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multtest and ALL data

Kasper Daniel Hansen
(adapted from the multtest vignettes)
Bioconductor 2. August 2006

1. Introduction

We will look at adjusting for multiple testing in an analysis of the ALL dataset. This analysis is very rudimental and quite similar to what is done in the multtest vignette. The ALL dataset was chosen because of its size and availability as a Bioconductor data package.

1. Getting started

You will need the following packages from Bioconductor: multtest, ALL and genefilter. First we load the packages and the data:

library(multtest)
library(ALL)
library(genefilter)
data(ALL)

ALL is an exprSet from a study with 128 samples using the Affymetrix hgu95av2 chip with 12,625 probesets. The data has been preprocessed using RMA. We start by following the original article and filter out probesets where i) the coefficient of variation is between 0.7 and 10 and where ii) at least 20% of the samples have a measured intensity of at least 100 (on the original scale, not the log2 scale which is the scale of the expr slot in the ALL data)

ffun <- filterfun(pOverA(p = 0.2, A = 100), cv(a = 0.7, b = 10))
filt <- genefilter(2^exprs(ALL), ffun)
filtALL <- ALL[filt, ]

filtALL now contains 431 genes (probesets). If you are unfamilar with the data, you can have a look at the covariates by

filtALL
summary(pData(filtALL))

3. Standard t-tests and marginal adjustments

We start by finding genes that are differentially expressed between B-cells and T-cells. We will be using the BT covariate which has no missing values. However, since the two types have been subdivided into T1, T2, etc. we start by defining a new variable which is TRUE when the sample comes from a B-cell and FALSE if coming from a T-cell:

BT <- is.element(pData(filtALL)$BT,
    c("B", "B1", "B2", "B3", "B4", "B5"))

Next we do a Welsh t-test (not assuming equal variances in the two groups) and save the p-vales. By looking at them, we see that without adjusting for multiple testing, 296 genes are significant at level 0.05.

rawp <- esApply(filtALL.sex, MARGIN = 1, FUN = function(x) {
    t.test(x[bt], x[!bt])$p.value
})
sum(rawp <= 0.05)

We will start by doing a marginal adjustment of the p-values. Marginal adjustments only take the vector of p-values into account and are therefore not as powerful as joint adjustments. On the other hand, they are easy to do and there are no restrictions on the sample size. A number of procedures have been suggested, we start be looking at the results for procedures controlling the familywise error rate (FWER). By doing so, we try to control the possibility of making a single ***.

fwer <- mt.rawp2adjp(rawp, c("Bonferroni", "Holm", "Hochberg"))
mt.reject(fwer$adjp, alpha = 0.05)$r

We see that the Bonferroni adjustment is the most conservative (yielding the least rejections) while the Holm and Hochberg adjustments yields similar results.

We can also try to control the false discory rate (FDR) which is very popular in genomics. By doing so, we try to control the expected number of false negatives amongst the total number of rejected hypotheses. This is a popular error rate because it is relative to how many rejections one has in total. Two popular marginal procedures exists for this: Benjamini-Hochberg and Bejamini-Yuketeli. The first procedure assumes weak dependence amongst the test-statistics, but is also the least conservative.

fwer <- mt.rawp2adjp(rawp, c("BH", "BY"))
mt.reject(fwer$adjp, alpha = 0.01)$r

Note that the alpha parameter this time has a different interpretation because the error rate (FDR vs. FWER) is different.

It would be a good idea to try out the methodology on another covariate. While the mechanical steps are very similar, try to interpret the results. I would suggest sex. Note that sex (surprisingly) has a few missing values, so you will need to subset the filtALL exprSet to remove these sample, an example would be

filtALL.sex <- filtALL[, !is.na(pData(filtALL)$sex)]

4. Standard t-tests and joint adjustments

We will now be using a joint procedure. These types of procedures have the advantage that it is possible to take the dependency structure of the data into account. In general they are preferred to marginal adjustments, with the caveat that one needs a reasonable number of samples to use them. They are also much slower.

We will be using the bootstrap estimator and use a step-down minP procedure. In the following we are using 5,000 bootstrap replicates (which is a reasonable number). That may take too long to complete on your machine. We have supplied a complete run in the multtestPractical.rda file which can be loaded by the command below, given that the working directory contains the data file.

seed <- 675
MTP.BT <- MTP(X = filtALL, Y = BT, alternative = "two.sided",
    typeone = "fdr", alpha = 0.01, B = 5000, method = "sd.minP", seed = seed)
getwd()
list.files()
setwd()
load("multtestPractical.rda")
The workhorse function MTP contains a seed argument, which explicitely allows for reproducibility. Note that we are considering a two-sided alternative. This means we are searching for genes which are different between the two groups. By using a one-sided alternative (less/greater) we would be looking for genes that are higher expressed in one of the groups (of course, in that case care needs to be taken that the groups are in the right order).

MTP produces a member of the S4 class MTP. You can examine this class a bit by

slotNames("MTP")
The class has an update method which allows the user to try out different error rates, different levels and so on, without recomputing the boostrap procedure. Let us say we want to try out the family-wise error rate. 
update(MTP.BT, alpha = 0.05, typeone = "fwer")