- Table of Contents

Large-scale genome sequencing and gene expression analyses can result in lists of genes whose immediate relevance to the study objective may be unclear. Approaches collectively referred to as ‘enrichment’ or ‘overrepresentation’ analyses all aim to distill from more meaningful biological themes from reams of biological data.

Enrichment methods source candidate sets of genes that share attributes such as membership in a pathway or location within a cell. Gene sets are defined in curated biological databases such as the classes described within the Gene Ontology GO (Ashburner 2000). These sets are tested one after another to determine whether the constituents are overrepresented (or depleted) in a target gene list typically derived from experiments.

Our overarching goal is to describe the theory underlying many of the enrichment software tools currently in use (Khatri 2012). We use the concrete example of assessing GO term enrichment in the output of a large-scale gene expression analysis although the approaches are found in many other types of analyses. By then end of this discussion you should:

- Be familiar with how an enrichment analysis is structured
- Be familiar with rationale underlying Fisher’s Exact Test
- Be aware of the meaning of a p-value arising from Fisher’s Exact Test

Data from experiments can often be classified in several ways, for example, by age, gender and treatment response. One may wonder if the proportions within one category are associated with the proportions of another category. For example, it is common to ask if individuals treated with a drug respond more often than those with a placebo. In the case where there are two categories of interest the data can be displayed as a 2-by-2 *contingency table*.

A

contingency tableshows the distribution of one variable in rows and another in columns, used to study the association between the two variables.

Suppose we undertake a gene expression analysis comparing subtypes of high-grade ovarian serous cancer (HGS-OvCa) and we derive a list of differentially expressed genes (DEGs). For the purposes of this discussion let us restrict an enrichment analysis to a single gene set, specifically genes involved in the GO biological process ‘translation initiation’. Table 1 depicts a contingency table corresponding to some hypothetical experimental results.

**Table 1. Contingency table for HGS-OvCa gene expression data**

In this case, the expression of 32 genes has been analyzed: 15 DEG were identified and 15 genes were associated with the GO annotation ‘translation initiation’. The totals for DEG and gene set membership are the *marginal values*, as they lie on the periphery of our 2-by-2 table.

The middle cells contain *joint values* because they represent genes falling under two categories (Table 1, light blue). Here, 12 genes are both DEG and tagged with ‘translation initiation’. This seems like a large proportion of the marginal DEG total (12/15 DEGs) and our intuition might lead us to the opinion that this is a result worth following up on…

We seemed quite confident that observing 12 DEG out of 15 in the gene set seemed like enough evidence to suggest that our ovarian cancer subtypes differed in the state of their translation initiation gene expression. However, what would we think if only 9 met both criteria? Furthermore, a more realistic large-scale expression analysis might involved thousands of genes observed rather than just 32. It is clear that we yearn for a way to make our decisions less arbitrary and more scaleable.

Fisher’s exact test is a statistical procedure developed by R. A. Fisher in the mid 1930’s (Fisher 1935). Strictly speaking, the test is used to determine the probabilities of observing the various joint values within a contingency table under two important assumptions:

- The marginal values are fixed
- There is no association between categorical values

These assumptions constitute the ‘null hypothesis’ (): We take the *a priori* stance that the categories are independent. We simply don’t know the ground truth of whether there exists a relationship between HGS-OvCa subtype gene expression and genes involved in ‘translation initiation’. Consequently, we take our actual contingency table data and calculate the probability that this or any table with more extreme joint values (unobserved) would occur under the null hypothesis. A small probability is interpreted as a discrepancy between the data and the null hypothesis of no association between variables. These calculated probabilities are referred to as ‘p-values’.

Smaller p-values point to an interesting result only if all of the assumptions used to compute the p-value are valid

Let us return to the HGS-OvCa gene expression analysis example. Fisher’s Exact Test provides a statistical basis upon which to establish how extreme our particular table of observations are **in relation to all possible ones that could have given those same marginal totals given no association between categories**. With this in mind, we simply enumerate the different joint values that are possible for the same marginal totals (Figure 1).

