62-testing.Rmd

# Hypothesis testing {#sec-testing}

The goal of this chapter is to demonstrate some of the fundamentals of
hypothesis testing as used in bioinformatics. For prerequisites within
the Biomedical sciences masters degree at the UCLouvain, see
*WFARM1247* (Traitement statistique des données).

Parts of this chapter are based on chapter 6 from *Modern Statistics
for Modern Biology* [@MSMB].

## Refresher

```{r toss, echo = FALSE}
set.seed(2)
n <- 100
p <- 0.59
flips <- sample(c("H", "T"), size = n,
                replace = TRUE,
                prob = c(p, 1 - p))
```

We have flipped a coing 100 times and have obtained the following results.

```{r tossres, echo = FALSE}
table(flips)
```

If the coin was unbiased we expect roughly 50 heads. Can we make any
claims regarding the biaised or unbiaised nature of that coin?

A coin toss can be modeled by a binomial distribution. The histogram
below shows the binomial statistic for 0 to 100 heads; it represents
the binomial density of an unbiased coin. The vertical line shows the
number of head observed.


```{r tosshist, echo = FALSE, fig.cap = "Binomial density of an unbiased coin to get 0 to 100 heads. The full area of the histogram sums to 1."}
num_heads <- sum(flips == "H")
binomial_dens <-
    tibble(k = 0:n) %>%
    mutate(p = dbinom(k, size = n, prob = 0.5))

ggplot(binomial_dens, aes(x = k, y = p)) +
    geom_bar(stat = "identity") +
    geom_vline(xintercept = num_heads)
```

Above, we see that the most likely outcome would be 50 heads with a
probability of `r max(binomial_dens$p)`. No head and 100 heads have a
probability of $`r binomial_dens$p[binomial_dens$k == 0]`$.


We set

- $H_0$: the coin is fair
- $H_1$: the coin is baised

If `r num_heads` isn't deemed too *extreme*, then we won't reject
$H_0$ and conclude that the coin in fair. If `r num_heads` is deemed
too extreme, then we reject $H_0$ and accept $H_1$, and conclude that
the coin is baised.

To define *extreme*, we set $\alpha = 0.05$ and rejet $H_0$ if our result is
outside of the 95% most probably values.

```{r tossstat, echo = FALSE}
alpha <- 0.05
binomial_dens <-
    arrange(binomial_dens, p) %>%
    mutate(reject = (cumsum(p) <= alpha))
```

```{r tosshist2, echo = FALSE, fig.cap = "Binomial density of for an unbiased coin to get 0 to 100 heads. The areas in red sum to 0.05."}
ggplot(binomial_dens) +
    geom_bar(aes(x = k, y = p, fill = reject), stat = "identity") +
    scale_fill_manual(
        values = c(`TRUE` = "red", `FALSE` = "#5a5a5a")) +
    geom_vline(xintercept = num_heads, col = "blue") +
    theme(legend.position = "none")
```

We can also compute the p-value, the tells us how likely we are to see
such a extreme or more extreme value under $H_0$.

```{r binom_test}
binom.test(x = 62, n = 100, p = 0.5)
```

Whenever we make such a decision, we will be in one of the following situations:

|                     | $H_0$ is true           | $H_1$ is true            |
|---------------------|-------------------------|--------------------------|
| Reject $H_0$        | Type I (false positive) | True positive            |
| Do not reject $H_0$ | True negative           | Type II (false negative) |


See below for a step by step guide to this example.

## A biological example

A typical biological example consists in measuring a gene of interest
in two populations of interest (say control and group), represented by
biological replicates. The figure below represents the distribution of
the gene of interest in the two populations, with the expression
intensities of triplicates in each population.

```{r exdists, echo = FALSE, fig.cap = "Expression of a gene in two populations with randomly chosen triplicates."}
set.seed(123)
s1 <- rnorm(3, 6, 1.2)
s2 <- rnorm(3, 11, 1.5)
tibble(groups = rep(c("control", "group"), each = 100),
       expression = c(rnorm(100, 6, 1.2), rnorm(100, 11, 1.5))) %>%
    ggplot(aes(x = expression, fill = groups, colour = groups)) +
    geom_density(alpha = 0.5) +
    geom_vline(xintercept = s1, colour = "#F8766D") +
    geom_vline(xintercept = s2, colour = "#00BFC4")
```

