QC and MDS plot of nuclei RNA-seq
Evangelyn Sim
2/19/2021
Last updated: 2021-02-19
Checks: 6 1
Knit directory: 2021_UoM_Yap_shRNA_nuclei_RNAseq_ATACseq/
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Introduction
In the GEO submission 1 processed files were uploaded.
- mrna_fulllen_pe_strrev.mx.fix_filt
They have been uploaded in the /output folder and will be used below to generate different figures.
Used libraries and functions
library(edgeR)Loading required package: limmalibrary(limma)
library(Glimma)
library(gplots)
Attaching package: 'gplots'The following object is masked from 'package:stats':
lowess1. QC
Count per million reads
rm1 <- read.delim("/group/card2/Evangelyn_Sim/Collaboration_Kev_UoM/Sequencing_ATAC_RNA/Github/2021_UoM_Yap_shRNA_nuclei_RNAseq_ATACseq/output/mrna_fulllen_pe_strrev.mx.fix_filt", header = TRUE, row.names = 1)
rm2 = rm1[,c(1:6)]
mycpm = cpm(rm2)
summary(mycpm) Lacz.1 Lacz.2 Lacz.3 Yap.1
Min. : 0.00 Min. : 0.00 Min. : 0.00 Min. : 0.00
1st Qu.: 0.77 1st Qu.: 0.79 1st Qu.: 0.91 1st Qu.: 1.05
Median : 6.07 Median : 6.04 Median : 6.50 Median : 6.42
Mean : 60.84 Mean : 60.84 Mean : 60.84 Mean : 60.84
3rd Qu.: 30.14 3rd Qu.: 29.92 3rd Qu.: 30.42 3rd Qu.: 28.70
Max. :87479.04 Max. :91051.03 Max. :82580.58 Max. :98985.15
Yap.2 Yap.3
Min. : 0.02 Min. : 0.01
1st Qu.: 1.27 1st Qu.: 0.99
Median : 6.93 Median : 5.88
Mean : 60.84 Mean : 60.84
3rd Qu.: 28.78 3rd Qu.: 25.23
Max. :137655.22 Max. :193645.35 x <- DGEList(rm2)
names(x)[1] "counts" "samples"logcountsx = cpm(x, log = T)
write.csv(logcountsx, file = "/group/card2/Evangelyn_Sim/Collaboration_Kev_UoM/Sequencing_ATAC_RNA/Github/2021_UoM_Yap_shRNA_nuclei_RNAseq_ATACseq/output/logCPM_mrna_fulllen_pe_strrev.mx.fix_filt.csv")par(mfrow=c(1,2))
barplot(x$samples$lib.size, names=colnames(x), las=2, col = "blue", main = "Library size")
boxplot(logcountsx, xlab="", ylab="Log2 counts per million", las=2, col = "blue")
abline(h=median(logcountsx), col="navy")
2. MDS plot
Read files
sampleinfo = as.data.frame(colnames(rm2))
colnames(sampleinfo) = c("SampleName")
sampleinfo$group = gsub(".[0-9]", "", sampleinfo$SampleName)
write.table(sampleinfo, file = "/group/card2/Evangelyn_Sim/Collaboration_Kev_UoM/Sequencing_ATAC_RNA/Github/2021_UoM_Yap_shRNA_nuclei_RNAseq_ATACseq/output/sampleinfo.txt", sep = "\t", row.names = F, col.names = T)
sampleinfo = read.delim("/group/card2/Evangelyn_Sim/Collaboration_Kev_UoM/Sequencing_ATAC_RNA/Github/2021_UoM_Yap_shRNA_nuclei_RNAseq_ATACseq/output/sampleinfo.txt", header = T)
levels(sampleinfo$group)[1] "Lacz" "Yap" sampleinfo$colour = c("blue","red")[sampleinfo$group]
table(colnames(rm2)==sampleinfo$SampleName)
TRUE
6 y <- DGEList(rm2)Make plot
par(mfrow=c(1,2))
plotMDS(y, pch=c(0,1)[sampleinfo$group], col=sampleinfo$colour, cex = 1.5)
legend("topright", legend =levels(sampleinfo$group), pch=c(0,1), col = c("blue","red"), cex=1)
plotMDS(y, cex = 0.8)
sessionInfo()R version 3.6.1 (2019-07-05)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)
Matrix products: default
BLAS: /hpc/software/installed/R/3.6.1/lib64/R/lib/libRblas.so
LAPACK: /hpc/software/installed/R/3.6.1/lib64/R/lib/libRlapack.so
locale:
[1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] gplots_3.1.0 Glimma_1.12.0 edgeR_3.26.8 limma_3.40.6
[5] workflowr_1.6.2
loaded via a namespace (and not attached):
[1] Rcpp_1.0.5 pillar_1.4.6 compiler_3.6.1 later_1.1.0.1
[5] git2r_0.27.1 highr_0.8 bitops_1.0-6 tools_3.6.1
[9] digest_0.6.27 jsonlite_1.7.0 evaluate_0.14 lifecycle_0.2.0
[13] tibble_3.0.3 lattice_0.20-41 pkgconfig_2.0.3 rlang_0.4.7
[17] rstudioapi_0.11 yaml_2.2.1 xfun_0.18 stringr_1.4.0
[21] knitr_1.30 caTools_1.18.0 gtools_3.8.2 fs_1.5.0
[25] vctrs_0.3.2 locfit_1.5-9.4 rprojroot_1.3-2 grid_3.6.1
[29] glue_1.4.2 R6_2.5.0 rmarkdown_2.5 magrittr_1.5
[33] whisker_0.4 backports_1.1.10 promises_1.1.1 ellipsis_0.3.1
[37] htmltools_0.5.0 httpuv_1.5.4 KernSmooth_2.23-17 stringi_1.5.3
[41] crayon_1.3.4