BRGenomics 1.12.0

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:

```
library(BRGenomics)
data("PROseq")
data("txs_dm6_chr4")
txs_pr <- promoters(txs_dm6_chr4, 0, 100)
```

```
countmatrix_pr <- getCountsByPositions(PROseq, txs_pr, ncores = 1)
dim(countmatrix_pr)
## [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")
```

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))) {
plot_meds(i)
}
```

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 for your data. However, it’s important to keep in mind how unexpressed genes can affect the median. A common rule of thumb is that a given cell is only apt to express around half of its genes; if that holds true, the median of an unfiltered genelist will be close to zero, and could be insensitive to changes occurring at expressed genes.

A robust alternative to plotting mean or median signal profiles is to plot
**bootstrapped** mean signal profiles, known as metaprofile plots or metaplots.

To bootstrap the mean signal by position, a small number of genes are randomly sampled from a genelist, and the mean signal at each position is calculated for that group of genes. This process of randomly sampling genes and calculating mean signals is repeated over many iterations, and the final bootstrapped mean for each position is the median of the sampled means.

One feature of bootstrapping is that it is robust to outliers. But more than
that, the a bootstrapped mean provides an *expectation* for what the mean signal
would be for any arbitrary group of genes, and associated with that expectation
is a measure of uncertainty. By taking quantiles of the sub-sampled means *other
than* the median (the expected value), we can estimate the extent to which the
mean varies across arbitrary groups of genes.

For example, the 75th percentile of the sub-sampled means is a number for which, 25% of the time, we calculated a higher mean. Similarly, a 90% confidence interval about the bootstrapped mean encompasses all values between the 5th and 95th percentiles of the sub-sampled means.

We can use the `metaSubsample()`

function to bootstrap mean values by position
for a genelist. The function accepts the same arguments as
`getCountsByPositions()`

, in addition to other arguments related to the
bootstrapping.

By default, 10% of the genelist is randomly sampled 1000 times, and confidence bands are returned for the 12.5th and 87.5th percentiles (i.e. a 75% confidence interval).

Given the small size our dataset, we’ll reduce this to a 30% confidence interval, and we’ll additionally use 5 bp bins:

```
bootmeans.df <- metaSubsample(PROseq, txs_pr, binsize = 5,
lower = 0.35, upper = 0.65, ncores = 1)
head(bootmeans.df)
```

```
## x mean lower upper sample.name
## 1 3 0.03823529 0.02941176 0.05294118 PROseq
## 2 8 0.05294118 0.04117647 0.07058824 PROseq
## 3 13 0.07647059 0.05294118 0.10000000 PROseq
## 4 18 0.07058824 0.05088235 0.09411765 PROseq
## 5 23 0.12941176 0.10000000 0.16470588 PROseq
## 6 28 0.09411765 0.08235294 0.10794118 PROseq
```

A dataframe is returned for plotting, and notice how the x-values have been automatically adjusted to be the center of the bins.

Below, we show how to plot the confidence bands using base R plotting, as well
as `ggplot2`

.

```
plot(mean ~ x, data = bootmeans.df, type = "l",
main = "PRO-seq Signal", ylim = c(0, 1.4),
xlab = "Distance from TSS",
ylab = "Mean Signal + 30% CI")
# draw a polygon to add confidence bands,
# and use adjustcolor() to add transparency
polygon(c(bootmeans.df$x, rev(bootmeans.df$x)),
c(bootmeans.df$lower, rev(bootmeans.df$upper)),
col = adjustcolor("black", 0.1), border = FALSE)
```

```
require(ggplot2)
ggplot(bootmeans.df, aes(x, mean)) +
geom_line() +
geom_ribbon(aes(x, ymin = lower, ymax = upper),
alpha = 0.1) +
labs(title = "PRO-seq Signal",
x = "Distance from TSS",
y = "Mean Signal + 30% CI") +
theme_bw()
```