We don't have access to the whole population and thus use a sample
thereof (the replicated measurements) to estimate the population
parameters. We have

```{r extab, echo = FALSE}
df <- data.frame(control = s1, group = s2)
df <- rbind(df,
            c(mean(s1), mean(s2)),
            c(sd(s1), sd(s2)))
rownames(df) <- c(paste("rep", 1:3, sep = "."),
                  "mean", "sd")
knitr::kable(df)
```

We set our hypothesis as

- $H_0$: the means of the two groups are the same, $\mu_{1} = \mu_{2}$.
- $H_1$: the means of the two groups are different, $\mu_{1} \neq \mu_{2}$.

and calculate a two-sided, two-sample t-test (assuming unequal
variances) with

$$
t = \frac{ \bar X_{1} - \bar X_{2} }
         { \sqrt{ \frac{ s_{1}^{2} }{ N_{1} } + \frac{ s_{2}^{2} }{ N_{2} } } }
$$


where $\bar X_i$, $s_{i}^{2}$ and $N_i$ are the mean, variance and
size of samples 1 and 2.


A t-test has the following assumptions:

- the data are normally distributed;
- the data are independent and identically distributed;
- equal or un-equal (Welch test) variances.

Note that the t-test is robust to deviations[^models].

[^models]: All models are wrong. Some are useful.


In R, we do this with

```{r}
s1
s2
t.test(s1, s2)
```


`r msmbstyle::question_begin()`

Note that this result, this p-value, is specific to our sample, the
measured triplicates, that are assumed to be representative of the
population. Can you imagine other sets of triplicates that are
compatible with the population distributions above, but that could
lead to non-significant results?

`r msmbstyle::question_end()`


In practice, we would apply moderated versions of the tests, such as
the one provided in the `r Biocpkg("limma")` package and also widely
applied to RNA-Seq count data.



## A more realistic biological example

Let's now use the `tdata1` dataset from the `rWSBIM1322` package that
provide gene expression data for 100 genes and 6 samples, three in
group A and 3 in group B.

```{r}
library("rWSBIM1322")
data(tdata1)
head(tdata1)
```

`r msmbstyle::question_begin()`
Visualise the distribution of the `tdata1` data and, if necessary,
log-transform it.
`r msmbstyle::question_end()`

`r msmbstyle::solution_begin()`

```{r tex1}
log_tdata1 <- log2(tdata1)
par(mfrow = c(1, 2))
limma::plotDensities(tdata1)
limma::plotDensities(log_tdata1)
```

`r msmbstyle::solution_end()`

We are now going to apply a t-test to feature (row) 73, comparing the
expression intensities in groups A and B. As we have seen, this can be
done with the `t.test` function:


```{r}
x <- log_tdata1[73, ]
t.test(x[1:3], x[4:6])
```

`r msmbstyle::question_begin()`
- Interpret the results of the test above.
- Repeat it with another features.
`r msmbstyle::question_end()`


`r msmbstyle::question_begin()`
We would now like to repeat the same analysis on the 100 genes.

- Write a function that will take a vector as input and perform a
  t-test of the first values (our group A) against the 3 last values
  (our group B) and returns the p-values.

- Apply the test to all the genes.

- How many significantly differentically expressed genes do you find?
  What features are of possible biological interest?

`r msmbstyle::question_end()`


`r msmbstyle::solution_begin()`
```{r}
my_t_test <- function(x) {
    t.test(x[1:3], x[4:6])$p.value
}
pvals <- apply(log_tdata1, 1, my_t_test)
table(pvals < 0.05)
head(sort(pvals))
```
`r msmbstyle::solution_end()`


`r msmbstyle::question_begin()`
The data above have been generated with the `rnorm` function for all
samples.

- Do you still think any of the features show significant differences?

- Why are there still some features (around 5%) that show a
  significant p-value at an alpha of 0.05?

`r msmbstyle::question_end()`


To answer these questions, let's refer to [this xkcd
cartoon](https://xkcd.com/882/) that depicts scientists testing
whether eating jelly beans causes acne.


```{r, results='markup', fig.cap="Do jelly beans cause acne? Scientists investigate. From [xkcd](https://xkcd.com/882/).", echo=FALSE, purl=FALSE, fig.align='center'}
knitr::include_graphics("./figs/jellybeans.png")
```

The data that was used to calculate the p-values was all drawn from the same distribution $N(10, 2)$.

