So far we have used P-values for inference based on false positive rate (this is the definition of P-value itself), on family-wise error rate (FWER; Bonferroni and Holm) and on false discovery rate (FDR; Benjamini-Hochberg and Benjamini-Yekutieli). See slides.

Here we will consider a quantity called Q-value that can be attached to each test and that gives an empirical estimate of FDR among all tests whose Q-values are smaller or equal to the given Q-value. We will follow Storey and Tibshirani (2003, PNAS 100: 9440-9445), whose work led to `qvalue`

R-package. Before defining Q-value we will first refine BH method by empirically estimating \(p_0\), the number of null tests. Remember that the proof of BH theorem showed that BH(\(\alpha_F\)) method actually controls FDR at level \(p_0/p \cdot \alpha_F.\) When \(p_0\) is considerably smaller than \(p\), we can improve the accuracy of the FDR method by estimating \(p_0.\)

Letâ€™s define, for each P-value threshold \(t\in[0,1],\) \[\textrm{FDR}(t) = \textrm{E}\left(\frac{\textrm{FD}(t)}{D(t)}\right),\] where random variables \(\textrm{FD}(t)=\#\{\textrm{null P-values} \leq t\}\) and \(D(t)=\#\{\textrm{P-values} \leq t\}\) in an experiment where in total \(p\) P-values are available.

To refine our understanding of FDR methods, letâ€™s think that random variables \(\textrm{FD}(t)\) and \(D(t)\) result from \(p\) draws of P-values from a mixture distribution between Uniform(0,1) (for null P-values) and an alternative distribution with cdf \(\Phi_1\) and pdf \(\phi_1\) (for non-null P-values), with mixture proportion \(\pi_0\) for the null. In other words, cdf \(\Phi\) and pdf \(\phi\) of the P-values are \[\begin{eqnarray} \Phi(t)&=&\pi_0 \cdot t + (1-\pi_0) \Phi_1(t), \qquad t\in[0,1],\\ \phi(t)&=&\pi_0 \cdot 1 + (1-\pi_0) \phi_1(t), \qquad t\in[0,1]. \end{eqnarray}\] We can interpret sampling from such a mixture distribution as a 2-step process. Namely, we first choose between the null distribution (with probability \(\pi_0\)) and the alternative distribution (with probability \(1-\pi_0\)), and second, conditional on the chosen distribution, we sample a P-value from the chosen distribution.

**Example 3.1.** Suppose we do \(p=10000\) tests of which \(m=2000\) are non-null, i.e., we can model this as a mixture distribution with \(\pi_0=\frac{p-m}{p} = 0.80\). Null P-values come from Uniform(0,1) and non-null P-values come from distribution Beta(0.1,4.9). (Beta(0.1,4.9) gives values in [0,1] that have mean \(0.1/(0.1+4.9)=0.02\) and a skew to right; there is no particular reason why in real data non-null P-values had exactly this distribution, but this is used for a demonstration purpose here; see figures below.) Letâ€™s plot theoretical (orange) and empirical (green) density functions for (1) null tests, (2) non-null tests and (3) for all tests combined (that is, the mixture distribution of the null and alternative distributions).

```
p = 10000
m = 2000
beta.1 = 0.1 # weight for unit interval's end point 1
beta.0 = 4.9 # weight for unit interval's end point 0
null.pval = runif(p-m, 0, 1)
alt.pval = rbeta(m, beta.1, beta.0) #non-null = alternative distribution
pval = c(alt.pval, null.pval) #all P-values together
eff = c(rep(T, m), rep(F, p - m)) #indicator for non-null effects
par(mfrow = c(1,3)) #Empirical histogram and theoretical curve for (1), (2), (3)
hist(null.pval, breaks = 20, prob = T, col = "limegreen", main = "null", xlab = "",
xlim = c(0,1), sub="", lwd = 1.5, lty = 2, xaxs="i", border = NA)
curve(dunif(x, 0, 1), 0, 1, add = T, col = "orange", lwd = 2)
hist(alt.pval, breaks = 20, prob = T, col = "limegreen", main = "non-null", xlab = "",
xlim = c(0,1), sub="", lwd = 1.5, lty = 2, xaxs="i", border = NA)
curve(dbeta(x, shape1 = beta.1, shape2 = beta.0), 0, 1, add = T, col = "orange", lwd = 2)
legend("topright", fill = c("limegreen","orange"),
legend = c("empir","theor"), cex = 1.5)
hist(pval, breaks = 20, prob = T, col = "limegreen", main = "combined", xlab = "",
xlim = c(0,1), sub="", lwd = 1.5, lty = 2, xaxs="i", border = NA)
curve((p-m)/p*1 + m/p*dbeta(x, shape1 = beta.1, shape2 = beta.0), 0, 1, add = T,
col = "orange", lwd = 2)
```

Density plots show how the null P-values follow the uniform distribution and non-null P-values are enriched for small values near 0.

