1 Conventional Profiles

As an example, we’ll plot PRO-seq signal in promoter-proximal regions by calculating the mean signal intensity at each base in the first 100 bases of a transcript.

First, calculate signal at each base for all promoter-proximal regions:

txs_pr <- promoters(txs_dm6_chr4, 0, 100)
countmatrix_pr <- getCountsByPositions(PROseq, txs_pr, ncores = 1)
## [1] 339 100
dim(countmatrix_pr) == c(length(txs_pr), unique(width(txs_pr)))
## [1] TRUE TRUE

For each position (each column of the matrix), calculate the mean, and plot:

plot(x = 1:ncol(countmatrix_pr),
     y = colMeans(countmatrix_pr),
     type = "l", 
     xlab = "Distance to TSS (bp)",
     ylab = "Mean PRO-seq Reads")


1.1 Drawbacks

One drawback of using the arithmetic mean is that means are not robust to outliers. In other words, the mean signal at any position is liable to be determined by a small number of highly influential points. This is especially problematic for high dynamic range data like PRO-seq.

It’s common to see this issue addressed through the use medians/quantiles in place of arithmetic means. However, observe what happens as we plot the median signal across our gene list using several different gene-filtering thresholds:

plot_meds <- function(sig_thresh) {
  idx <- which(rowSums(countmatrix_pr) > sig_thresh)
  plot(x = 1:ncol(countmatrix_pr),
       y = apply(countmatrix_pr[idx, ], 2, median),
       type = "l", 
       main = sprintf("Regions with >%s reads", sig_thresh),
       xlab = "Distance to TSS (bp)",
       ylab = "Median PRO-seq Reads")

par(mfrow = c(3, 2))

for (i in c(0, 30*2^(0:4))) {

The paucity of transcription on Drosophila chromosome 4 makes this a somewhat extreme example, and you might find that the above situation is a non-issue