Example bulk RNA-seq analysis 4
Mark Ziemann
2024-05-29
Source: https://github.com/markziemann/background
Intro
Here we are performing an analysis of some gene expression data to demonstrate the difference between ORA and FCS methods and to highlight the differences caused by improper background gene set use.
The dataset being used is SRP038101 and we are comparing the cells expressing a set7kd shRNA construct (case) compared to the scrambled construct (control).
Data are obtained from http://dee2.io/
suppressPackageStartupMessages({
library("getDEE2")
library("DESeq2")
library("clusterProfiler")
library("mitch")
library("kableExtra")
library("eulerr")
library("biomaRt")
})Get expression data and make an MDS plot
name = "SRP096177"
mdat <- getDEE2Metadata("hsapiens")
samplesheet <- mdat[grep("SRP096177",mdat$SRP_accession),]
samplesheet <- samplesheet[order(samplesheet$SRR_accession),]
samplesheet$trt <- as.factor(c(1,1,1,0,0,0))
s1 <- samplesheet
s1 %>% kbl(caption = "sample sheet") %>% kable_paper("hover", full_width = F)| SRR_accession | QC_summary | SRX_accession | SRS_accession | SRP_accession | Experiment_title | GEO_series | trt | |
|---|---|---|---|---|---|---|---|---|
| 330280 | SRR5150592 | PASS | SRX2468682 | SRS1901000 | SRP096177 | GSM2448982: Set7KD_rep1; Homo sapiens; RNA-Seq | GSE93236 | 1 |
| 330281 | SRR5150593 | PASS | SRX2468683 | SRS1901005 | SRP096177 | GSM2448983: Set7KD_rep2; Homo sapiens; RNA-Seq | GSE93236 | 1 |
| 330282 | SRR5150594 | PASS | SRX2468684 | SRS1901001 | SRP096177 | GSM2448984: Set7KD_rep3; Homo sapiens; RNA-Seq | GSE93236 | 1 |
| 330283 | SRR5150595 | PASS | SRX2468685 | SRS1901002 | SRP096177 | GSM2448985: NTC_rep1; Homo sapiens; RNA-Seq | GSE93236 | 0 |
| 330284 | SRR5150596 | PASS | SRX2468686 | SRS1901003 | SRP096177 | GSM2448986: NTC_rep2; Homo sapiens; RNA-Seq | GSE93236 | 0 |
| 330285 | SRR5150597 | PASS | SRX2468687 | SRS1901004 | SRP096177 | GSM2448987: NTC_rep3; Homo sapiens; RNA-Seq | GSE93236 | 0 |
w <- getDEE2("hsapiens",samplesheet$SRR_accession,metadata=mdat,legacy = TRUE)## For more information about DEE2 QC metrics, visit
## https://github.com/markziemann/dee2/blob/master/qc/qc_metrics.mdx <- Tx2Gene(w)
x <- x$Tx2Gene
# save the genetable for later
gt <- w$GeneInfo[,1,drop=FALSE]
gt$accession <- rownames(gt)
# counts
x1 <- x[,which(colnames(x) %in% samplesheet$SRR_accession)]Here show the number of genes in the annotation set, and those detected above the detection threshold.
# filter out lowly expressed genes
x1 <- x1[which(rowSums(x1)/ncol(x1)>=(10)),]
nrow(x)## [1] 39297nrow(x1)## [1] 15607Now multidimensional scaling (MDS) plot to show the correlation between the datasets. If the control and case datasets are clustered separately, then it is likely that there will be many differentially expressed genes with FDR<0.05.
plot(cmdscale(dist(t(x1))), xlab="Coordinate 1", ylab="Coordinate 2", pch=19, col=s1$trt, main="MDS")
Differential expression
Now run DESeq2 for control vs case.
y <- DESeqDataSetFromMatrix(countData = round(x1), colData = s1, design = ~ trt)## converting counts to integer modey <- DESeq(y)## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testingde <- results(y)
de <- as.data.frame(de[order(de$pvalue),])
rownames(de) <- sapply(strsplit(rownames(de),"\\."),"[[",1)
head(de) %>% kbl() %>% kable_paper("hover", full_width = F)| baseMean | log2FoldChange | lfcSE | stat | pvalue | padj | |
|---|---|---|---|---|---|---|
| ENSG00000168542 | 1259.1001 | 2.7325696 | 0.0952226 | 28.69666 | 0 | 0 |
| ENSG00000164692 | 11379.8760 | 2.2961488 | 0.0887089 | 25.88407 | 0 | 0 |
| ENSG00000172531 | 2130.9201 | -1.7175060 | 0.0712043 | -24.12082 | 0 | 0 |
| ENSG00000106484 | 4549.7360 | 1.2253274 | 0.0581660 | 21.06603 | 0 | 0 |
| ENSG00000163017 | 300.4833 | 4.8785541 | 0.2741929 | 17.79242 | 0 | 0 |
| ENSG00000130508 | 10277.9876 | -0.9442904 | 0.0530794 | -17.79015 | 0 | 0 |
Now let’s have a look at some of the charts showing differential expression. In particular, an MA plot and volcano plot.
