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Here are three experiments to illustrate when the different approaches to statistics are appropriate. In the first experiment, you are testing a plant extract on rabbits to see if it will lower their blood pressure. You already know that the plant extract is a diuretic (makes the rabbits pee more) and you already know that diuretics tend to lower blood pressure, so you think there's a good chance it will work. If it does work, you'll do more low-cost animal tests on it before you do expensive, potentially risky human trials. Your prior expectation is that the null hypothesis (that the plant extract has no effect) has a good chance of being false, and the cost of a false positive is fairly low. So you should do frequentist hypothesis testing, with a significance level of 0.05.

Now instead of testing 1000 plant extracts, imagine that you are testing just one. If you are testing it to see if it kills beetle larvae, you know (based on everything you know about plant and beetle biology) there's a pretty good chance it will work, so you can be pretty sure that a *P* value less than 0.05 is a true positive. But if you are testing that one plant extract to see if it grows hair, which you know is very unlikely (based on everything you know about plants and hair), a *P* value less than 0.05 is almost certainly a false positive. In other words, *if you expect that the null hypothesis is probably true, a statistically significant result is probably a false positive.* This is sad; the most exciting, amazing, unexpected results in your experiments are probably just your data trying to make you jump to ridiculous conclusions. You should require a much lower *P* value to reject a null hypothesis that you think is probably true.

In the olden days, when people looked up *P* values in printed tables, they would report the results of a statistical test as "*P**P**P*>0.10", etc. Nowadays, almost all computer statistics programs give the exact *P* value resulting from a statistical test, such as *P*=0.029, and that's what you should report in your publications. You will conclude that the results are either significant or they're not significant; they either reject the null hypothesis (if *P* is below your pre-determined significance level) or don't reject the null hypothesis (if *P* is above your significance level). But other people will want to know if your results are "strongly" significant (*P* much less than 0.05), which will give them more confidence in your results than if they were "barely" significant (*P*=0.043, for example). In addition, other researchers will need the exact *P* value if they want to combine your results with others into a .

This criticism only applies to two-tailed tests, where the null hypothesis is "Things are exactly the same" and the alternative is "Things are different." Presumably these critics think it would be okay to do a one-tailed test with a null hypothesis like "Foot length of male chickens is the same as, or less than, that of females," because the null hypothesis that male chickens have smaller feet than females could be true. So if you're worried about this issue, you could think of a two-tailed test, where the null hypothesis is that things are the same, as shorthand for doing two one-tailed tests. A significant rejection of the null hypothesis in a two-tailed test would then be the equivalent of rejecting one of the two one-tailed null hypotheses.

You should decide whether to use the one-tailed or two-tailed probability before you collect your data, of course. A one-tailed probability is more powerful, in the sense of having a lower chance of false negatives, but you should only use a one-tailed probability if you really, truly have a firm prediction about which direction of deviation you would consider interesting. In the chicken example, you might be tempted to use a one-tailed probability, because you're only looking for treatments that decrease the proportion of worthless male chickens. But if you accidentally found a treatment that produced 87% male chickens, would you really publish the result as "The treatment did not cause a significant decrease in the proportion of male chickens"? I hope not. You'd realize that this unexpected result, even though it wasn't what you and your farmer friends wanted, would be very interesting to other people; by leading to discoveries about the fundamental biology of sex-determination in chickens, in might even help you produce more female chickens someday. Any time a deviation in either direction would be interesting, you should use the two-tailed probability. In addition, people are skeptical of one-tailed probabilities, especially if a one-tailed probability is significant and a two-tailed probability would not be significant (as in our chocolate-eating chicken example). Unless you provide a very convincing explanation, people may think you decided to use the one-tailed probability *after* you saw that the two-tailed probability wasn't quite significant, which would be cheating. It may be easier to always use two-tailed probabilities. **For this handbook, I will always use two-tailed probabilities, unless I make it very clear that only one direction of deviation from the null hypothesis would be interesting.**

