Inital RNA-seq analysis, Pairwise
ERM
20230414
Last updated: 2023-09-25
Checks: 7 0
Knit directory: Cardiotoxicity/
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This starts the documentation of the RNA-seq cardiotoxicity analysis for my cardiotoxicity data.
library(edgeR)#
library(limma)#
library(RColorBrewer)
library(gridExtra)#
library(reshape2)#
library(data.table)
library(tidyverse)
library(scales)
library(biomaRt)#
library(cowplot)#
library(ggrepel)#
library(corrplot)
library(Hmisc)
library(ggpubr)pca_plot <-
function(df,
col_var = NULL,
shape_var = NULL,
title = "") {
ggplot(df) + geom_point(aes_string(
x = "PC1",
y = "PC2",
color = col_var,
shape = shape_var
),
size = 5) +
labs(title = title, x = "PC 1", y = "PC2") +
scale_color_manual(values = c(
"#8B006D",
"#DF707E",
"#F1B72B",
"#3386DD",
"#707031",
"#41B333"
))
}
pca_var_plot <- function(pca) {
# x: class == prcomp
pca.var <- pca$sdev ^ 2
pca.prop <- pca.var / sum(pca.var)
var.plot <-
qplot(PC, prop, data = data.frame(PC = 1:length(pca.prop),
prop = pca.prop)) +
labs(title = 'Variance contributed by each PC',
x = 'PC', y = 'Proportion of variance')
}
calc_pca <- function(x) {
# Performs principal components analysis with prcomp
# x: a sample-by-gene numeric matrix
prcomp(x, scale. = TRUE, retx = TRUE)
}
get_regr_pval <- function(mod) {
# Returns the p-value for the Fstatistic of a linear model
# mod: class lm
stopifnot(class(mod) == "lm")
fstat <- summary(mod)$fstatistic
pval <- 1 - pf(fstat[1], fstat[2], fstat[3])
return(pval)
}
plot_versus_pc <- function(df, pc_num, fac) {
# df: data.frame
# pc_num: numeric, specific PC for plotting
# fac: column name of df for plotting against PC
pc_char <- paste0("PC", pc_num)
# Calculate F-statistic p-value for linear model
pval <- get_regr_pval(lm(df[, pc_char] ~ df[, fac]))
if (is.numeric(df[, f])) {
ggplot(df, aes_string(x = f, y = pc_char)) + geom_point() +
geom_smooth(method = "lm") + labs(title = sprintf("p-val: %.2f", pval))
} else {
ggplot(df, aes_string(x = f, y = pc_char)) + geom_boxplot() +
labs(title = sprintf("p-val: %.2f", pval))
}
}
x_axis_labels = function(labels, every_nth = 1, ...) {
axis(side = 1,
at = seq_along(labels),
labels = F)
text(
x = (seq_along(labels))[seq_len(every_nth) == 1],
y = par("usr")[3] - 0.075 * (par("usr")[4] - par("usr")[3]),
labels = labels[seq_len(every_nth) == 1],
xpd = TRUE,
...