As a result, we should not expect to find a statistically significant
result, unless we repeat the test enought times. Enough here depends
on the $\alpha$ we set to control the type I error. If we set $\alpha$
to 0.05, we accept that rejecting $H_{0}$
in 5% of the extreme cases where we shouldn't reject it. This is an
acceptable threshold that however doesn't hold when we repeat the test
many time.


An important visualistion when performing statistical test repeatedly,
is to visualise the distribution of computed p-values. Below, we see
the histogram of the `tdata1` data and 100 values drawn from a uniform
distribution between 0 and 1. Both are very similar; they are flat.


```{r pvalhist, fig.cap = "Distribution of p-values for the `tdata1` dataset (left) and 100 (p-)values uniformely distributed between 0 and 1 (right)."}
par(mfrow = c(1, 2))
hist(pvals)
hist(runif(100))
```

Below we see the expected trends of p-values for different
scenarios. This example comes from the [Variance
explained](http://varianceexplained.org/statistics/interpreting-pvalue-histogram/).

```{r pvalhists2, results='markup', fig.cap="Expected trends of p-values for different scenarios ([source](http://varianceexplained.org/statistics/interpreting-pvalue-histogram/)).", echo=FALSE, purl=FALSE, fig.align='center', out.width='100%', fig.align='center'}
knitr::include_graphics("./figs/plot_melted-1.png")
```

In an experiment with enough truly differentially expression features,
on expects to observe a substantial increase of small p-values
(anti-conservative). In other words, we expect to see more small
p-values that at random, or when no statistically significant are
present (uniform). All other scenarios warrant further inspection, as
they might point to issue with the data of the tests.

When many tests are preformed, the p-values need to be *adjusted*, to
take into account that many tests have been performed.

## Adjustment for multiple testing

There are two classes of multiple testing adjustment methods:

- **Family-wise error rate** (FWER) that gives the probability of one
  or more false positives. The **Bonferroni correction** for *m* tests
  multiplies each p-value by *m*. One then checks if any results still
  remains below significance threshold.

- **False discovery rate** (FDR) that computes the expected fraction
  of false positives among all discoveries. It allows us to choose *n*
  results with a given FDR. Widely used examples are
  Benjamini-Hochberg or q-values.


The figure below illustrates the principle behind the FDR
adjustment. The procedure estimate the proportion of hypothesis that
are null and then adjust the p-values accordingly.

```{r fdrhist, results='markup', fig.cap="Principle behind the false discovery rate p-value adjustment ([source](http://varianceexplained.org/statistics/interpreting-pvalue-histogram/)).", echo=FALSE, purl=FALSE, fig.align='center', out.width='100%', fig.align='center'}
knitr::include_graphics("./figs/fdr.png")
```

Back to our `tdata1` example, we need to take this into account and
adjust the p-values for multiple testing. Below we apply the
Benjamini-Hochberg FDR procedure using the `p.adjust` function and
confirm that none of the features are differentially expressed.

```{r}
adj_pvals <- p.adjust(pvals, method = "BH")
```

`r msmbstyle::question_begin()`
Are there any adjusted p-values that are still significant?
`r msmbstyle::question_end()`


`r msmbstyle::solution_begin()`
```{r}
any(adj_pvals < 0.05)
min(adj_pvals)
```
`r msmbstyle::solution_end()`


## Result visualisation

`r msmbstyle::question_begin()`
The `tdata4` dataset is already log2 and can be processed as
it is. Practice what we have see so far and identify differential
expressed genes in group A vs B.
`r msmbstyle::question_end()`


`r msmbstyle::solution_begin()`
```{r tdata4ex1}
library("rWSBIM1322")
data(tdata4)

my_t_test <- function(x)
    t.test(x[1:3], x[4:6])$p.value

pvals <- apply(tdata4, 1, my_t_test)
hist(pvals)
table(pvals < 0.05)

adjp <- p.adjust(pvals, method = "BH")
head(sort(adjp))
```
`r msmbstyle::solution_end()`


`r msmbstyle::question_begin()`
Calculate a log2 fold-change between groups A and B. To do so, you
need to subtract mean expression of group B from mean of group
A[^l2fc]. Repeat this for all genes. Visualise and interpret the distribution
of the log2 fold-changes.


