Bulk RNASeq: additional exploratory plots - CTL04
Date: April 28, 2026
1. Environment Set Up
knitr::opts_chunk$set(echo = TRUE, warning=FALSE)Values of RMarkdown parameters
for (i in 1:length(params))
print(paste('Parameter:', names(params)[i], ' - Value:', params[[i]], '- Class:', class(params[[i]])))
## [1] "Parameter: SEBio - Value: ~/DataDir/bulkRNASeq/3.DataExploration/CTL04/Output/FemaleLineSE_Bio.rds - Class: character"
## [1] "Parameter: OutputFolder - Value: ~/ - Class: character"
## [1] "Parameter: CpmFilt - Value: 2 - Class: integer"
## [1] "Parameter: SampleFilt - Value: 4 - Class: integer"library(RNASeqBulkExploratory) #Our Package
library(DT)
library(gridExtra)
library(SummarizedExperiment)
## Loading required package: MatrixGenerics
## Loading required package: matrixStats
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## Attaching package: 'MatrixGenerics'
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## colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse,
## colCounts, colCummaxs, colCummins, colCumprods, colCumsums,
## colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs,
## colMads, colMaxs, colMeans2, colMedians, colMins, colOrderStats,
## colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds,
## colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads,
## colWeightedMeans, colWeightedMedians, colWeightedSds,
## colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet,
## rowCollapse, rowCounts, rowCummaxs, rowCummins, rowCumprods,
## rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps,
## rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians, rowMins,
## rowOrderStats, rowProds, rowQuantiles, rowRanges, rowRanks,
## rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs, rowVars,
## rowWeightedMads, rowWeightedMeans, rowWeightedMedians,
## rowWeightedSds, rowWeightedVars
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## get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
## match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
## Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
## table, tapply, union, unique, unsplit, which.max, which.min
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## Loading required package: IRanges
## Loading required package: GenomeInfoDb
## Loading required package: Biobase
## Welcome to Bioconductor
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## Vignettes contain introductory material; view with
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## 'citation("Biobase")', and for packages 'citation("pkgname")'.
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library(ggplot2)SE_Bio <- readRDS(params$SEBio)
CpmFilt <- params$CpmFilt
SampleFilt <- params$SampleFilt
OutputFolder <- params$OutputFolder2. Data Upload
SE_Filt <- filterSE(SE_Bio, cpmTh=CpmFilt, sampleTh=SampleFilt)
## UPDATED library.size slot- Re-calculate library size and calculate TMM
- Calculate cpm and logCpm (with normalization) and store in SE_Filt
SE_Filt <- normTmmSE(SE_Filt, useNormFactors=TRUE, priorCount=0.25) 3. Sample-to-sample correlation heatmap
Correlation matrix across samples calculated on the basis of the Spearman correlation.
# Set heatmap size on the basis of the number of samples
Width <- 7 + (dim(SE_Filt)[2]- 8) * 0.24
Width <- ifelse(Width<=14, Width, 14)
Height <- 5 + (dim(SE_Filt)[2]- 8) * 0.22Cols <- c('DMSO' = 'grey30', 'CTL' = 'azure3',
'AhHyd_Ag'='#F8766D', 'AhHyd_Inh'='#F8766D50',
'Andr_Ag'='#fccb17', 'Andr_Inh'='#C49A0050',
"Estr_Ag"= '#53B400', "Estr_Inh"= '#53B40050',
'GC_Ag' = '#00C094', 'GC_Inh' = '#00C09450',
'LivX_Ag' = '#00B6EB', 'LivX_Inh' = '#00B6EB50',
'Ret_Ag' = '#A58AFF', 'Ret_Inh' = '#A58AFF50',
'Thyr_Ag' = '#FB61D7', 'Thyr_Inh' = '#FB61D750'
)sampleCorrHeatmap(
SE_Filt,
plotTitle = "Filtered dataset",
annotation_col = c("Condition"),
annotation_colors = list(
Condition = Cols
),
display_numbers = TRUE
)
## There are many samples. For readability "display_numbers" will be set to FALSE
cond_order <- c("CTL", "DMSO")
other_cond <- setdiff(unique(colData(SE_Filt)$Condition), cond_order)
cond_levels <- c(cond_order, sort(other_cond))
colData(SE_Filt)$Condition <- factor(
colData(SE_Filt)$Condition,
levels = cond_levels
)
ord <- order(colData(SE_Filt)$Condition)
SE_Filt_ord <- SE_Filt[, ord]sampleCorrHeatmap(
SE_Filt_ord,
plotTitle = "Filtered dataset",
annotation_col = c("Condition"),
annotation_colors = list(
Condition = Cols),
display_numbers = TRUE,
cluster_rows = FALSE,
cluster_cols = FALSE,
show_rownames = FALSE,
show_colnames = FALSE
)
## There are many samples. For readability "display_numbers" will be set to FALSE
SessionInfo <- sessionInfo()
Date <- date()SessionInfo## R version 4.2.1 (2022-06-23)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.4 LTS
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## attached base packages:
## [1] stats4 stats graphics grDevices utils datasets methods
## [8] base
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## other attached packages:
## [1] ggplot2_3.4.1 SummarizedExperiment_1.28.0
## [3] Biobase_2.58.0 GenomicRanges_1.50.2
## [5] GenomeInfoDb_1.34.9 IRanges_2.32.0
## [7] S4Vectors_0.36.1 BiocGenerics_0.44.0
## [9] MatrixGenerics_1.10.0 matrixStats_0.63.0
## [11] gridExtra_2.3 DT_0.27
## [13] RNASeqBulkExploratory_0.2.1
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## [4] digest_0.6.31 utf8_1.2.3 R6_2.5.1
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## [10] zlibbioc_1.44.0 rlang_1.1.1 rstudioapi_0.14
## [13] jquerylib_0.1.4 Matrix_1.5-3 rmarkdown_2.20
## [16] textshaping_0.3.6 htmlwidgets_1.6.1 pheatmap_1.0.12
## [19] RCurl_1.98-1.10 munsell_0.5.0 DelayedArray_0.24.0
## [22] compiler_4.2.1 xfun_0.37 pkgconfig_2.0.3
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## [46] bslib_0.4.2 ragg_1.2.3 generics_0.1.3
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## [55] colorspace_2.1-0 knitr_1.42 sass_0.4.5Date## [1] "Tue Apr 28 13:08:37 2026"