)
}Initial RNA-seq quality checks
drug_palc <- c("#8B006D","#DF707E","#F1B72B", "#3386DD","#707031","#41B333")
seq_info %>%
filter(type=="Total_reads") %>%
ggplot(., aes (x =samplenames, y=count, fill = drug, group_by=indv))+
geom_col()+
geom_hline(aes(yintercept=20000000))+
scale_fill_manual(values=drug_palc)+
ggtitle(expression("Total number of reads by sample"))+
xlab("")+
ylab(expression("RNA -sequencing reads"))+
theme_bw()+
theme(plot.title = element_text(size = rel(2), hjust = 0.5),
axis.title = element_text(size = 15, color = "black"),
axis.ticks = element_line(linewidth = 1.5),
axis.line = element_line(linewidth = 1.5),
axis.text.y = element_text(size =10, color = "black", angle = 0, hjust = 0.8, vjust = 0.5),
axis.text.x = element_text(size =10, color = "black", angle = 90, hjust = 1, vjust = 0.2),
#strip.text.x = element_text(size = 15, color = "black", face = "bold"),
strip.text.y = element_text(color = "white"))
seq_info %>%
filter(type=="Total_reads") %>%
ggplot(., aes (x =drug, y=count, fill = drug))+
geom_boxplot()+
scale_fill_manual(values=drug_palc)+
ggtitle(expression("Total number of reads by treatment"))+
xlab("")+
ylab(expression("RNA -sequencing reads"))+
theme_bw()+
theme(plot.title = element_text(size = rel(2), hjust = 0.5),
axis.title = element_text(size = 15, color = "black"),
axis.ticks = element_line(linewidth = 1.5),
axis.line = element_line(linewidth = 1.5),
axis.text.y = element_text(size =10, color = "black", angle = 0, hjust = 0.8, vjust = 0.5),
axis.text.x = element_text(size =10, color = "black", angle = 90, hjust = 1, vjust = 0.2),
#strip.text.x = element_text(size = 15, color = "black", face = "bold"),
strip.text.y = element_text(color = "white"))
seq_info %>%
filter(type=="Total_reads") %>%
ggplot(., aes (x =as.factor(indv), y=count))+
geom_boxplot(aes(fill=as.factor(indv)))+
scale_fill_brewer(palette = "Dark2", name = "Individual")+
ggtitle(expression("Total number of reads by individual"))+
xlab("")+
ylab(expression("RNA -sequencing reads"))+
theme_bw()+
theme(plot.title = element_text(size = rel(2), hjust = 0.5),
axis.title = element_text(size = 15, color = "black"),
axis.ticks = element_line(linewidth = 1.5),
axis.line = element_line(linewidth = 1.5),
axis.text.y = element_text(size =10, color = "black", angle = 0, hjust = 0.8, vjust = 0.5),
axis.text.x = element_text(size =10, color = "black", angle = 0, hjust = 1),
#strip.text.x = element_text(size = 15, color = "black", face = "bold"),
strip.text.y = element_text(color = "white"))
seq_info %>%
filter(type=="Mapped_reads") %>%
ggplot(., aes (x =samplenames, y=count, fill = drug, group_by=indv))+
geom_col()+
geom_hline(aes(yintercept=20000000))+
scale_fill_manual(values=drug_palc)+
ggtitle(expression("Total number of mapped reads by sample"))+
xlab("")+
ylab(expression("RNA -sequencing reads"))+
theme_bw()+
theme(plot.title = element_text(size = rel(2), hjust = 0.5),
axis.title = element_text(size = 15, color = "black"),
axis.ticks = element_line(linewidth = 1.5),
axis.line = element_line(linewidth = 1.5),
axis.text.y = element_text(size =10, color = "black", angle = 0, hjust = 0.8, vjust = 0.5),
axis.text.x = element_text(size =10, color = "black", angle = 90, hjust = 1, vjust = 0.2),
#strip.text.x = element_text(size = 15, color = "black", face = "bold"),
strip.text.y = element_text(color = "white"))
seq_info %>%
filter(type=="Mapped_reads") %>%