[^l2fc]: We need to subtract the means because the data are already log-transformed and $log\frac{a}{b} = log(a) - log(c)$.

`r msmbstyle::question_end()`


`r msmbstyle::solution_begin()`
```{r tdata4ex2}
my_lfc <- function(x)
    mean(x[1:3]) - mean(x[4:6])
lfc <- apply(tdata4, 1, my_lfc)
hist(lfc)

```
`r msmbstyle::solution_end()`


We generally want to consider both the statistical significance (the
p-value) and the magnitude of the difference of expression (the
fold-change) to provide a biological interpretation of the data. For
this, we use a *volcano plot* as shown below. The most interesting
features are those towards to top corners given that they have a small
p-value (i.e. a large value for *-log10(p-value)*) and large (in
absolute value) fold-changes.


```{r volc, echo = FALSE, fig.cap = "A volcano plot."}
plot(lfc, -log10(adjp))
grid()
abline(h = -log10(0.05))
abline(v = c(-1, 1))
```

`r msmbstyle::question_begin()`
Reproduce the volcano plot above using the `adjp` and `lfc` variables
computed above.
`r msmbstyle::question_end()`


`r msmbstyle::solution_begin()`
```{r volcex}
plot(lfc, -log10(adjp))
grid()
abline(h = -log10(0.05))
abline(v = c(-1, 1))
```
`r msmbstyle::solution_end()`


[Chapter 6 on
*testing*](https://www.huber.embl.de/msmb/Chap-Testing.html) of
*Modern Statistics for Modern Biology* [@MSMB], and in particlar
section 6.5, provides additional details about the t-test.

The t-test comes in multiple flavors, all of which can be chosen
through parameters of the `t.test` function. What we did above is
called a two-sided two-sample unpaired test with unequal
variance. **Two-sided** refers to the fact that we were open to reject
the null hypothesis if the expression in the first group was either
larger or smaller than that in the second one.

**Two-sample** indicates that we compared the means of two groups to
each other; another option is to compare the mean of one group against
a given, fixed number.

**Unpaired** means that there was no direct 1:1 mapping between the
measurements in the two groups. If, on the other hand, the data had
been measured on the same patients before and after treatment, then a
paired test would be more appropriate, as it looks at the change of
expression within each patient, rather than their absolute expression.

**Equal variance** refers to the way the statistic is calculated. That
expression is most appropriate if the variances within each group are
about the same. If they are very different, an alternative form,
called the Welch t-test exist.

An important assumption when performing a t-test is the **independence
assumption** among the observations in a sample. An additional exercise
below demonstrate the risk of dependence between samples.


Finally, it is important to highlight that all the methods and
concepts described above will only be relevant if the problem is
properly stated. Hence the importance of clearly laying out the
**experimental design** and stating the **biological question** of
interest before running experiment. Indeed, quoting the mathematician
Richard Hamming:

> It is better to solve the right problem the wrong way than to solve
> the wrong problem the right way.

To this effect, a [type III
error](https://en.wikipedia.org/wiki/Type_III_error) has been defined
as errors that occur when researchers provide the right answer to the wrong
question.


<!-- ## Linear regression -->

<!-- Suggested TODO  -->

<!-- ## Empirical approximations  -->
<!-- - Bootstrapping to generate null distributions -->


## A word of caution

Summary of statistical inference:

1. Set up a model of reality: null hypothesis $H_0$ (no difference) and *alternative hypothesis*
   $H_1$ (there is a difference).

2. Do an experiment to collect data.

3. Perform the statistical inference with the collected data.

4. Make a decision: reject $H_0$ if the computed probability is deemed too small.

What not to do:

### p-hacking {-}

Instead of setting up one statistical hypothesis before data
collection and then testing it, collect data at large, then try lots
of different tests until one give a statistically significant result
[@Head:2015].

### p-harking {-}

HARKing (Hypothesizing After the Results are Known) is the fraudulous
practice of presenting a post hoc hypothesis, i.e., one based on prior
result, as a priori hypotheses [@Kerr:1998].

## Additional exercises


`r msmbstyle::question_begin()`

- Draw three values from two distributions $N(6, 2)$ and $N(8, 2)$, perform a t-test and interpret the results.