There may exist 16 possible arrangements of joint values for fixed marginal totals, however, this does not imply that each is equally likely. To see why, consider the arrangement in the first row and second column of (Figure 1; R1-C2): This arrangement is reproduced in Table 2 and shows 1 DEG that is a member of the gene set leaving 14 DEGs outside. To calculate the probability of this (unobserved) arrangement under the null hypothesis, we will make use of the rules for combinations.

**Table 2. Contingency table for arrangement R1-C2**

R.A. Fisher’s insight was to leverage the rules for enumerating combinations to derive an exact probability for any given contingency table under the null hypothesis. For our 32 genes of which 15 are DEG and 15 are within a gene set, we must first calculate the ways that each of the joint values could have arisen simply by randomly selecting genes. We illustrate the process for the arrangement in Table 2 and the process proceeds by calculating three values, one for each row:

- Ways to select 1 DEG
*without replacement*out of 15 tagged IN the gene set - Ways to select 14 DEGs
*without replacement*from 17 tagged NOT IN the gene set - Ways to select 15 DEGs
*without replacement*from 32 total

These three values are sufficient to calculate the probability of any particular contingency table. The first number represents the ways 1 DEG can be selected from 15 possible ‘IN Translation Initiation’ (Figure 2, top left). In statistical jargon, this is described as ‘15 Choose 1’. There are exactly 15 ways this can be done: One for each of the 15 genes with the tag.

Similarly, there are ‘17 Choose 14’ or 680 ways that 14 DEGs can be selected from 17 ‘NOT IN Translation Initiation’. To see this, consider the 17 genes labeled 16 through 32 in the top right group of Figure 2. We can choose 14 genes by selecting those labelled 16 through 29. Alternatively, one can also select 16 through 28 then 30 or for that matter 16 through 28 then 31 and so on. The third number ‘32 Choose 15’ is precisely 565 722 720.

The probability of observing this arrangement then is given by the quotient:

Some of you may recognize the expression on the left as the probability function for the hypergeometric distribution.

At this point we have the basic building blocks to describe Fisher’s Exact Test. Referring to our HGS-OvCa example, we have in hand the probability of our contingency table of observed joint values (Table 2). Fisher’s Exact Test amounts to summing the probability of observing our table of observed joint values **in addition to those more extreme than our table**. The possible tables and their respective probabilities are displayed in red Figure 3.

Figure 3 shows that our observed result in Table 1 has a probability equal to 0.0005469 (Figure 3, R4-C1). The one-sided test requires us to sum the probabilities of the observed table and those unobserved tables which possess more than 12 genes the are DE and IN the gene set (Figure 3, red)

From this result, we claim that the probability of our observed data or that more extreme under the assumption that there is no association between expression and gene set membership is 0.0005726. Whether this represents an interesting discrepancy from the null hypothesis, an experiment worth repeating, or an ‘enrichment’ of genes in the set amongst DE genes is left up to the researcher’s interest and expertise.

We have not yet considered the possibility that DEGs may contain fewer members of the gene set than would be expected if genes were sampled randomly, that is, DEGs are depleted for ‘IN Translation Initiation’. The two-sided Fisher’s Exact Test accounts for both enrichment and depletion. Although there are several flavours of the test (Rivals 2006), we demonstrate an approach whereby the tables with probabilities smaller than our observed data () are summed

This result is just under double the one-sided p-value, resulting from an ‘uneven’ distribution of p-values about the mean. Once again, whether this represents a valuable discrepancy from the null hypothesis and an observation worth following up on is left up to the discretion of the researcher.

There has been much discussion with regards to the assumptions, limitations, and overall relevance of this class of procedures (Khatri 2005, Goeman 2007). We leave these more nuanced discussions for the reader to follow up on.

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