ggplot(., aes (x =as.factor(indv), y=count))+
geom_boxplot(aes(fill=as.factor(indv)))+
scale_fill_brewer(palette = "Dark2", name = "Individual")+
ggtitle(expression("Total mapped reads by individual"))+
xlab("")+
ylab(expression("Number of reads"))+
theme_bw()+
theme(plot.title = element_text(size = rel(2), hjust = 0.5),
axis.title = element_text(size = 15, color = "black"),
axis.ticks = element_line(linewidth = 1.5),
axis.line = element_line(linewidth = 1.5),
axis.text.y = element_text(size =10, color = "black", angle = 0, hjust = 0.8, vjust = 0.5),
axis.text.x = element_text(size =10, color = "black", angle = 0, hjust = 0.5),
#strip.text.x = element_text(size = 15, color = "black", face = "bold"),
strip.text.y = element_text(color = "white"))
seq_info %>%
filter(type=="Mapped_reads") %>%
ggplot(., aes (x =drug, y=count))+
geom_boxplot(aes(fill=drug))+
scale_fill_manual(values=drug_palc)+
ggtitle(expression("Total mapped reads by treatment"))+
xlab("")+
ylab(expression("Number of reads"))+
theme_bw()+
theme(plot.title = element_text(size = rel(2), hjust = 0.5),
axis.title = element_text(size = 15, color = "black"),
axis.ticks = element_line(linewidth = 1.5),
axis.line = element_line(linewidth = 1.5),
axis.text.y = element_text(size =10, color = "black", angle = 0, hjust = 0.8, vjust = 0.5),
axis.text.x = element_text(size =10, color = "black", angle = 0, hjust = 0.5),
#strip.text.x = element_text(size = 15, color = "black", face = "bold"),
strip.text.y = element_text(color = "white"))
Initial histograms from count matrix
cpm <- cpm(mymatrix)
lcpm <- cpm(mymatrix, log=TRUE) ### for determining the basic cutoffs
dim(cpm)[1] 28395 72row_means <- rowMeans(lcpm)
x <- mymatrix[row_means > 0,]
dim(x)[1] 14084 72filcpm_counts <- cpm(x$counts, log = TRUE)
label <- (interaction(drug, indv, time))
colnames(filcpm_counts) <- label
hist(lcpm, main = "Histogram of total counts (unfiltered)",
xlab =expression("Log"[2]*" counts-per-million"), col =4 )
hist(cpm(x$counts, log = TRUE), main = "Histogram of filtered counts using rowMeans > 0 method",
xlab =expression("Log"[2]*" counts-per-million"), col =2 ) 
boxplot(lcpm, main = "Boxplots of log cpm per sample",xaxt = "n", xlab= "")
x_axis_labels(labels = label, every_nth = 1, adj=0.7, srt =90, cex =0.4)
boxplot(filcpm_counts, main ="boxplots of log cpm per sample filtered",xaxt = "n", xlab="")
x_axis_labels(labels = label, every_nth = 1, adj=0.7, srt =90, cex =0.4)
PCA by treatment and as a whole
### VEH 
### DNR 
### DOX 
### EPI 
### MTX 
### TRZ 
samplenames indv drug time RIN group PC1 PC2 PC3
DNR.1.3h MCW_RM_R_11 1 DNR 3h 9.3 1 -18.33154 61.71013 44.039139
DOX.1.3h MCW_RM_R_12 1 DOX 3h 9.8 2 -12.36280 73.97678 24.576395
EPI.1.3h MCW_RM_R_13 1 EPI 3h 9.8 3 -11.16205 66.48794 33.025628
MTX.1.3h MCW_RM_R_14 1 MTX 3h 10 4 -10.19948 73.48343 19.016766
TRZ.1.3h MCW_RM_R_15 1 TRZ 3h 9.6 5 -12.17619 80.01454 2.640624
VEH.1.3h MCW_RM_R_16 1 VEH 3h 9.9 6 -14.98226 76.62199 12.706808
PC4 PC5 PC6
DNR.1.3h -4.547031 24.642107 -35.03245
DOX.1.3h -8.626528 -19.908580 -18.97447
EPI.1.3h -9.349549 18.083569 -43.06551
MTX.1.3h -14.639651 -9.065324 -24.29908
TRZ.1.3h -17.019296 -34.253925 -11.77881
VEH.1.3h -4.173412 -39.846595 -17.16213
| Version | Author | Date |
|---|---|---|
| b936755 | reneeisnowhere | 2023-09-25 |
| aeeade4 | reneeisnowhere | 2023-09-25 |
| 8daa38a | reneeisnowhere | 2023-05-26 |
| bdbf1c2 | reneeisnowhere | 2023-04-21 |
