Source: TBA

Introduction

baseDir=getwd()
dataDir=paste(baseDir,"/him_chart_idats/CHART_idats",sep="")

suppressPackageStartupMessages({
  library("missMethyl")
  library("limma")
  library("minfi")
  library("IlluminaHumanMethylation450kanno.ilmn12.hg19")
  library("IlluminaHumanMethylationEPICanno.ilm10b2.hg19")
  library("ruv")
  library("RColorBrewer")
  library("matrixStats")
  library("gplots")
  library("FlowSorted.Blood.450k")
  library("reshape2")
  library("ggplot2")
  library("DMRcate")
  library("FlowSorted.Blood.EPIC")
  library("mitch")
  library("kableExtra")
  library("vioplot")
  library("WGCNA")
})

#source("meth_functions.R")

Load data

Load the annotation data and the Epic methylation data.

This analysis is to be conducted on Ubuntu with R4.

ann = getAnnotation(IlluminaHumanMethylationEPICanno.ilm10b2.hg19)

ann_sub = ann[,c("chr","pos","strand","Name","Islands_Name",
    "Relation_to_Island","UCSC_RefGene_Name","UCSC_RefGene_Group")]

targets_gen = read.metharray.sheet(dataDir, pattern = "CHART_sample_sheet_adults.csv")
## [read.metharray.sheet] Found the following CSV files:
## [1] "/analysis/him_chart_idats/CHART_idats/CHART_sample_sheet_adults.csv"
#targets$ID = paste(targets$Sample_Group,targets_gen$Sample_Name,sep=".")
rgSet = read.metharray.exp(targets = targets_gen)
## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls

## Warning in readChar(con, nchars = n): truncating string with embedded nuls
sampleNames(rgSet) = targets_gen$sample_id

rgSet$Slide <- as.numeric(rgSet$Slide)
rgSet$gender_m_f <- as.character(rgSet$gender_m_f)
rgSet$Sex <- as.character(rgSet$gender_m_f)
rgSet$sample_id <- as.character(rgSet$sample_id)


targets_gen$gender_m_f <- factor(targets_gen$gender_m_f)
targets_gen$status <- factor(targets_gen$status,levels=c("control","case"))
targets_gen$birth <- factor(targets_gen$birth,levels=c("Vaginal","Elective caesarian","Emergency caesarean"))
targets_gen$gest <- as.numeric(targets_gen$gest)
## Warning: NAs introduced by coercion
targets_gen$sga2 <-  factor(paste(targets_gen$sga,targets_gen$lrga),levels=c("Not small for gestational age - above 10th percentile Large for gestational age - above 90th percentile","Not small for gestational age - above 10th percentile Not large for gestational age - below 90th percentile","Small for gestational age - below 10th percentile Not large for gestational age - below 90th percentile"))

Testing familial structure

snpBetas = getSnpBeta(rgSet)
## Loading required package: IlluminaHumanMethylationEPICmanifest
d = dist(t(snpBetas))
hr = hclust(d, method = "complete", members=NULL)
plot(hr)

Quality control

detP = detectionP(rgSet)
qcReport(rgSet, sampNames = targets_gen$sample_id,
  pdf="qc-report_chart.pdf")
## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
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## caller; using TRUE

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## caller; using TRUE

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## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
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## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
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## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
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## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
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## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
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## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

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## caller; using TRUE

## Warning in type.convert.default(X[[i]], ...): 'as.is' should be specified by the
## caller; using TRUE

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## png 
##   2
cols=brewer.pal(4,"Set1")

barplot(apply(detP,2,mean),
  col=as.numeric(factor(targets_gen$Sample_Name)),
  las=2,cex.names= 0.8, cex.axis=0.75,
  main="Mean detection p-values of probe signals",
  ylab="Mean detection p-value")

barplot(apply(detP,2,mean),
  col=as.numeric(factor(targets_gen$Sample_Name)),
  las=2,cex.names= 0.8, cex.axis=0.75,ylim=c(0,0.010),
  main="Mean detection p-values of probe signals",
  ylab="Mean detection p-value")

Preprocessing

mset.raw = preprocessRaw(rgSet)

Data exploration

Using Multi-dimensional scaling (MDS) plots before filtering.

mdsPlot(mset.raw, sampGroups = targets_gen$Sample_Name, 
  sampNames=targets_gen$Social_interaction_on_ADOS,legendPos="bottom")

mdsPlot(mset.raw, sampGroups = targets_gen$Sex, 
  sampNames=targets_gen$SampleID,legendPos="bottom")