- Repeat the above experiment multiple times. Do you get *similar* results? Why?

- Repeat with values from distributions $N(5, 1)$ and $N(8, 1)$.

`r msmbstyle::question_end()`

```{r, include = FALSE}
s1 <- rnorm(3, 6, 2)
s2 <- rnorm(3, 8, 2)
t.test(s1, s2)

s1 <- rnorm(3, 5, 1)
s2 <- rnorm(3, 8, 1)
t.test(s1, s2)
```

`r msmbstyle::question_begin()`
- Generate random data using `rnorm` for 100 genes and 6 samples and
  test for differential expression, comparing for each gene the 3
  first samples against the 3 last samples. Verify that you identify
  about 5 p-values smaller that 0.05. Visualise and interprete the
  histogram of these p-values.

- Adjust the 100 p-values for multiple testing and compare the initial
  and adjusted p-values.
`r msmbstyle::question_end()`

`r msmbstyle::question_begin()`
We have used two-sided two-sample t-tests above. Also familiarise
yourselves with one-sided, one-sample and paired tests. In particular,
verify how to implement these with the `t.test` function.
`r msmbstyle::question_end()`


`r msmbstyle::question_begin()`

Simulate a dataset of log2 fold-changes measured in triplicate for
1000 genes.

- What function would you use to generate these data?

- What test would you use to test for differential expression? Apply
  it to calculate 1000 p-values.

- Visualise and interpret the histogram of p-values.

- FDR-adjust the p-values.

`r msmbstyle::question_end()`



`r msmbstyle::question_begin()`

Load the `tdata2` dataset from the `rWSBIM1322` package. These data
represent the measurement of an inflammation biomarker in the blood of
15 patients, before and after treatment for a acute liver
inflammation.  Visualise the data and run a test to verify if the
treatment has had an effect or not.

`r msmbstyle::question_end()`

```{r, include = FALSE}
library("rWSBIM1322")
data(tdata2)
tdata2$patient <- 1:15

library("tidyverse")
pivot_longer(tdata2,
             names_to = "time",
             values_to = "biomarker",
             c(before, after)) %>%
    ggplot(aes(x = factor(time, levels = c("before", "after")),
               y = biomarker)) +
    geom_boxplot() +
    geom_point(aes(colour = factor(patient)), size = 3) +
    geom_segment(data = tdata2,
                 aes(x = 1, xend = 2,
                     y = before, yend = after,
                     colour = factor(patient)))

t.test(tdata2$before, tdata2$after, paired = TRUE)
```

`r msmbstyle::question_begin()`

Load the `tdata3` data from the `rWSBIM1322` package (version >=
0.1.5). 100 genes have been measures in 2 groups (A and B) in
triplicates.

- Perform a t-test, visualise the p-value on a histogram. Do you
  expect to see any genes significantly differentially expressed?
  Verify your expectation by adjusting the p-values for multiple
  comparisions. Are there any significant results after adjustment?

- Produce a volcano plot for these results and interpret what you see.

- Unless you haven't already done so, visualise the distributions of
  the genes for the 6 samples. What do you observe? Re-interpret the
  results obtained above in the light of these findings.

- Normalise your data using centring. Visualise the distributions
  before and after transformation and make sure you understand the
  differences.

- Repeat the first point above on the normalised data. Do you still
  find any differentially expressed genes? Explain why.

`r msmbstyle::question_end()`

```{r, include = FALSE}
library("rWSBIM1322")
data(tdata3)

my_t_test <- function(x) t.test(x[1:3], x[4:6])$p.value

pvals1 <- apply(tdata3, 1, my_t_test)
hist(pvals1)
## Anti-conservative distribution, hence expecting significant result(s).
pvals1_adj <- p.adjust(pvals1, method = "BH")
head(sort(pvals1_adj))

my_lfc <- function(x) mean(x[1:3]) - mean(x[4:6])
lfc <- apply(tdata3, 1, my_lfc)
plot(lfc, -log10(pvals1_adj))
grid()

tdata3_cent <- scale(tdata3, scale = FALSE, center = TRUE)
par(mfrow = c(1, 2))
boxplot(tdata3)
boxplot(tdata3_cent)
## Systematic effect in group B. Cancelled out by centring.

pvals2 <- apply(tdata3_cent, 1, my_t_test)
hist(pvals2)
## Uniform distribution, expecting no significant results.
head(sort(p.adjust(pvals2, method = "BH")))
```


`r msmbstyle::question_begin()`
- Load the `cptac_se_prot` data and extract the first feature,
  `P00918ups|CAH2_HUMAN_UPS`, a protein in this case. Perform a
  t-test between the groups 6A and 6B.