| 1f4237c | reneeisnowhere | 2023-04-20 |
| 5ef62c9 | reneeisnowhere | 2023-04-17 |
| 7f62d67 | reneeisnowhere | 2023-04-17 |
| 21fd945 | reneeisnowhere | 2023-04-17 |
| 8221ec3 | reneeisnowhere | 2023-04-16 |
| 8d08bd2 | reneeisnowhere | 2023-04-11 |
| 4cd8ac4 | reneeisnowhere | 2023-04-11 |
| 08936e7 | reneeisnowhere | 2023-04-10 |
| 85526c5 | reneeisnowhere | 2023-04-10 |
| b266b76 | reneeisnowhere | 2023-04-10 |
| f0a75e1 | reneeisnowhere | 2023-04-10 |
| Version | Author | Date |
|---|---|---|
| b936755 | reneeisnowhere | 2023-09-25 |
| aeeade4 | reneeisnowhere | 2023-09-25 |
| 8daa38a | reneeisnowhere | 2023-05-26 |
| bdbf1c2 | reneeisnowhere | 2023-04-21 |
| 1f4237c | reneeisnowhere | 2023-04-20 |
| 5ef62c9 | reneeisnowhere | 2023-04-17 |
| 7f62d67 | reneeisnowhere | 2023-04-17 |
| 21fd945 | reneeisnowhere | 2023-04-17 |
| 8221ec3 | reneeisnowhere | 2023-04-16 |
| 8d08bd2 | reneeisnowhere | 2023-04-11 |
| 4cd8ac4 | reneeisnowhere | 2023-04-11 |
| 08936e7 | reneeisnowhere | 2023-04-10 |
| 85526c5 | reneeisnowhere | 2023-04-10 |
| b266b76 | reneeisnowhere | 2023-04-10 |
| f0a75e1 | reneeisnowhere | 2023-04-10 |
PC1 PC2 PC3 PC4 PC5 PC6
Standard deviation 63.98853 47.11608 34.21502 32.58775 28.22245 23.90977
Proportion of Variance 0.29072 0.15762 0.08312 0.07540 0.05655 0.04059
Cumulative Proportion 0.29072 0.44834 0.53146 0.60687 0.66342 0.70401
PC7
Standard deviation 21.56133
Proportion of Variance 0.03301
Cumulative Proportion 0.73702Typical genes expressed in iPSC-CMS
genecardiccheck <- c("MYH7", "TNNT2","MYH6","ACTN2","BMP3","TNNI3","RYR2","CACNA1C","KCNQ1", "HCN1", "ADRB1", "ADRB2")
#ensembl <- useMart("ensembl", dataset="hsapiens_gene_ensembl")
#saveRDS(ensembl, "data/ensembl_backup.RDS")
#ensemble <- readRDS("data/ensembl_backup.RDS")
#my_chr <- c(1:22, 'M', 'X', 'Y') ## creates a filter for each database
#my_attributes <- c('entrezgene_id', 'ensembl_gene_id', 'hgnc_symbol')
#heartgenes <- getBM(attributes=my_attributes,filters ='hgnc_symbol',
# values = genecardiccheck, mart = ensembl)
#write.csv(heartgenes, "data/heartgenes.csv")
heartgenes <-read.csv("data/heartgenes.csv")
fungraph <- as.data.frame(filcpm_counts[rownames(filcpm_counts) %in% heartgenes$entrezgene_id,])
fungraph %>%
rownames_to_column("entrezgene_id") %>%
pivot_longer(-entrezgene_id, names_to = "samples",values_to = "counts") %>%
mutate(gene = case_match(entrezgene_id,"88"~"ACTN2","153"~"ADRB1",
"154"~"ADRB2","651"~"BMP3","775"~"CACNA1C", "100874369"~"CACNA1C","348980"~"HCN1",
"3784"~"KCNQ1", "4624"~"MYH6","4625"~"MYH7","6262"~"RYR2",
"7137"~"TNNI3","7139"~"TNNT2",.default = entrezgene_id)) %>%
ggplot(., aes(x=reorder(gene,counts,decreasing=TRUE), y=counts))+
geom_boxplot()+
ggtitle(expression("Expression of typical cardiac tissue genes"))+
xlab("")+
ylab(expression("log"[2]~"cpm"))+
theme_bw()+
theme(plot.title = element_text(size = rel(2), hjust = 0.5),
axis.title = element_text(size = 15, color = "black"),
axis.ticks = element_line(linewidth = 1.5),
axis.line = element_line(linewidth = 1.5),
axis.text = element_text(size =10, color = "black", angle = 0),
strip.text.y = element_text(color = "white"))
correlation heatmap of counts matrix
colnames(filcpm_counts) <- label
# saveRDS(filcpm_counts,"data/filcpm_counts.RDS")
mcort <- cor(filcpm_counts)
pheatmap::pheatmap(mcort , cluster_rows = TRUE, cluster_cols = TRUE)
now to get the counts set for DEG!!
