05-statisticalInference-twosampleT.Rmd

---
title: "5. Statistical Inference: Two-sample t-test"
author: "Lieven Clement"
date: "statOmics, Ghent University (https://statomics.github.io)"
---

<a rel="license" href="https://creativecommons.org/licenses/by-nc-sa/4.0"><img alt="Creative Commons License" style="border-width:0" src="https://i.creativecommons.org/l/by-nc-sa/4.0/88x31.png" /></a>

```{r setup, include=FALSE, cache=FALSE}
knitr::opts_chunk$set(
  include = TRUE, comment = NA, echo = TRUE,
  message = FALSE, warning = FALSE, cache = TRUE
)
library(tidyverse)
```

<iframe width="560" height="315" src="https://www.youtube.com/embed/gghS0czPKGY?si=VsPkue-f7GgVeILc" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" referrerpolicy="strict-origin-when-cross-origin" allowfullscreen></iframe>

# Smelly armpit example

- Smelly armpits are not caused by sweat, itself. The smell is caused by specific micro-organisms belonging to the group of *Corynebacterium spp.* that metabolise sweat.
Another group of abundant bacteria are the *Staphylococcus spp.*, these bacteria do not metabolise sweat in smelly compounds.

- The CMET-group at Ghent University does research to on transplanting the armpit microbiome to save people with smelly armpits.

- Proposed Therapy:
  	1. Remove armpit-microbiome with antibiotics
    2. Influence armpit microbiome with microbial  transplant (https://youtu.be/9RIFyqLXdVw)

- Experiment:

    - 20 subjects with smelly armpits are attributed to one of two treatment groups
    - placebo (only antibiotics)
    - transplant (antibiotica followed by microbial transplant).
    - The microbiome is sampled 6 weeks upon the treatment
    - The relative abundance of *Staphylococcus spp.* on *Corynebacterium spp.* + *Staphylococcus spp.* in the microbiome is measured via DGGE (*Denaturing Gradient Gel Electrophoresis*).

---

## Import the data

```{r}
ap <- read_csv("https://raw.githubusercontent.com/GTPB/PSLS20/master/data/armpit.csv")
ap
```

## Data exploration

We plot the direct relative abundances in function of the treatment group. With the ggplot2 library we can easily build plots by adding layers.

```{r}
ap %>% ggplot(aes(x = trt, y = rel)) +
  geom_boxplot(outlier.shape = NA) +
  geom_point(position = "jitter")

ap %>% ggplot(aes(sample = rel)) +
  geom_qq() +
  geom_qq_line() +
  facet_wrap(~trt)
```

---

# Two sample T-test
## Notation

Suppose that $Y_{ij}$ is the response for subjects $i=1,\ldots, n_j$ from population $j=1,2$.

Use of the term **treatment** or **group** instead of population

Here the treatment is $j=1$ microbial transplant vs $j=2$ placebo.

We assume

$$Y_{ij}\text{ i.i.d. } N(\mu_j,\sigma^2)\;\;\;i=1,\ldots,n_i\;j=1,2.$$

Note, that we assume equal variances **homoscedastic**

(Unequal variances are referred to as  **heteroscedastic**)

---

## Hypotheses

Test $$ H_0: \mu_1 = \mu_2 $$
against
$$  H_1: \mu_1 \neq \mu_2 .$$

$H_1$ is again the research hypothesis: the average relative abundance of *Staphylococcus spp.* is different upon microbial transplant then upon placebo treatment.

$H_0$ and $H_1$ can also be specified in terms of the effect size between the two treatments, $\mu_1-\mu_2$
$$H_0: \mu_1-\mu_2 = 0,$$
$$H_1: \mu_1-\mu_2 \neq 0.$$

We can estimate the effect size using the difference in sample means:
$$\hat \mu_1-\hat \mu_2=\bar Y_1 -\bar Y_2.$$

---

## Variance estimator

The experimental units are independent so the sample means are also independent and the variance on the difference is
$$\text{Var}_{\bar Y_1 -\bar Y_2}=\frac{\sigma^2}{n_1}+\frac{\sigma^2}{n_2}=\sigma^2 \left(\frac{1}{n_1}+\frac{1}{n_2}\right).$$

And the standard error becomes
$$\sigma_{\bar Y_1 -\bar Y_2}=\sigma\sqrt{\frac{1}{n_1}+\frac{1}{n_2}}.$$

The variance can be estimated within each group using the sample variance:

$$S_1^2 = \frac{1}{n_1-1}\sum_{i=1}^{n_1} (Y_{i1}-\bar{Y}_1)^2.$$

$$S_2^2 = \frac{1}{n_2-1}\sum_{i=1}^{n_2} (Y_{i2}-\bar{Y}_2)^2.$$

But, if we assume equal variances $\sigma_1^2=\sigma_2^2=\sigma^2$ than we can estimate the variance more precise by using all observations in both groups. This variance estimator is also referred to as the *pooled variance estimator: $S^2_p$*.

So $S_1^2$ en $S_2^2$ are estimators of the same parameter $\sigma^2$.

And we can combine them into one estimator based on all $n_1+n_2$ observations:

$$  S_p^2 = \frac{n_1-1}{n_1+n_2-2} S_1^2 + \frac{n_2-1}{n_1+n_2-2} S_2^2 = \frac{1}{n_1+n_2-2}\sum_{j=1}^2\sum_{i=1}^{n_j} (Y_{ij} - \bar{Y}_j)^2.$$

$$ S_p^2= \sum\limits_{j=1}^2\sum\limits_{i=1}^{n_j} \frac{(Y_{ij}-\bar{Y}_{.j})^2}{n_1+n_2-2}$$


The pooled variance estimator uses the squared deviations of the observations from their group mean and has $n_1+n_2-2$ degrees of freedom.