- Duplicate the data of that first feature to obtain a vector of 12
  values and repeat the t-test (now 6 against 6) above. Interpret the
  new results.


`r msmbstyle::question_end()`


```{r, include = FALSE}
library("SummarizedExperiment")
data(cptac_se_prot)
x1 <- assay(cptac_se_prot)["P00918ups|CAH2_HUMAN_UPS", 1:3]
x2 <- assay(cptac_se_prot)["P00918ups|CAH2_HUMAN_UPS", 4:6]

t.test(x1, x2)
t.test(rep(x1, 2), rep(x2, 2))
## - The power of the t-test depends on the sample size. Even if the
##   underlying biological differences are the same, a dataset with
##   more observations tends to give more significant results. This can
##   also be seen from the way the numbers N1 and N2 appear in the t-test
##   formula.
## - The assumption of independence between the measurements is really
##   important, and in the case above, the duplicated data are clearly
##   dependent.
```

### The coin example, step by step {-}

The following code chunk simulates 100 coin toss from a biased coin.

```{r}
set.seed(2)
n <- 100
p <- 0.59
flips <- sample(c("H", "T"), size = n,
                replace = TRUE,
                prob = c(p, 1 - p))
```

If the coin were unbiased we expect roughly 50 heads. Let us see how
many heads and tails there are.

```{r}
table(flips)
```

We calculate the binomial statistic for a number of flips between 0
and 100. This is the binomial density for an unbiased coin.

```{r}
library("dplyr")
num_heads <- sum(flips == "H")
binomial_dens <-
    tibble(k = 0:n) %>%
    mutate(p = dbinom(k, size = n, prob = 0.5))
```

`r msmbstyle::question_begin()`
- Write some code to check that the probabilties from the binomial
  statistic sum to $1$.

- Change the `prob` argument and show that the probabilities still sum
  to $1$.
`r msmbstyle::question_end()`

The following code chunk plots the binomial statistic and the number
of heads observed is marked in blue.


```{r,}
library("ggplot2")
ggplot(binomial_dens, aes(x = k, y = p)) +
    geom_bar(stat = "identity") +
    geom_vline(xintercept = num_heads)
```

`r msmbstyle::question_begin()`
- Change the prob argument above and re-plot the binomial statistic,
  what do you notice about how the distribution is centered?
`r msmbstyle::question_end()`

Now, we set the size of the reject threshold, this is a choice and
corresponds to how many false discoveries we are happy to allow.

```{r}
alpha <- 0.05
```

`r msmbstyle::question_begin()`
- Without looking below use the arrange function from `dplyr` to order
  the probabilities, smallest first.

- Looking at the output, what is the most unlikely number of heads to
  observe?

- Looking at the output, what is the most likely number of heads to
  observe?
`r msmbstyle::question_end()`

`r msmbstyle::solution_begin()`

```{r}
binomial_dens %>%
    filter(p == max(p))

binomial_dens %>%
    filter(p == min(p))
```

`r msmbstyle::solution_end()`


`r msmbstyle::question_begin()`

- What does the following code chunk do?


```{r}
binomial_dens <-
    arrange(binomial_dens, p) %>%
    mutate(reject = (cumsum(p) <= alpha))

```
`r msmbstyle::question_end()`


Let us plot the reject region in red

```{r}
ggplot(binomial_dens) +
    geom_bar(aes(x = k, y = p, fill = reject), stat = "identity") +
    scale_fill_manual(
        values = c(`TRUE` = "red", `FALSE` = "#5a5a5a")) +
    geom_vline(xintercept = num_heads, col = "blue") +
    theme(legend.position = "none")
```

`r msmbstyle::question_begin()`
- Is there evidence that our coin is biased?
- Change the size of the reject region to a smaller value, what is our
  conclusion now?
`r msmbstyle::question_end()`


The above test already has an easy to use function in R:

```{r}
binom.test(x = num_heads, n = n, p = 0.5)
```