Using ENCODE methylation data (RRBS)

ENCODE project has generated reduced-representation bilsulfite sequencing data for multiple cell lines. The data is organized in an extended bed format with additional columns denoting % methylation and coverage per base. Luckily, this sort of generic % methylation information can be read in by R package methylKit, which is a package for analyzing basepair resolution 5mC and 5hmC data. 

The code snippets below show how to read RRBS bed file produced by ENCODE. But, let's first download the files.

wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/wgEncodeHaibMethylRrbsA549Dm002p7dHaibSitesRep1.bed.gz

wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/wgEncodeHaibMethylRrbsAg04449UwstamgrowprotSitesRep1.bed9.gz

Unfortunately, methylKit currently can not read them directly because the track definition line causes a problem. It should be deleted from each bed file. Ideally, methylKit should be able to skip over lines (this issue should be fixed in later versions)
For now, we have to use some unix tools to remove the first lines from the bed files. You run the code below in your terminal. This set of commands will delete the first line from every *.gz file in the directory so be careful.

for files in *.gz
do
  gzip -dc "$files" | tail +2 | gzip -c > "$files".tmp
  if [ "$?" -eq 0 ]; then
    mv "$files".tmp "$files"
  fi
done

Now we can read the files using methylKit. The pipeline argument defines which columns in the file are corresponding to chr,start,end,strand, percent methylation and coverage:

1234567

library(methylKit) obj = read("wgEncodeHaibMethylRrbsA549Dm002p7dHaibSitesRep1.bed.gz", sample.id = "test", assembly = "hg19", header = FALSE, context = "CpG", resolution = "base", pipeline = list(fraction = FALSE, chr.col = 1, start.col = 3, end.col = 3,coverage.col = 5, strand.col = 6, freqC.col = 11))

view rawread.rrbs.bed.R hosted with ❤ by GitHub

You can also read multiple files at a time:

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file.list = list("wgEncodeHaibMethylRrbsA549Dm002p7dHaibSitesRep1.bed.gz", "wgEncodeHaibMethylRrbsAg04449UwstamgrowprotSitesRep1.bed9.gz") obj = read(file.list, sample.id = list("test", "control"), treatment = c(1,0), assembly = "hg19", header = FALSE, context = "CpG", resolution = "base", pipeline = list(fraction = FALSE, chr.col = 1, start.col = 3, end.col = 3, coverage.col = 5, strand.col = 6, freqC.col = 11)) obj ## methylRawList object with 2 methylRaw objects ## ## methylRaw object with 1464031 rows ## -------------- ## id chr start end strand coverage numCs numTs ## 1 chr1.100002990 chr1 100002990 100002990 + 3 1 2 ## 2 chr1.100003154 chr1 100003154 100003154 - 19 1 18 ## 3 chr1.1000171 chr1 1000171 1000171 + 26 7 19 ## 4 chr1.1000191 chr1 1000191 1000191 + 26 14 12 ## 5 chr1.1000192 chr1 1000192 1000192 - 22 9 13 ## 6 chr1.1000199 chr1 1000199 1000199 + 26 15 11 ## -------------- ## sample.id: test ## assembly: hg19 ## context: CpG ## resolution: base ## ## methylRaw object with 1233588 rows ## -------------- ## id chr start end strand coverage numCs numTs ## 1 chr1.1000171 chr1 1000171 1000171 + 22 1 21 ## 2 chr1.1000191 chr1 1000191 1000191 + 22 4 18 ## 3 chr1.1000192 chr1 1000192 1000192 - 12 4 8 ## 4 chr1.1000199 chr1 1000199 1000199 + 22 11 11 ## 5 chr1.1000200 chr1 1000200 1000200 - 12 4 8 ## 6 chr1.1000207 chr1 1000207 1000207 - 12 4 8 ## -------------- ## sample.id: control ## assembly: hg19 ## context: CpG ## resolution: base ## ## treament: 1 0

view rawread.bed.files.rrbs.R hosted with ❤ by GitHub

Since we have read the files and now they are methylKit objects, we can use all of the methylKit functionality on these objects. For example, the code below plots the distribution of methylation % for covered bases.

getMethylationStats(obj[[1]], plot = TRUE)

You can check the methylKit vignette and the website for the rest of the functionality and details.