  24.642107 -35.03245
Do.1.3h 24.576395  -8.626528 -19.908580 -18.97447
Ep.1.3h 33.025628  -9.349549  18.083569 -43.06551
Mi.1.3h 19.016766 -14.639651  -9.065324 -24.29908
Tr.1.3h  2.640624 -17.019296 -34.253925 -11.77881
Ve.1.3h 12.706808  -4.173412 -39.846595 -17.16213

Importance of components:
                           PC1     PC2      PC3     PC4      PC5      PC6
Standard deviation     63.9885 47.1161 34.21502 32.5878 28.22245 23.90977
Proportion of Variance  0.2907  0.1576  0.08312  0.0754  0.05655  0.04059
Cumulative Proportion   0.2907  0.4483  0.53146  0.6069  0.66342  0.70401
                            PC7      PC8      PC9     PC10     PC11     PC12
Standard deviation     21.56133 20.74271 19.52290 17.47711 16.26239 15.49112
Proportion of Variance  0.03301  0.03055  0.02706  0.02169  0.01878  0.01704
Cumulative Proportion   0.73702  0.76757  0.79463  0.81632  0.83510  0.85213
                           PC13     PC14     PC15     PC16     PC17    PC18
Standard deviation     14.03690 13.01039 11.78935 11.40888 10.15831 9.76192
Proportion of Variance  0.01399  0.01202  0.00987  0.00924  0.00733 0.00677
Cumulative Proportion   0.86612  0.87814  0.88801  0.89725  0.90458 0.91135
                          PC19    PC20    PC21   PC22    PC23    PC24   PC25
Standard deviation     8.77010 8.66331 8.08961 7.8745 7.45127 7.07877 6.6073
Proportion of Variance 0.00546 0.00533 0.00465 0.0044 0.00394 0.00356 0.0031
Cumulative Proportion  0.91681 0.92214 0.92678 0.9312 0.93513 0.93869 0.9418
                          PC26    PC27    PC28    PC29    PC30    PC31    PC32
Standard deviation     6.30345 6.04266 5.85249 5.72714 5.52443 5.49059 5.26497
Proportion of Variance 0.00282 0.00259 0.00243 0.00233 0.00217 0.00214 0.00197
Cumulative Proportion  0.94461 0.94720 0.94963 0.95196 0.95413 0.95627 0.95824
                          PC33    PC34    PC35    PC36    PC37    PC38    PC39
Standard deviation     5.18567 5.07323 4.83532 4.80110 4.65658 4.52528 4.46361
Proportion of Variance 0.00191 0.00183 0.00166 0.00164 0.00154 0.00145 0.00141
Cumulative Proportion  0.96014 0.96197 0.96363 0.96527 0.96681 0.96826 0.96968
                          PC40    PC41    PC42    PC43    PC44    PC45    PC46
Standard deviation     4.39495 4.30843 4.22660 4.20932 4.12976 4.07652 4.04834
Proportion of Variance 0.00137 0.00132 0.00127 0.00126 0.00121 0.00118 0.00116
Cumulative Proportion  0.97105 0.97237 0.97363 0.97489 0.97610 0.97728 0.97845
                          PC47    PC48    PC49    PC50    PC51    PC52   PC53
Standard deviation     3.99248 3.89519 3.86841 3.81632 3.80862 3.78710 3.7466
Proportion of Variance 0.00113 0.00108 0.00106 0.00103 0.00103 0.00102 0.0010
Cumulative Proportion  0.97958 0.98066 0.98172 0.98275 0.98378 0.98480 0.9858
                          PC54    PC55    PC56    PC57   PC58    PC59    PC60
Standard deviation     3.70669 3.65110 3.64619 3.59910 3.5568 3.50638 3.46507
Proportion of Variance 0.00098 0.00095 0.00094 0.00092 0.0009 0.00087 0.00085
Cumulative Proportion  0.98677 0.98772 0.98866 0.98958 0.9905 0.99136 0.99221
                          PC61    PC62    PC63    PC64    PC65   PC66    PC67
Standard deviation     3.44803 3.40615 3.34532 3.27967 3.22481 3.1318 3.11900
Proportion of Variance 0.00084 0.00082 0.00079 0.00076 0.00074 0.0007 0.00069
Cumulative Proportion  0.99305 0.99388 0.99467 0.99543 0.99617 0.9969 0.99756
                          PC68    PC69    PC70   PC71     PC72
Standard deviation     3.10497 3.05639 2.88429 2.6597 9.29e-14
Proportion of Variance 0.00068 0.00066 0.00059 0.0005 0.00e+00
Cumulative Proportion  0.99824 0.99891 0.99950 1.0000 1.00e+00