proportions or distributions refer to data sets where outcomes are divided into three or more discrete categories. A common textbook example involves the analysis of genetic crosses where either genotypic or phenotypic results are compared to what would be expected based on Mendel's laws. The standard prescribed statistical procedure in these situations is the test, an approximation method that is analogous to the normal approximation test for binomials. The basic requirements for multinomial tests are similar to those described for binomial tests. Namely, the data must be acquired through random sampling and the outcome of any given trial must be independent of the outcome of other trials. In addition, a minimum of five outcomes is required for each category for the Chi-square goodness-of-fit test to be valid. To run the Chi-square goodness-of-fit test, one can use standard software programs or websites. These will require that you enter the number of expected or control outcomes for each category along with the number of experimental outcomes in each category. This procedure tests the null hypothesis that the experimental data were derived from the same population as the control or theoretical population and that any differences in the proportion of data within individual categories are due to chance sampling.

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Regardless of the method used, the -value derived from a test for differences between proportions will answer the following question: What is the probability that the two experimental samples were derived from the same population? Put another way, the null hypothesis would state that both samples are derived from a single population and that any differences between the sample proportions are due to chance sampling. Much like statistical tests for differences between means, proportions tests can be one- or two-tailed, depending on the nature of the question. For the purpose of most experiments in basic research, however, two-tailed tests are more conservative and tend to be the norm. In addition, analogous to tests with means, one can compare an experimentally derived proportion against a historically accepted standard, although this is rarely done in our field and comes with the possible caveats discussed in . Finally, some software programs will report a 95% CI for the difference between two proportions. In cases where no statistically significant difference is present, the 95% CI for the difference will always include zero.

Entire books are devoted to the statistical method known as . This section will contain only three paragraphs. This is in part because of the view of some statisticians that ANOVA techniques are somewhat dated or at least redundant with other methods such as (see ). In addition, a casual perusal of the worm literature will uncover relatively scant use of this method. Traditionally, an ANOVA answers the following question: are any of the mean values within a dataset likely to be derived from populations^{} that are truly different? Correspondingly, the null hypothesis for an ANOVA is that all of the samples are derived from populations, whose means are identical and that any difference in their means are due to chance sampling. Thus, an ANOVA will implicitly compare all possible pairwise combinations of samples to each other in its search for differences. Notably, in the case of a positive finding, an ANOVA will not directly indicate which of the populations are different from each other. An ANOVA tells us only that at least one sample is likely to be derived from a population that is different from at least one other population.

The rationale behind using the paired -test is that it takes meaningfully linked data into account when calculating the -value. The paired -test works by first calculating the difference between each individual pair. Then a mean and variance are calculated for all the differences among the pairs. Finally, a one-sample -test is carried out where the null hypothesis is that the mean of the differences is equal to zero. Furthermore, the paired -test can be one- or two-tailed, and arguments for either are similar to those for two independent means. Of course, standard programs will do all of this for you, so the inner workings are effectively invisible. Given the enhanced power of the paired -test to detect differences, it is often worth considering how the statistical analysis will be carried out at the stage when you are developing your experimental design. Then, if it's feasible, you can design the experiment to take advantage of the paired -test method.

It is also worth pointing out that there is another way in which the -test could be used for this analysis. Namely, we could take the ratios from the first three blots (3.33, 3.41, and 2.48), which average to 3.07, and carry out a one-sample two-tailed -test. Because the null hypothesis is that there is no difference in the expression of protein X between wild-type and backgrounds, we would use an expected ratio of 1 for the test. Thus, the -value will tell us the probability of obtaining a ratio of 3.07 if the expected ratio is really one. Using the above data points, we do in fact obtain = 0.02, which would pass our significance cutoff. In fact, this is a perfectly reasonable use of the -test, even though the test is now being carried out on ratios rather than the unprocessed data. Note, however, that changing the numbers only slightly to 3.33, 4.51, and 2.48, we would get a mean of 3.44 but with a corresponding -value of 0.054. This again points out the problem with -tests when one has very small sample sizes and moderate variation within samples.

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