`summary(null.pval)`

```
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 0.0000108 0.2521960 0.4999947 0.5002426 0.7467176 0.9998005
```

`summary(alt.pval)`

```
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 0.0000000 0.0000001 0.0001608 0.0196240 0.0081581 0.7202532
```

When these two distributions are combined in the rightmost panel, the enrichment of small P-values is still present but has a smaller weight of 20% compared to the weight of 80% given to the null distribution.

Since \(\Phi(t)\) is the probability that a particular P-value from this mixture distribution \(\leq t\), the random variables \(D(t)\) and \(\textrm{FD}(t)\) are distributed as \[\begin{eqnarray} D(t) &\sim& \textrm{Bin}(p,\Phi(t)) \\ \textrm{FD}(t)\,|\,D(t)&\sim& \textrm{Bin}(D(t), \theta_t), \text{ where } \\ \theta_{t} &=& \textrm{Pr}(\textrm{NULL} \, | \, P \leq t ) = \frac{\textrm{Pr}(\textrm{NULL})\textrm{Pr}(P \leq t \, | \, \textrm{NULL})}{\textrm{Pr}(P \leq t)}\\ &=&\frac{\pi_0 t} {\pi_0 t + (1-\pi_0)\Phi_1(t)}. \end{eqnarray}\] Using the law of total expectation: \(\textrm{E}_Y(Y) = \textrm{E}_X(\textrm{E}_Y(Y\,|\,X))\), we have that \[\textrm{FDR}(t) = \textrm{E}\left(\frac{\textrm{FD}(t)}{D(t)}\right)= \textrm{E}\left(\textrm{E}\left(\frac{\textrm{FD}(t)}{D(t)}\,\middle|\,D(t)\right)\right)= \textrm{E}\left(\frac{1}{D(t)} \textrm{E}(\textrm{FD}(t)\,|\,D(t)) \right)= \textrm{E}\left(\frac{1}{D(t)} \theta_{t}\,D(t) \right)= \theta_{t}.\] On the other hand, \(\textrm{E}(\textrm{FD}(t)) = \textrm{E}(\textrm{E}(\textrm{FD}(t)\,|\,D(t))) =\textrm{E}(D(t)\theta_t) = \theta_t\cdot \textrm{E}(D(t))\) Thus \[\frac{\textrm{E}(\textrm{FD}(t))}{\textrm{E}(D(t))} = \frac{\theta_t\cdot\textrm{E}(D(t))}{\textrm{E}(D(t))} = \theta_t= \textrm{FDR}(t). \] So we can estimate \(\textrm{FDR}(t)\) by the ratio of expectations of \(\textrm{FD}(t)\) and \(D(t)\). And what could we use as estimates for these expectations?

For each P-value threshold \(t\), denote the number of all discoveries at threshold \(t\) by \(\widehat{D}(t) = \#\{\textrm{P-values} \leq t\}.\) We use this to estimate \(\textrm{E}(D(t)) \approx \widehat{D}(t)\).

To estimate \(\textrm{E}(FD(t))\), we remember that the null P-values are uniformly distributed and hence \(\textrm{E}(FD(t)) = p_0 \cdot t = \pi_0\cdot p \cdot t.\) In estimating \(\pi_0\) we again rely on the fact that the null P-values are uniformly distributed and that most P-values near 1 are expected to be from the null distribution. Letâ€™s remind us how our P-value distribution looked like.

```
hist(pval, breaks = 40, prob = T, col = "limegreen", main = "All P-values", xlab = "",
xlim = c(0,1), sub = "", lwd = 1.5, lty = 2, xaxs = "i", border = NA)
abline(h = 1, lty = 2, lwd = 2)
```

