## ----setup, message=FALSE, warning=FALSE-------------------------------------- library(chipseq) library(GenomicFeatures) library(lattice) ## ----preprocess, eval=FALSE--------------------------------------------------- # qa_list <- lapply(sampleFiles, qa) # report(do.call(rbind, qa_list)) # ## spend some time evaluating the QA report, then procede # filter <- compose(chipseqFilter(), alignQualityFilter(15)) # cstest <- GenomicRangesList(lapply(sampleFiles, function(file) { # as(readAligned(file, filter), "GRanges") # })) # cstest <- cstest[seqnames(cstest) %in% c("chr10", "chr11", "chr12")] ## ----------------------------------------------------------------------------- data(cstest) cstest ## ----convert-cstest, echo=FALSE, eval=FALSE----------------------------------- # ## code used to convert the GenomeDataList to a GRangesList # cstest <- GenomicRangesList(lapply(cstest, function(gd) # do.call(c, lapply(names(gd), function(chr) # pos <- gd[[chr]] # starts <- c( pos[["-"]] - 23L, pos[["+"]]) # GRanges(chr, IRanges(starts, width = 24), # rep(c("-", "+"), elementNROWS(pos))) )) # )) ## ----------------------------------------------------------------------------- cstest$ctcf ## ----estimate.mean.fraglen---------------------------------------------------- fraglen <- estimate.mean.fraglen(cstest$ctcf, method="correlation") fraglen[!is.na(fraglen)] ## ----------------------------------------------------------------------------- ctcf.ext <- resize (cstest$ctcf, width = 200) ctcf.ext ## ----------------------------------------------------------------------------- cov.ctcf <- coverage(ctcf.ext) cov.ctcf ## ----------------------------------------------------------------------------- islands <- slice(cov.ctcf, lower = 1) islands ## ----------------------------------------------------------------------------- viewSums(islands) viewMaxs(islands) nread.tab <- table(viewSums(islands) / 200) depth.tab <- table(viewMaxs(islands)) nread.tab[,1:10] depth.tab[,1:10] ## ----------------------------------------------------------------------------- islandReadSummary <- function(x) { g <- resize(x, 200) s <- slice(coverage(g), lower = 1) tab <- table(viewSums(s) / 200) df <- DataFrame(tab) colnames(df) <- c("chromosome", "nread", "count") df$nread <- as.integer(df$nread) df } ## ----------------------------------------------------------------------------- head(islandReadSummary(cstest$ctcf)) ## ----------------------------------------------------------------------------- nread.islands <- DataFrameList(lapply(cstest, islandReadSummary)) nread.islands <- stack(nread.islands, "sample") nread.islands ## ----fig.height=10------------------------------------------------------------ xyplot(log(count) ~ nread | sample, as.data.frame(nread.islands), subset = (chromosome == "chr10" & nread <= 40), layout = c(1, 2), pch = 16, type = c("p", "g")) ## ----fig.height=8------------------------------------------------------------- xyplot(log(count) ~ nread | sample, data = as.data.frame(nread.islands), subset = (chromosome == "chr10" & nread <= 40), layout = c(1, 2), pch = 16, type = c("p", "g"), panel = function(x, y, ...) { panel.lmline(x[1:2], y[1:2], col = "black") panel.xyplot(x, y, ...) }) ## ----------------------------------------------------------------------------- islandDepthSummary <- function(x) { g <- resize(x, 200) s <- slice(coverage(g), lower = 1) tab <- table(viewMaxs(s) / 200) df <- DataFrame(tab) colnames(df) <- c("chromosome", "depth", "count") df$depth <- as.integer(df$depth) df } depth.islands <- DataFrameList(lapply(cstest, islandDepthSummary)) depth.islands <- stack(depth.islands, "sample") plt <- xyplot(log(count) ~ depth | sample, as.data.frame(depth.islands), subset = (chromosome == "chr10" & depth <= 20), layout = c(1, 2), pch = 16, type = c("p", "g"), panel = function(x, y, ...){ lambda <- 2 * exp(y[2]) / exp(y[1]) null.est <- function(xx) { xx * log(lambda) - lambda - lgamma(xx + 1) } log.N.hat <- null.est(1) - y[1] panel.lines(1:10, -log.N.hat + null.est(1:10), col = "black") panel.xyplot(x, y, ...) }) ## depth.islands <- summarizeReads(cstest, summary.fun = islandDepthSummary) ## ----fig.height=10, echo=FALSE------------------------------------------------ plt ## ----islandDepthPlot, eval=FALSE---------------------------------------------- # islandDepthPlot(cov.ctcf) ## ----peakCutoff--------------------------------------------------------------- peakCutoff(cov.ctcf, fdr = 0.0001) ## ----------------------------------------------------------------------------- peaks.ctcf <- slice(cov.ctcf, lower = 8) peaks.ctcf ## ----peakSummary-------------------------------------------------------------- peaks <- peakSummary(peaks.ctcf) ## ----------------------------------------------------------------------------- peak.depths <- viewMaxs(peaks.ctcf) cov.pos <- coverage(ctcf.ext[strand(ctcf.ext) == "+"]) cov.neg <- coverage(ctcf.ext[strand(ctcf.ext) == "-"]) peaks.pos <- Views(cov.pos, ranges(peaks.ctcf)) peaks.neg <- Views(cov.neg, ranges(peaks.ctcf)) wpeaks <- tail(order(peak.depths$chr10), 4) wpeaks ## ----fig.height=5------------------------------------------------------------- coverageplot(peaks.pos$chr10[wpeaks[1]], peaks.neg$chr10[wpeaks[1]]) ## ----fig.height=5------------------------------------------------------------- coverageplot(peaks.pos$chr10[wpeaks[2]], peaks.neg$chr10[wpeaks[2]]) ## ----fig.height=5------------------------------------------------------------- coverageplot(peaks.pos$chr10[wpeaks[3]], peaks.neg$chr10[wpeaks[3]]) ## ----fig.height=5------------------------------------------------------------- coverageplot(peaks.pos$chr10[wpeaks[4]], peaks.neg$chr10[wpeaks[4]]) ## ----------------------------------------------------------------------------- ## find peaks for GFP control cov.gfp <- coverage(resize(cstest$gfp, 200)) peaks.gfp <- slice(cov.gfp, lower = 8) peakSummary <- diffPeakSummary(peaks.gfp, peaks.ctcf) plt <- xyplot(asinh(sums2) ~ asinh(sums1) | seqnames, data = as.data.frame(peakSummary), panel = function(x, y, ...) { panel.xyplot(x, y, ...) panel.abline(median(y - x), 1) }, type = c("p", "g"), alpha = 0.5, aspect = "iso") ## ----fig.height=5, echo=FALSE------------------------------------------------- plt ## ----------------------------------------------------------------------------- mcols(peakSummary) <- within(mcols(peakSummary), { diffs <- asinh(sums2) - asinh(sums1) resids <- (diffs - median(diffs)) / mad(diffs) up <- resids > 2 down <- resids < -2 change <- ifelse(up, "up", ifelse(down, "down", "flat")) }) ## ----------------------------------------------------------------------------- library(TxDb.Mmusculus.UCSC.mm9.knownGene) gregions <- transcripts(TxDb.Mmusculus.UCSC.mm9.knownGene) gregions ## ----------------------------------------------------------------------------- promoters <- flank(gregions, 1000, both = TRUE) ## ----------------------------------------------------------------------------- peakSummary$inPromoter <- peakSummary %over% promoters xtabs(~ inPromoter + change, peakSummary) ## ----------------------------------------------------------------------------- peakSummary$inUpstream <- peakSummary %over% flank(gregions, 20000) peakSummary$inGene <- peakSummary %over% gregions ## ----------------------------------------------------------------------------- sumtab <- as.data.frame(rbind(total = xtabs(~ change, peakSummary), promoter = xtabs(~ change, subset(peakSummary, inPromoter)), upstream = xtabs(~ change, subset(peakSummary, inUpstream)), gene = xtabs(~ change, subset(peakSummary, inGene)))) ## cbind(sumtab, ratio = round(sumtab[, "down"] / sumtab[, "up"], 3)) ## ----rtracklayer-upload, eval=FALSE------------------------------------------- # library(rtracklayer) # session <- browserSession() # genome(session) <- "mm9" # session$gfpCov <- cov.gfp # session$gfpPeaks <- peaks.gfp # session$ctcfCov <- cov.ctcf # session$ctcfPeaks <- peaks.ctcf ## ----rtracklayer-view, eval=FALSE--------------------------------------------- # peak.ord <- order(unlist(peak.depths), decreasing=TRUE) # peak.sort <- as(peaks.ctcf, "GRanges")[peak.ord] # view <- browserView(session, peak.sort[1], full = c("gfpCov", "ctcfCov")) ## ----tracklayer-view-5, eval=FALSE-------------------------------------------- # views <- browserView(session, head(peak.sort, 5), full = c("gfpCov", "ctcfCov")) ## ----------------------------------------------------------------------------- sessionInfo()