group1 <- interaction(drug,time)
mm <- model.matrix(~0 + group1)
colnames(mm) <- c("A3", "X3", "E3","M3","T3", "V3","A24", "X24", "E24","M24","T24", "V24")
y <- voom(x, mm,plot =TRUE)
corfit <- duplicateCorrelation(y, mm, block = indv)
v <- voom(x, mm, block = indv, correlation = corfit$consensus)
fit <- lmFit(v, mm, block = indv, correlation = corfit$consensus)
cm <- makeContrasts(
V.DA = V3 - A3,
V.DX = V3 - X3,
V.EP = V3 - E3,
V.MT = V3 - M3,
V.TR = V3 - T3,
V.DA24 = V24-A24,
V.DX24= V24-X24,
V.EP24= V24-E24,
V.MT24= V24-M24,
V.TR24= V24-T24,
levels = mm)
vfit <- lmFit(y, mm)
vfit<- contrasts.fit(vfit, contrasts=cm)
efit2 <- eBayes(vfit)
# saveRDS(efit2,"data/efit2_final.RDS")DEG analysis
summary
efit2 <- readRDS("data/efit2_final.RDS")
sum <- summary(decideTests(efit2))
sum 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 0Volcano plots from pairwise gene analysis
library(cowplot)
siglist_final <- readRDS("data/siglist_final.RDS")
list2env(siglist_final,envir = .GlobalEnv)<environment: R_GlobalEnv>volcanosig <- function(df, psig.lvl,topg) {
df <- df %>%
mutate(threshold = ifelse(adj.P.Val > psig.lvl, "A", ifelse(adj.P.Val <= psig.lvl & logFC<=0,"B","C")))
ggplot(df, aes(x=logFC, y=-log10(adj.P.Val))) +
geom_point(aes(color=threshold))+
xlab(expression("Log"[2]*" FC"))+
ylim(0,30)+
ylab(expression("-log"[10]*"P Value"))+
scale_color_manual(values = c("black", "red","blue"))+
theme_cowplot()+
theme(legend.position = "none",
plot.title = element_text(size = rel(0.8), hjust = 0.5),
axis.title = element_text(size = rel(0.8)))
}
v1 <- volcanosig(V.DA.top, 0.01,0)+ ggtitle("Daunorubicin\n 3 hour")
v2 <- volcanosig(V.DA24.top, 0.01,0)+ ggtitle("Daunorubicin\n 24 hour")
v3 <- volcanosig(V.DX.top, 0.01,0)+ ggtitle("Doxorubicin\n 3 hour")+ylab("")
v4 <- volcanosig(V.DX24.top, 0.01,0)+ ggtitle("Doxorubicin\n 24 hour")+ylab("")
v5 <- volcanosig(V.EP.top, 0.01,0)+ ggtitle("Epirubicin\n 3 hour")+ylab("")
v6 <- volcanosig(V.EP24.top, 0.01,0)+ ggtitle("Epirubicin\n 24 hour")+ylab("")
v7 <- volcanosig(V.MT.top, 0.01,0)+ ggtitle("Mitoxatrone\n 3 hour")+ylab("")
v8 <- volcanosig(V.MT24.top, 0.01,0)+ ggtitle("Mitoxatrone\n 24 hour")+ylab("")
v9 <- volcanosig(V.TR.top, 0.01,0)+ ggtitle("Trastuzumab\n 3 hour")+ylab("")
v10 <- volcanosig(V.TR24.top, 0.01,0)+ ggtitle("Trastuzumab\n 24 hour")+ylab("")
Volcanoplots <- plot_grid(v1,v3,v5,v7,v9,v2,v4,v6,v8,v10, nrow = 2, ncol = 5)