We can see that the density of P-values > 0.2 looks fairly flat. If we assume that the density of non-null P-values is \(\approx 0\) in this region, then the density function of P-values in this region is approximately \(\pi_0\cdot 1 + (1-\pi_0)\cdot 0 = \pi_0\). Thus the value of the density function in this flat part gives an estimate of the overall proportion of null P-values \(\pi_0\). The constant density function over any interval \((\lambda, 1]\) is the probability mass in the interval divided by the length of the interval (\(1-\lambda\)). With empirical data, this probability mass is

\(\#\{P_j >\lambda \,|\, j=1,\ldots,p\}/p\). We can apply such an estimate for any value \(\lambda\) to yield an estimator \[
\widehat{\pi}_0(\lambda) = \frac{\#\{P_j >\lambda\, |\, j=1,\ldots,p\}}{(1-\lambda) p}.
\] Parameter \(\lambda\) should be large enough that there are not many true effects with P-values \(>\lambda\) but simultaneously \(\lambda\) should be small enough that there are enough null P-values \(> \lambda\) so that the estimate has a good precision. An inclusion of a few non-null P-values \(>\lambda\) makes this estimate conservative, that is, an overestimate of \(\pi_0\). That keeps us on the safe side and leads to an overestimate \(\textrm{FDR}(t)\).

If we take \(\lambda = 0\), then \(\widehat{\pi}_0(\lambda)=1\) which is usually much too conservative when a considerable proportion of tests are expected to be truly non-null. On the other hand, as we set \(\lambda\) closer to 1, the variance of \(\widehat{\pi}_0(\lambda)\) increases due to decreasing number of P-values exceeding \(\lambda\), which makes the estimate more unreliable. Therefore, as a simple, practical choice for \(\lambda\) we could use 0.5, but optimal threshold would depend on the data set. Letâ€™s see see how the choice of \(\lambda\) affects the estimate \(\widehat{\pi}_0(\lambda)\) in our ongoing example data.

```
lambda = seq(0, 1, by = 0.05)
pi.0 = sapply(lambda, function(x) {sum(pval > x)}) / p / (1 - lambda)
plot(lambda, pi.0, t = "b", xlab = expression(lambda), ylab = expression(hat(pi)[0]))
abline(h = 1 - m/p, col = "red", lty = 2) #this is the true value
```

Indeed, we see that with values near \(\lambda = 0.5\), the estimate \(\widehat{\pi}_0(\lambda)\) is a good one whereas its quality deteriorates towards either of the endpoints of the interval.

With this estimator for \(\widehat{\pi}_0\), we have the estimate \[\widehat{\textrm{FDR}}(t) = \frac{\widehat{\textrm{FD}}(t)}{\widehat{D}(t)}= \frac{p\cdot \widehat{\textrm{Pr}}(\textrm{NULL P-value})\cdot \textrm{Pr}(P \leq t \,|\, \textrm{NULL P-value})}{\widehat{D}(t)} = \frac{p\cdot \widehat{\pi}_0 \cdot t}{\widehat{D}(t)}.\]

Letâ€™s plot our estimate \(\widehat{\textrm{FDR}}(t)\) in orange as function of \(t\) by using the value \(\widehat{\pi}_0(0.5)\) estimated above. Letâ€™s also plot a curve that corresponds to conservative assumption \(\pi_0=1\) in red and letâ€™s add the true empirical \(\textrm{FDP}(t)\) curve in blue, which we are able to do since we know which P-values were simulated from the null.

```
pval.sorted = sort(pval)
pi.0 = pi.0[which(lambda == 0.5)]
par(mar = c(5,6,2,1)) # widen left margin for text
plot(pval.sorted, p * pi.0 * pval.sorted / (1:p), xlab = "t",
ylab = expression(paste(hat(FDR),"(t) or FDP(t)")), t = "l", col = "orange",
xlim = c(0,1), ylim = c(0,1), lwd = 1.5)
#Add line for conservative assumption pi.0 = 1:
lines(pval.sorted, p * 1 * pval.sorted / (1:p), xlab = "t",
col = "red", lwd = 1.5)
#Add empirical FDP line
fdp = cumsum(!eff[order(pval)]) / (1:p) #true false discovery proportion in data
lines(pval.sorted, fdp, xlab = "t", lty = 3, col = "blue", lwd = 1)
legend("bottomright", leg = expression(hat(pi)[0], pi[0] == 1 ,paste("true fdp")),
col = c("orange", "red", "blue"), lty = c(1, 1, 3), lwd = c(2, 2, 1))
```