Cell type composition analysis

Try estimatecellcounts2.

cells <- estimateCellCounts2(rgSet, referencePlatform= "IlluminaHumanMethylationEPIC", 
  returnAll = TRUE)
## snapshotDate(): 2021-10-19
## see ?FlowSorted.Blood.EPIC and browseVignettes('FlowSorted.Blood.EPIC') for documentation
## downloading 1 resources
## retrieving 1 resource
## loading from cache
## [estimateCellCounts2] Combining user data with reference (flow sorted) data.
## [estimateCellCounts2] Processing user and reference data together.
## [estimateCellCounts2] Picking probes for composition estimation.
## [estimateCellCounts2] Estimating composition.
mset <- cells$normalizedData

cellCounts_new <- cells[[1]]
#plot cell type composition by sample group
a = cellCounts_new[targets_gen$Diagnosis == "0",]
b = cellCounts_new[targets_gen$Diagnosis == "1",]
c = cellCounts_new[targets_gen$Diagnosis == "2",]
age.pal <- brewer.pal(8,"Set1")

cellCounts_long <- melt(cellCounts_new, id = "celltype")

cellCounts_long$Var1 <- targets_gen[match(cellCounts_long$Var1,  targets_gen$sample_id),"group_exp_new"]

colnames(cellCounts_long) <- c("ART_group","celltype","value")

head(cellCounts_long)
##      ART_group celltype      value
## 1 Fresh embryo     CD8T 0.14527618
## 2 Fresh embryo     CD8T 0.23424722
## 3 Fresh embryo     CD8T 0.09020196
## 4         GIFT     CD8T 0.06489822
## 5      non-ART     CD8T 0.17094843
## 6      non-ART     CD8T 0.13801103

Filtering

detP <- detectionP(rgSet)

# exclude bad samples
sample_failed_probes <- apply(detP,2, function(x) { length(which(x>0.01)) / length(x) } )
barplot(sample_failed_probes[order(sample_failed_probes)])

bad_samples <- sample_failed_probes[sample_failed_probes>0.01]
bad_samples
##    4014_A    4051_A    4439_A    5156_A 
## 0.7714432 0.9624439 0.9735132 0.9720976
bad_samples <- names(bad_samples)
good_samples <- setdiff(names(sample_failed_probes),bad_samples)
detP <- detP[,good_samples]
mset <- mset[,good_samples]
targets_gen <- targets_gen[which(targets_gen$sample_id %in% good_samples),]


# exclude bad probes
failed_prope_frac <- apply(detP,1, function(x) { length(which(x>0.01)) / length(x) } )
probes_to_keep <- names(which(failed_prope_frac<0.05))
detP <- detP[probes_to_keep,]
mset <- mset[which(rownames(mset) %in% probes_to_keep),]

dim(mset)
## [1] 864281    233
gmset <- mapToGenome(mset)

#remove SNPs
gmset_flt = dropLociWithSnps(gmset, snps = c("CpG", "SBE"))

#Removing cross-reactive probes
XURL="https://raw.githubusercontent.com/sirselim/illumina450k_filtering/master/EPIC/13059_2016_1066_MOESM1_ESM.csv"
Xreact <- read.csv(XURL)

noXreact <-  !(featureNames(gmset) %in% Xreact$X)

gmset <- gmset[noXreact,]

#Removing probes on X and Y chromosomes
autosomes <- !(featureNames(gmset) %in% ann$Name[ann$chr %in% c("chrX","chrY")])
gmset_flt <- gmset[autosomes,]

#Relative log expression (RLE plot)
mvals = getM(gmset_flt)
dim(mvals)
## [1] 803094    233
medSq = apply(mvals, 1, median)
YSq = mvals - medSq

boxplot(YSq,outline=FALSE,ylim=c(-1.5,1.5), 
  ylab="Relative Log Methylation Value", 
  cols=as.character(factor(targets_gen$Social_interaction_on_ADOS,)) )