---

## Test statistic
Two-sample $t$-teststatistiek:

$$T = \frac{\bar{Y}_1-\bar{Y}_2}{\sqrt{\frac{S_p^2}{n_1}+\frac{S_p^2}{n_2}}} =
  \frac{\bar{Y}_1 - \bar{Y}_2}{S_p\sqrt{\frac{1}{n_1}+\frac{1}{n_2}}}.$$

The statistic T follows a t-distribution with $n_1+n_2-2$ under $H_0$ is all data are independent, normally distributed and have equal variances.

---

## Armpit example

We can implement the test in R:

```{r}
t.test(rel ~ trt, data = ap, var.equal = TRUE)
```

On the $5\%$ significance level we reject the null hypothesis in favor of the alternative hypothesis and conclude that the relative abundance of  *Staphylococcus spp.* is on average extreme significant larger is in transplantation group than in the placebo group.

If there is no effect of the transplant we have a probability of less then 9 in $100000$ to observe a test statistic in a random sample that is at least as extreme as what we observed in the armpit experiment.

This is extremely rare under $H_0$.

If $H_1$ is correct, we expect that the test statistic is larger in absolute value and expect small p-values. Hence we decide that there is a lot of evidence against $H_0$ in favour of $H_1$.

**Good statistical practice** is to report the $p$-value, but also  effect size along with its confidence interval. So that we can judge the statistical significance and the biological relevance.

### Conclusion

On average the relative abundance of *Staphylococcus spp.* in the microbiome of the armpit in the transplant group is extremely significantly different from that in the placebo group ($p<<0.001$). The relative abundance of *Staphylococcus spp.* is on average `r round(diff(t.test(rel~trt,data=ap,var.equal=TRUE)$estimate),1)`% larger in the transplant group than in the placebo group (95\% CI [`r paste(format(-t.test(rel~trt,data=ap,var.equal=TRUE)$conf.int[2:1],digits=2,nsmall=1),collapse=",")`]%).

---

# Assumptions

Validity of t-test depends on distributional assumptions:

- Independence (design)
- One-sample t-test: normality of the observations
- Paired t-test: normality of the difference
- Two-sample t-test: Normality of the observations in both groups, and equal variances.

If the assumptions are not met, the null distribution does not follow a t-distribution, and, the p-values and critical values are incorrect.

To construct confidence intervals we also rely on these assumptions.

- We used quantiles from the t-distribution to calculate the lower and upper limit.

- The correct coverage of the CI depends on these assumptions

---

## Evaluate normality

 - Boxplots and histograms: shape of distribution and outliers

 - QQ-plots

There also exist hypothesis tests (goodness-of-fit test), but their null hypothesis is that the data are normally distributed so we make a weak conclusion!

  - Kolmogorov-Smirnov, Shapiro-Wilk en Anderson-Darling.
  - In small samples they have a low power
  - In large samples they often flag very small deviations as significant

Recommendation

- Start with graphical exploration of the data and keep the sample size in mind to avoid overinterpretation of the plots.

- If you have doubts, use simulation where you simulate data with the same sample size from a Normal distribution with the same mean and variance as the one that you observed in the sample

- If you observed deviations of normality check in the literature how sensitive your method is such deviations of normality. (e.g. T-tests for instance are rather insensitive to deviations as long as the distribution of the data is symmetric.)

- In large samples you can resort to the central limit theorem.

- You might resort to transformations of the response.

---

## Homoscedasticity

- Boxplots: The box size is the inter quartile range (IQR) a robust estimator of the variance.

- If the differences are not large $\rightarrow$ homoscedasticiteit

- Again you can use simulation to get insight in the differences you can expect.

- Formal F-test can be used to compare the variances, but again under the null you assume equal variances, so the same criticism as for normality tests applies here.

---

## Welch modified t-test

If the data are heteroscedastic, you can use a Welch two-sample T-test, which no longer uses the pooled variance estimator.

$$T =  \frac{\bar{Y}_1 - \bar{Y}_2}{\sqrt{\frac{S^2_1}{n_1}+\frac{S^2_2}{n_2}}}$$
with $S^2_1$ en $S^2_2$ the sample variances in both groups.

This statistic follows approximately a t-distribution with a number of degrees of freedom between  $\text{min}(n_1-1,n_2-1)$ and $n_1+n_2-2$.

In R the degrees of freedom are estimated using the Welch- Satterthwaite approximation. You can do this by using the `t.test` function with argument `var.equal=FALSE`.

```{r}
t.test(rel ~ trt, data = ap, var.equal = FALSE)
```

Note that you can see that the Welch T-test is adopted in the title. The adjusted degrees of freedom are $df = 17.876$ $\pm$ to that of the conventional T-test, because the variances are approximately equal.

---

# How to report?

- In the scientific literature there is too much attention for p-values

- It is much more informative to combine an estimate with its confidence interval.

**Rule of thumb**:

Report an estimate together with its  confidence interval (and its p-value)

1. The result of the test can be derived of the confidence interval
2. It allows the reader to judge **scientific relevance**.

```{r}
t.test(rel ~ trt, data = ap)
```

The result of an $\alpha$-level t-test is equivalent with comparing the effect size under $H_0$ with the $1-\alpha$ CI.

An effect can be extremely statistically significant, but scientifically irrelevant. With a CI you will spot this.