Verrijking beoordelen

ChIP-seq met Bioconductor in R

Peter Humburg

Statistician, Macquarie University

Reads verlengen

Laad de data:

reads <- readGAlignments(bam)
reads_gr <- granges(reads[[1]])

Bepaal de gemiddelde fragmentlengte:

frag_length <- fragmentlength(qc_report)["GSM1598218"]

Verleng reads en bereken coverage:

reads_ext <- resize(reads_gr, width=frag_length)
cover_ext <- coverage(reads_ext)

Coverage voor pieken

Maak bins van 200 bp over het genoom.

bins <- tileGenome(seqinfo(reads), tilewidth=200, 
                   cut.last.tile.in.chrom=TRUE)

Zoek alle bins die overlappen met pieken.

peak_bins_overlap <- findOverlaps(bins, peaks)
peak_bins <- bins[from(peak_bins_overlap), ]

Tel het aantal reads dat elke piek-bin overlapt.

peak_bins$score <- countOverlaps(peak_bins, reads)

Gefractioneerde coverage-functie

count_bins <- function(reads, target, bins){
  # Find all bins overlapping peaks
  overlap <- from(findOverlaps(bins, target))
  target_bins <- bins[overlap, ]

  # Count the number of reads overlapping each peak bin
  target_bins$score <- countOverlaps(target_bins, reads)
  target_bins
}

Coverage voor geblackliste regio's

peak_bins <- count_bins(reads_ext, peaks, bins)
bl_bins <- count_bins(reads_ext, blacklist.hg19, bins)

Achtergrondcoverage

Verwijder alle bins die al zijn meegenomen.

bkg_bins <- subset(bins, !bins %in% peak_bins & !bins %in% bl_bins)

Tel het aantal reads dat met elke overgebleven bin overlapt.

bkg_bins$score <- countOverlaps(bkg_bins, reads_ext)

Laten we oefenen!

ChIP-seq met Bioconductor in R