MDS plots generation after filtering

pal = brewer.pal(8, "Dark2")
mds1Sq = plotMDS(mvals, top=1000, gene.selection="common",dim.plot=c(1,2))

mds2Sq = plotMDS(mvals, top=1000, gene.selection="common",dim.plot=c(1,3))

mds3Sq = plotMDS(mvals, top=1000, gene.selection="common",dim.plot=c(2,3))

mds4Sq = plotMDS(mvals, top=1000, gene.selection="common",dim.plot=c(3,4))

plotMDS(mds1Sq, xlab="Dimension 1", ylab="Dimension 2",
  col=pal[as.factor(targets_gen$group_exp_new)],
  dim=c(1,2), labels=targets_gen$sample_id)
legend("bottomright",bg="white",col=pal,cex=.7,pch=1,legend=0:1)

plotMDS(mds2Sq, xlab="Dimension 1", ylab="Dimension 3",
  col=pal[as.factor(targets_gen$group_exp_new)],dim=c(1,3),
  labels=targets_gen$sample_id)
legend("bottomright",bg="white",col=pal,cex=.7,pch=1,legend=0:1)

plotMDS(mds3Sq, xlab="Dimension 2", ylab="Dimension 3",
  col=pal[as.factor(targets_gen$group_exp_new)],dim=c(2,3),
  labels=targets_gen$sample_id)
legend("bottomright",bg="white",col=pal,cex=.7,pch=1,legend=0:1)

plotMDS(mds4Sq, xlab="Dimension 3", ylab="Dimension 4",
  col=pal[as.factor(targets_gen$group_exp_new)],dim=c(3,4),
  labels=targets_gen$sample_id)
legend("bottomright",bg="white",col=pal,cex=.7,pch=1,legend=0:1)

Principal Component Analysis (PCA)

fit <- prcomp(t(mvals),center = TRUE, scale = TRUE,retx=TRUE)
loadings = fit$x
plot(fit,type="lines")

nGenes = nrow(mvals)
nSamples = ncol(mvals)
datTraits =  targets_gen[,c("gender_m_f","status","birth","gest","sga2")]

datTraits$gender_m_f <-  as.numeric(datTraits$gender_m_f)
datTraits$status <-  as.numeric(datTraits$status)
datTraits$birth <- as.numeric(datTraits$birth)
datTraits$sga2 <- as.numeric(datTraits$sga2)
moduleTraitCor = cor(loadings[,1:6], datTraits, use = "p")

moduleTraitPvalue = corPvalueStudent(moduleTraitCor, nSamples)
par(cex=0.75, mar = c(6, 8.5, 3, 3))
textMatrix = paste(signif(moduleTraitCor, 2), "\n(", 
  signif(moduleTraitPvalue, 1), ")", sep = "");
dim(textMatrix) = dim(moduleTraitCor)

labeledHeatmap(Matrix = t(moduleTraitCor), 
  xLabels = colnames(loadings)[1:ncol(t(moduleTraitCor))],
  yLabels = names(datTraits), colorLabels = FALSE, colors = blueWhiteRed(6),
  textMatrix = t(textMatrix), setStdMargins = FALSE, cex.text = 0.5,
  cex.lab.y = 0.6, zlim = c(-1,1), 
  main = paste("PCA-trait relationships: Top principal components"))

pdf("pca_chart.pdf")

par(mar=c(3,8,2,2))

labeledHeatmap(Matrix = t(moduleTraitCor), 
  xLabels = colnames(loadings)[1:ncol(t(moduleTraitCor))],
  yLabels = names(datTraits), colorLabels = FALSE, colors = blueWhiteRed(6),
  textMatrix = t(textMatrix), setStdMargins = FALSE, cex.text = 0.5,
  cex.lab.y = 0.6, zlim = c(-1,1), 
  main = paste("PCA-trait relationships: Top principal components"))

dev.off()
## png 
##   2

Limma analysis looking for differences caused by ART corrected for sex

targets_gen$art <- targets_gen$Tx_new!=".c"
art <- targets_gen$art
sex <- targets_gen$sex

design <- model.matrix(~ sex + art )
fit <- lmFit(mvals, design)
## Coefficients not estimable: artTRUE
## Warning: Partial NA coefficients for 803094 probe(s)
fit2 <- eBayes(fit)
## Warning in .ebayes(fit = fit, proportion = proportion, stdev.coef.lim =
## stdev.coef.lim, : Estimation of var.prior failed - set to default value
summary(decideTests(fit2))
##        (Intercept) sexF_control sexM_case sexM_control artTRUE
## Down        262001           68     18829        11158       0
## NotSig        6938       802948    778016       767757       0
## Up          534155           78      6249        24179       0
top <- topTable(fit2,coef=ncol(design),num=Inf, sort.by = "P")

nsig <- sum(top$adj.P.Val < 0.05)
sum(top$P.Value< 0.05)
## [1] NA
output <-merge(ann_sub,top,by.x="Name",by.y="row.names")
output <- output[order(output$P.Value),]
write.csv(output, file="limma_CHART_art.csv",row.names=FALSE)
output <- subset(output,P.Value<1e-4)
head(output,30) %>% kbl() %>% kable_paper("hover", full_width = F)
Name chr pos strand Islands_Name Relation_to_Island UCSC_RefGene_Name UCSC_RefGene_Group logFC AveExpr t P.Value adj.P.Val B
saveRDS(design, "CHART_art_des.rds")
saveRDS(mvals, "CHART_art_mvals.rds")

