Last updated: 2021-02-19

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Knit directory: Mouse_PRKO_RNAseq_bulk/

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Introduction

In the GEO submission 2 processed files were uploaded.

  1. mrna_fulllen_pe_strrev_q30.mx.PRKO.unfiltered
  2. 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: limma
library(limma)
library(Glimma)
library(gplots)

Attaching package: 'gplots'
The following object is masked from 'package:stats':

    lowess

Count 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")

Version Author Date
440019f evangelynsim 2021-02-16 
boxplot(logcountsx, xlab="", ylab="Log2 counts per million", las=2, col = c("maroon1","turquoise1","red","royalblue1")[info$BinSex])
abline(h=median(logcountsx), col="navy")

Version Author Date
440019f evangelynsim 2021-02-16 

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