Volcanoplots
# saveRDS(Volcanoplots,"output/Volcanoplot_10.RDS")
sessionInfo()R version 4.3.1 (2023-06-16 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19045)
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] stats graphics grDevices utils datasets methods base
other attached packages:
[1] ggpubr_0.6.0 Hmisc_5.1-1 corrplot_0.92 ggrepel_0.9.3
[5] cowplot_1.1.1 biomaRt_2.56.1 scales_1.2.1 lubridate_1.9.2
[9] forcats_1.0.0 stringr_1.5.0 dplyr_1.1.3 purrr_1.0.2
[13] readr_2.1.4 tidyr_1.3.0 tibble_3.2.1 ggplot2_3.4.3
[17] tidyverse_2.0.0 data.table_1.14.8 reshape2_1.4.4 gridExtra_2.3
[21] RColorBrewer_1.1-3 edgeR_3.42.4 limma_3.56.2 workflowr_1.7.1
loaded via a namespace (and not attached):
[1] DBI_1.1.3 bitops_1.0-7 rlang_1.1.1
[4] magrittr_2.0.3 git2r_0.32.0 compiler_4.3.1
[7] RSQLite_2.3.1 getPass_0.2-2 png_0.1-8
[10] callr_3.7.3 vctrs_0.6.3 pkgconfig_2.0.3
[13] crayon_1.5.2 fastmap_1.1.1 backports_1.4.1
[16] dbplyr_2.3.3 XVector_0.40.0 labeling_0.4.3
[19] utf8_1.2.3 promises_1.2.1 rmarkdown_2.24
[22] tzdb_0.4.0 ps_1.7.5 bit_4.0.5
[25] xfun_0.40 zlibbioc_1.46.0 cachem_1.0.8
[28] GenomeInfoDb_1.36.1 jsonlite_1.8.7 progress_1.2.2
[31] blob_1.2.4 later_1.3.1 broom_1.0.5
[34] cluster_2.1.4 prettyunits_1.1.1 R6_2.5.1
[37] bslib_0.5.1 stringi_1.7.12 car_3.1-2
[40] rpart_4.1.19 jquerylib_0.1.4 Rcpp_1.0.11
[43] knitr_1.44 base64enc_0.1-3 IRanges_2.34.1
[46] nnet_7.3-19 httpuv_1.6.11 timechange_0.2.0
[49] tidyselect_1.2.0 abind_1.4-5 rstudioapi_0.15.0
[52] yaml_2.3.7 curl_5.0.2 processx_3.8.2
[55] lattice_0.21-8 plyr_1.8.8 Biobase_2.60.0
[58] withr_2.5.0 KEGGREST_1.40.0 evaluate_0.21
[61] foreign_0.8-85 BiocFileCache_2.8.0 xml2_1.3.5
[64] Biostrings_2.68.1 pillar_1.9.0 filelock_1.0.2
[67] carData_3.0-5 whisker_0.4.1 checkmate_2.2.0
[70] stats4_4.3.1 generics_0.1.3 rprojroot_2.0.3
[73] RCurl_1.98-1.12 S4Vectors_0.38.1 hms_1.1.3
[76] munsell_0.5.0 glue_1.6.2 pheatmap_1.0.12
[79] tools_4.3.1 ggsignif_0.6.4 locfit_1.5-9.8
[82] fs_1.6.3 XML_3.99-0.14 grid_4.3.1
[85] AnnotationDbi_1.62.2 colorspace_2.1-0 GenomeInfoDbData_1.2.10
[88] htmlTable_2.4.1 Formula_1.2-5 cli_3.6.1
[91] rappdirs_0.3.3 fansi_1.0.4 gtable_0.3.4
[94] rstatix_0.7.2 sass_0.4.7 digest_0.6.33
[97] BiocGenerics_0.46.0 farver_2.1.1 htmlwidgets_1.6.2
[100] memoise_2.0.1 htmltools_0.5.6 lifecycle_1.0.3
[103] httr_1.4.7 statmod_1.5.0 bit64_4.0.5