maplot <- function(de,contrast_name) {
sig <-subset(de, padj < 0.05 )
up <-rownames(subset(de, padj < 0.05 & log2FoldChange > 0))
dn <-rownames(subset(de, padj < 0.05 & log2FoldChange < 0))
GENESUP <- length(up)
GENESDN <- length(dn)
DET=nrow(de)
SUBHEADER = paste(GENESUP, "up, ", GENESDN, "down", DET, "detected")
ns <-subset(de, padj > 0.05 )
plot(log2(de$baseMean),de$log2FoldChange,
xlab="log2 basemean", ylab="log2 foldchange",
pch=19, cex=0.5, col="dark gray",
main=contrast_name, cex.main=0.7)
points(log2(sig$baseMean),sig$log2FoldChange,
pch=19, cex=0.5, col="red")
mtext(SUBHEADER,cex = 0.7)
}
make_volcano <- function(de,name) {
sig <- subset(de,padj<0.05)
N_SIG=nrow(sig)
N_UP=nrow(subset(sig,log2FoldChange>0))
N_DN=nrow(subset(sig,log2FoldChange<0))
DET=nrow(de)
HEADER=paste(N_SIG,"@5%FDR,", N_UP, "up", N_DN, "dn", DET, "detected")
plot(de$log2FoldChange,-log10(de$padj),cex=0.5,pch=19,col="darkgray",
main=name, xlab="log2 FC", ylab="-log10 pval", xlim=c(-6,6))
mtext(HEADER)
grid()
points(sig$log2FoldChange,-log10(sig$padj),cex=0.5,pch=19,col="red")
}
maplot(de,name)
make_volcano(de,name)
Need to add gene symbol
mart <- useDataset("hsapiens_gene_ensembl", useMart("ensembl"))
genes <- getBM(filters= "ensembl_gene_id",
attributes= c("ensembl_gene_id","hgnc_symbol"),
values=rownames(de), mart= mart)## Batch submitting query [=======>-----------------------] 25% eta: 1mBatch
## submitting query [===============>---------------] 50% eta: 48sBatch submitting
## query [======================>--------] 75% eta: 22sm <- merge(de,genes,by.x=0,by.y="ensembl_gene_id")
rownames(m) <- paste(m$Row.names,m$hgnc_symbol)
m$Row.names = m$hgnc_symbol = NULL
dim(de)## [1] 15607 6dim(m)## [1] 15483 6Save table
saveRDS(m,"bulkrna4.Rds")Session information
sessionInfo()## R version 4.4.0 (2024-04-24)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 22.04.4 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.10.0
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
##
## locale:
## [1] LC_CTYPE=en_AU.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_AU.UTF-8 LC_COLLATE=en_AU.UTF-8
## [5] LC_MONETARY=en_AU.UTF-8 LC_MESSAGES=en_AU.UTF-8
## [7] LC_PAPER=en_AU.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C
##
## time zone: Australia/Melbourne
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats4 stats graphics grDevices utils datasets methods
## [8] base
##
## other attached packages:
## [1] biomaRt_2.58.0 eulerr_7.0.2
## [3] kableExtra_1.4.0 mitch_1.14.0
## [5] clusterProfiler_4.10.0 DESeq2_1.42.0
## [7] SummarizedExperiment_1.32.0 Biobase_2.62.0
## [9] MatrixGenerics_1.14.0 matrixStats_1.3.0
## [11] GenomicRanges_1.54.1 GenomeInfoDb_1.38.5
## [13] IRanges_2.36.0 S4Vectors_0.40.2
## [15] BiocGenerics_0.48.1 getDEE2_1.12.0
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## loaded via a namespace (and not attached):
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## [4] ggplotify_0.1.2 filelock_1.0.3 tibble_3.2.1
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## [10] lattice_0.22-6 MASS_7.3-60.2 magrittr_2.0.3
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## [25] RCurl_1.98-1.14 yulab.utils_0.1.4 tweenr_2.0.3
## [28] rappdirs_0.3.3 GenomeInfoDbData_1.2.11 enrichplot_1.22.0
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