Typical genes expressed in iPSC-CMS

correlation heatmap of counts matrix

now to get the counts set for DEG!!

DEG analysis

summary

        V.DA  V.DX  V.EP  V.MT  V.TR V.DA24 V.DX24 V.EP24 V.MT24 V.TR24
Down     109     3    30    24     0   3540   3336   3105    428      0
NotSig 13552 14065 13874 14009 14084   7067   7439   7756  12969  14084
Up       423    16   180    51     0   3477   3309   3223    687      0

cormotif analysis

[1] 14084    10

Response sets look similar to previous results. This data is based on the filtered count matrix (using rowmeans>0 of cpm(log=true)). Classification of patterns appear to be:

motif 1- No Response set: 7490 (gene list made by filtering each column by posterior probability of <0.5) motif 2- Time-independent Top2Bi response cluster: 116

motif 3- Early Top2Bi response cluster: 424

motif 4- All BC drug response set (only 1!) (I could not really isolate this gene from the p.post data)

motif 5- Late Top2Bi response cluster:1615 **these are based on the most recent plots (motif numbers have changed a little)


R version 4.2.2 (2022-10-31 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19044)

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    

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

other attached packages:
 [1] Cormotif_1.42.0                        
 [2] affy_1.74.0                            
 [3] ggpubr_0.6.0                           
 [4] Hmisc_4.8-0                            
 [5] Formula_1.2-5                          
 [6] survival_3.5-3                         
 [7] corrplot_0.92                          
 [8] ggrepel_0.9.3                          
 [9] cowplot_1.1.1                          
[10] Homo.sapiens_1.3.1                     
[11] TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2
[12] org.Hs.eg.db_3.15.0                    
[13] GO.db_3.15.0                           
[14] OrganismDbi_1.38.1                     
[15] GenomicFeatures_1.48.4                 
[16] GenomicRanges_1.48.0                   
[17] GenomeInfoDb_1.32.4                    
[18] AnnotationDbi_1.58.0                   
[19] IRanges_2.30.1                         
[20] S4Vectors_0.34.0                       
[21] Biobase_2.56.0                         
[22] biomaRt_2.52.0                         
[23] scales_1.2.1                           
[24] lubridate_1.9.2                        
[25] forcats_1.0.0                          
[26] stringr_1.5.0                          
[27] dplyr_1.1.0                            
[28] purrr_1.0.1                            
[29] readr_2.1.4                            
[30] tidyr_1.3.0                            
[31] tibble_3.1.8                           
[32] tidyverse_2.0.0                        
[33] AnnotationHub_3.4.0                    
[34] BiocFileCache_2.4.0                    
[35] dbplyr_2.3.1                           
[36] BiocGenerics_0.42.0                    
[37] reshape2_1.4.4                         
[38] gridExtra_2.3                          
[39] mixOmics_6.20.0                        
[40] ggplot2_3.4.1                          
[41] lattice_0.20-45                        
[42] MASS_7.3-58.2                          
[43] RColorBrewer_1.1-3                     
[44] edgeR_3.38.4                           
[45] limma_3.52.4                           
[46] workflowr_1.7.0                        