Session information

sessionInfo()
## R version 4.1.3 (2022-03-10)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.4 LTS
## 
## Matrix products: default
## BLAS/LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.8.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] parallel  stats4    stats     graphics  grDevices utils     datasets 
## [8] methods   base     
## 
## other attached packages:
##  [1] IlluminaHumanMethylationEPICmanifest_0.3.0         
##  [2] WGCNA_1.72-1                                       
##  [3] fastcluster_1.2.3                                  
##  [4] dynamicTreeCut_1.63-1                              
##  [5] vioplot_0.4.0                                      
##  [6] zoo_1.8-11                                         
##  [7] sm_2.2-5.7.1                                       
##  [8] kableExtra_1.3.4                                   
##  [9] mitch_1.6.0                                        
## [10] FlowSorted.Blood.EPIC_1.12.1                       
## [11] ExperimentHub_2.2.1                                
## [12] AnnotationHub_3.2.2                                
## [13] BiocFileCache_2.2.1                                
## [14] dbplyr_2.3.2                                       
## [15] nlme_3.1-155                                       
## [16] quadprog_1.5-8                                     
## [17] genefilter_1.76.0                                  
## [18] DMRcate_2.8.5                                      
## [19] ggplot2_3.4.1                                      
## [20] reshape2_1.4.4                                     
## [21] FlowSorted.Blood.450k_1.32.0                       
## [22] gplots_3.1.3                                       
## [23] RColorBrewer_1.1-3                                 
## [24] ruv_0.9.7.1                                        
## [25] IlluminaHumanMethylationEPICanno.ilm10b2.hg19_0.6.0
## [26] limma_3.50.3                                       
## [27] missMethyl_1.28.0                                  
## [28] IlluminaHumanMethylationEPICanno.ilm10b4.hg19_0.6.0
## [29] IlluminaHumanMethylation450kanno.ilmn12.hg19_0.6.0 
## [30] minfi_1.40.0                                       
## [31] bumphunter_1.36.0                                  
## [32] locfit_1.5-9.7                                     
## [33] iterators_1.0.14                                   
## [34] foreach_1.5.2                                      
## [35] Biostrings_2.62.0                                  
## [36] XVector_0.34.0                                     
## [37] SummarizedExperiment_1.24.0                        
## [38] Biobase_2.54.0                                     
## [39] MatrixGenerics_1.6.0                               
## [40] matrixStats_0.63.0                                 
## [41] GenomicRanges_1.46.1                               
## [42] GenomeInfoDb_1.30.1                                
## [43] IRanges_2.28.0                                     
## [44] S4Vectors_0.32.4                                   
## [45] BiocGenerics_0.40.0                                
## 
## loaded via a namespace (and not attached):
##   [1] rappdirs_0.3.3                rtracklayer_1.54.0           
##   [3] GGally_2.1.2                  R.methodsS3_1.8.2            
##   [5] tidyr_1.3.0                   bit64_4.0.5                  
##   [7] knitr_1.37                    DelayedArray_0.20.0          
##   [9] R.utils_2.12.2                data.table_1.14.8            
##  [11] rpart_4.1.16                  doParallel_1.0.17            
##  [13] KEGGREST_1.34.0               RCurl_1.98-1.10              
##  [15] GEOquery_2.62.2               AnnotationFilter_1.18.0      
##  [17] generics_0.1.2                GenomicFeatures_1.46.5       
##  [19] preprocessCore_1.56.0         RSQLite_2.3.0                
##  [21] bit_4.0.5                     tzdb_0.3.0                   
##  [23] webshot_0.5.4                 xml2_1.3.3                   
##  [25] httpuv_1.6.9                  xfun_0.37                    
##  [27] hms_1.1.3                     jquerylib_0.1.4              
##  [29] evaluate_0.15                 promises_1.2.0.1             
##  [31] fansi_1.0.2                   restfulr_0.0.15              
##  [33] scrime_1.3.5                  progress_1.2.2               
##  [35] caTools_1.18.2                DBI_1.1.3                    
##  [37] htmlwidgets_1.6.2             