QC filtered sequencing reads
Evangelyn Sim
2/15/2021
Last updated: 2021-02-16
Checks: 6 1
Knit directory: Mouse_PRKO_RNAseq_bulk/
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Introduction
In the GEO submission 2 processed files were uploaded.
- mrna_fulllen_pe_strrev_q30.mx.PRKO.unfiltered
- mrna_fulllen_pe_strrev_q30.mx.PRKO.all.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':
lowessCount per million reads
rm1 <- read.delim("/group/card2/Evangelyn_Sim/Transcriptome_chromatin_human/Sequencing_ATAC_RNA/GITHUB/Mouse_PRKO_RNAseq_bulk/output/mrna_fulllen_pe_strrev_q30.mx.PRKO.all.fix_filt", row.names = 1)
info = read.delim("/group/card2/Evangelyn_Sim/Transcriptome_chromatin_human/Sequencing_ATAC_RNA/GITHUB/Mouse_PRKO_RNAseq_bulk/output/20200211_PRKO_RNA_samplesheet.txt", header = TRUE, sep = "\t")
info$ID = gsub("-",".",info$ID)
names(rm1) = info$ID[match(names(rm1),info$ID)]
m = match(info$ID,names(rm1))
rm2 = rm1[,m]
rm1 = rm2
###calculate cpm
mycpm = cpm(rm1)
summary(mycpm) PR.KO.F.1 PR.KO.F.2 PR.KO.F.3 PR.KO.F.4
Min. : 0.00 Min. : 0.000 Min. : 0.00 Min. : 0.000
1st Qu.: 1.45 1st Qu.: 1.679 1st Qu.: 0.80 1st Qu.: 0.821
Median : 8.08 Median : 8.667 Median : 5.89 Median : 5.969
Mean : 60.20 Mean : 60.205 Mean : 60.20 Mean : 60.205
3rd Qu.: 33.38 3rd Qu.: 35.712 3rd Qu.: 30.41 3rd Qu.: 30.841
Max. :41591.39 Max. :26979.638 Max. :38634.02 Max. :30994.242
PR.KO.M.1 PR.KO.M.2 PR.KO.M.3 PR.KO.M.4
Min. : 0.00 Min. : 0.01 Min. : 0.00 Min. : 0.00
1st Qu.: 1.56 1st Qu.: 1.53 1st Qu.: 0.85 1st Qu.: 0.97
Median : 8.27 Median : 8.25 Median : 6.31 Median : 6.68
Mean : 60.20 Mean : 60.20 Mean : 60.20 Mean : 60.20
3rd Qu.: 34.53 3rd Qu.: 33.92 3rd Qu.: 31.03 3rd Qu.: 30.76
Max. :36859.69 Max. :36011.74 Max. :39536.31 Max. :39661.96
PR.WT.F.1 PR.WT.F.2 PR.WT.F.3 PR.WT.M.1
Min. : 0.00 Min. : 0.00 Min. : 0.00 Min. : 0.02
1st Qu.: 1.56 1st Qu.: 0.87 1st Qu.: 0.99 1st Qu.: 1.68
Median : 8.33 Median : 5.82 Median : 6.91 Median : 8.98
Mean : 60.20 Mean : 60.20 Mean : 60.20 Mean : 60.20
3rd Qu.: 34.04 3rd Qu.: 29.23 3rd Qu.: 32.86 3rd Qu.: 35.43
Max. :42162.06 Max. :33512.06 Max. :35055.03 Max. :41445.30
PR.WT.M.2 PR.WT.M.3 PR.WT.M.4
Min. : 0.00 Min. : 0.00 Min. : 0.00
1st Qu.: 1.01 1st Qu.: 0.71 1st Qu.: 0.96
Median : 6.16 Median : 5.39 Median : 6.99
Mean : 60.20 Mean : 60.20 Mean : 60.20
3rd Qu.: 30.23 3rd Qu.: 28.98 3rd Qu.: 31.87
Max. :32040.19 Max. :32678.54 Max. :37133.85 x <- DGEList(rm1)
names(x)[1] "counts" "samples"logcountsx = cpm(x, log = T)
write.csv(logcountsx, "logCPM_mrna_fulllen_pe_strrev_q30.mx.PRKO.all.fix_filt.csv")
barplot(x$samples$lib.size, names=colnames(x), las=2, col = c("maroon1","turquoise1","red","royalblue1")[info$BinSex], main = "Library size")
boxplot(logcountsx, xlab="", ylab="Log2 counts per million", las=2, col = c("maroon1","turquoise1","red","royalblue1")[info$BinSex])
abline(h=median(logcountsx), col="navy")
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