loaded via a namespace (and not attached):
  [1] utf8_1.2.3                    tidyselect_1.2.0             
  [3] RSQLite_2.3.0                 htmlwidgets_1.6.1            
  [5] grid_4.2.2                    BiocParallel_1.30.4          
  [7] munsell_0.5.0                 preprocessCore_1.58.0        
  [9] codetools_0.2-19              statmod_1.5.0                
 [11] interp_1.1-3                  withr_2.5.0                  
 [13] colorspace_2.1-0              filelock_1.0.2               
 [15] highr_0.10                    knitr_1.42                   
 [17] rstudioapi_0.14               ggsignif_0.6.4               
 [19] MatrixGenerics_1.8.1          labeling_0.4.2               
 [21] git2r_0.31.0                  GenomeInfoDbData_1.2.8       
 [23] pheatmap_1.0.12               farver_2.1.1                 
 [25] bit64_4.0.5                   rprojroot_2.0.3              
 [27] vctrs_0.5.2                   generics_0.1.3               
 [29] xfun_0.37                     timechange_0.2.0             
 [31] R6_2.5.1                      locfit_1.5-9.7               
 [33] bitops_1.0-7                  cachem_1.0.7                 
 [35] DelayedArray_0.22.0           promises_1.2.0.1             
 [37] BiocIO_1.6.0                  nnet_7.3-18                  
 [39] gtable_0.3.1                  processx_3.8.0               
 [41] rlang_1.0.6                   splines_4.2.2                
 [43] rtracklayer_1.56.1            rstatix_0.7.2                
 [45] broom_1.0.3                   checkmate_2.1.0              
 [47] BiocManager_1.30.20           yaml_2.3.7                   
 [49] abind_1.4-5                   backports_1.4.1              
 [51] httpuv_1.6.9                  RBGL_1.72.0                  
 [53] tools_4.2.2                   affyio_1.66.0                
 [55] ellipsis_0.3.2                jquerylib_0.1.4              
 [57] Rcpp_1.0.10                   plyr_1.8.8                   
 [59] base64enc_0.1-3               progress_1.2.2               
 [61] zlibbioc_1.42.0               RCurl_1.98-1.10              
 [63] ps_1.7.2                      prettyunits_1.1.1            
 [65] rpart_4.1.19                  deldir_1.0-6                 
 [67] SummarizedExperiment_1.26.1   cluster_2.1.4                
 [69] fs_1.6.1                      magrittr_2.0.3               
 [71] data.table_1.14.8             RSpectra_0.16-1              
 [73] whisker_0.4.1                 matrixStats_0.63.0           
 [75] hms_1.1.2                     mime_0.12                    
 [77] evaluate_0.20                 xtable_1.8-4                 
 [79] XML_3.99-0.13                 jpeg_0.1-10                  
 [81] compiler_4.2.2                ellipse_0.4.3                
 [83] crayon_1.5.2                  htmltools_0.5.4              
 [85] corpcor_1.6.10                later_1.3.0                  
 [87] tzdb_0.3.0                    DBI_1.1.3                    
 [89] rappdirs_0.3.3                Matrix_1.5-3                 
 [91] car_3.1-1                     cli_3.6.0                    
 [93] parallel_4.2.2                igraph_1.4.1                 
 [95] pkgconfig_2.0.3               getPass_0.2-2                
 [97] GenomicAlignments_1.32.1      foreign_0.8-84               
 [99] xml2_1.3.3                    rARPACK_0.11-0               
[101] bslib_0.4.2                   XVector_0.36.0               
[103] callr_3.7.3                   digest_0.6.31                
[105] graph_1.74.0                  Biostrings_2.64.1            
[107] rmarkdown_2.20                htmlTable_2.4.1              
[109] restfulr_0.0.15               curl_5.0.0                   
[111] shiny_1.7.4                   Rsamtools_2.12.0             
[113] rjson_0.2.21                  lifecycle_1.0.3              
[115] jsonlite_1.8.4                carData_3.0-5                
[117] fansi_1.0.4                   pillar_1.8.1                 
[119] KEGGREST_1.36.3               fastmap_1.1.1                
[121] httr_1.4.5                    interactiveDisplayBase_1.34.0
[123] glue_1.6.2                    png_0.1-8                    
[125] BiocVersion_3.15.2            bit_4.0.5                    
[127] stringi_1.7.12                sass_0.4.5                   
[129] blob_1.2.3                    latticeExtra_0.6-30          
[131] memoise_2.0.1