reshape_0.8.9                
##  [39] purrr_1.0.1                   ellipsis_0.3.2               
##  [41] dplyr_1.1.0                   backports_1.4.1              
##  [43] permute_0.9-7                 annotate_1.72.0              
##  [45] biomaRt_2.50.3                deldir_1.0-6                 
##  [47] sparseMatrixStats_1.6.0       vctrs_0.6.0                  
##  [49] ensembldb_2.18.4              cachem_1.0.6                 
##  [51] withr_2.5.0                   Gviz_1.38.4                  
##  [53] BSgenome_1.62.0               checkmate_2.1.0              
##  [55] GenomicAlignments_1.30.0      prettyunits_1.1.1            
##  [57] mclust_6.0.0                  svglite_2.1.1                
##  [59] cluster_2.1.2                 lazyeval_0.2.2               
##  [61] crayon_1.5.0                  edgeR_3.36.0                 
##  [63] pkgconfig_2.0.3               ProtGenerics_1.26.0          
##  [65] nnet_7.3-17                   rlang_1.1.0                  
##  [67] lifecycle_1.0.3               filelock_1.0.2               
##  [69] dichromat_2.0-0.1             rngtools_1.5.2               
##  [71] base64_2.0.1                  Matrix_1.4-0                 
##  [73] Rhdf5lib_1.16.0               base64enc_0.1-3              
##  [75] beeswarm_0.4.0                png_0.1-8                    
##  [77] viridisLite_0.4.1             rjson_0.2.21                 
##  [79] bitops_1.0-7                  R.oo_1.25.0                  
##  [81] KernSmooth_2.23-20            rhdf5filters_1.6.0           
##  [83] blob_1.2.4                    DelayedMatrixStats_1.16.0    
##  [85] doRNG_1.8.6                   stringr_1.5.0                
##  [87] nor1mix_1.3-0                 readr_2.1.4                  
##  [89] jpeg_0.1-10                   scales_1.2.1                 
##  [91] memoise_2.0.1                 magrittr_2.0.2               
##  [93] plyr_1.8.8                    zlibbioc_1.40.0              
##  [95] compiler_4.1.3                BiocIO_1.4.0                 
##  [97] illuminaio_0.36.0             Rsamtools_2.10.0             
##  [99] cli_3.6.0                     DSS_2.42.0                   
## [101] htmlTable_2.4.1               Formula_1.2-5                
## [103] MASS_7.3-55                   tidyselect_1.2.0             
## [105] stringi_1.7.6                 highr_0.9                    
## [107] yaml_2.3.5                    askpass_1.1                  
## [109] latticeExtra_0.6-30           grid_4.1.3                   
## [111] sass_0.4.5                    VariantAnnotation_1.40.0     
## [113] tools_4.1.3                   rstudioapi_0.13              
## [115] foreign_0.8-82                bsseq_1.30.0                 
## [117] gridExtra_2.3                 digest_0.6.29                
## [119] BiocManager_1.30.16           shiny_1.7.4                  
## [121] Rcpp_1.0.10                   siggenes_1.68.0              
## [123] BiocVersion_3.14.0            later_1.3.0                  
## [125] org.Hs.eg.db_3.14.0           httr_1.4.2                   
## [127] AnnotationDbi_1.56.2          biovizBase_1.42.0            
## [129] colorspace_2.1-0              rvest_1.0.3                  
## [131] XML_3.99-0.14                 splines_4.1.3                
## [133] statmod_1.5.0                 multtest_2.50.0              
## [135] systemfonts_1.0.4             xtable_1.8-4                 
## [137] jsonlite_1.8.0                R6_2.5.1                     
## [139] echarts4r_0.4.4               Hmisc_5.0-1                  
## [141] pillar_1.7.0                  htmltools_0.5.4              
## [143] mime_0.12                     glue_1.6.2                   
## [145] fastmap_1.1.0                 BiocParallel_1.28.3          
## [147] interactiveDisplayBase_1.32.0 beanplot_1.3.1               
## [149] codetools_0.2-18              utf8_1.2.2                   
## [151] lattice_0.20-45               bslib_0.4.2                  
## [153] tibble_3.1.6                  curl_4.3.2                   
## [155] gtools_3.9.4                  GO.db_3.14.0                 
## [157] openssl_2.0.0                 interp_1.1-3                 
## [159] survival_3.3-1                rmarkdown_2.20               
## [161] munsell_0.5.0                 rhdf5_2.38.1                 
## [163] GenomeInfoDbData_1.2.7        impute_1.68.0                
## [165] HDF5Array_1.22.1              gtable_0.3.3