Between-individual variation, replication, and sampling (cont'd)

M. Drew LaMar
March 30, 2016

“There are no null results; there are only insufficiently clever choices of \( H_0 \). ”

- @richarddmorey

Proper randomization

Assigning treatments to subjects (one possibility):

  1. List all \( n \) subjects, one per row, in a spreadsheet.
  2. Use the computer to give each subject a random number.
  3. Assign treatment A or B to those subjects receiving the lowest or highest numbers, respectively.

Randomization in time

Remember, randomization is important in all processes of the experiment, including preparation, setup, and measurement.

Randomize measurement of replicates in time:

  • Watching 50 hours of great tit courtship behaviour on video increases your ability to observe
  • After 10 hours of counting through a microscope, tiredeness kicks in
  • Aging equipment

This shows time of measurement could be a confounding factor.

Examples, examples, examples

Birdsongs and attractiveness

Question: How do we measure relationship between male birdsongs and attractiveness to females?

Experimental Design: Record the complex song of one male and the simple song of another male, and then play these same two songs to each of 40 different females. Compute a confidence interval for the mean attractiveness of the two male songs.

Discuss: What is wrong with this design so far?

Answer: Each measure of female choice is a pseudoreplicate (\( n=40 \)).

Examples, examples, examples

Discuss: What is wrong with this design so far?

Answer: Each measure of female choice is a pseudoreplicate (\( n=40 \)).

Discuss: What can we do to correct for this pseudoreplication?

Answer: Record songs of 40 males with complex songs, and 40 separate males with simple songs. Each female should listen to a unique pair of songs, one simple and one complex. Design can get even more complicated than this.

Discuss: What are examples of confounding variables in the pseudoreplicated case?

Examples, examples, examples

Blood sugar levels

Experimental Design: Phlebotomist takes 15 samples from each of 10 patients, yielding a total of 150 measurements.

Discuss: What is the replicate and sample size in this situation? Why?

Examples, examples, examples

Antibiotics and bacterial growth rates

Experimental Design: Two agar plates: one with antibiotic, one without. Spread bacteria on both plates, let them grow for 24 hours, then measure diameter of 100 colonies on each plate?

Discuss: What is the replicate and sample size in this situation? Why?

What sample size should I use?

Three things:

  • Plan for precision (estimation)
  • Plan for power (hypothesis testing)
  • Plan for data loss

We'll use a two-sample \( t \)-test as the example in this section.

Plan for precision

We would like to compute a 95% confidence interval for \( \mu_{1}-\mu_{2} \).

\[ \bar{Y}_{1}-\bar{Y}_{2} \pm \mathrm{margin \ of \ error}, \]

where “margin of error” is the half-width of the 95% confidence interval.

In this case, the following formula is an approximation to the number of samples needed to achieve the desired margin of error (assuming balanced design, i.e. \( n_{1}=n_{2}=n \)):

\[ n \approx 8\left(\frac{\mathrm{margin \ of \ error}}{\sigma}\right)^{-2} \]

Plan for precision

Plan for power

Two-sample \( t \)-test:

\[ H_{0}: \mu_{1} - \mu_{2} = 0. \] \[ H_{A}: \mu_{1} - \mu_{2} \neq 0. \]

A conventional power to aim for is 0.80, i.e. we aim to prove \( H_{0} \) is false in 80% of experiments.

Assuming a significance level of 0.05, a quick approximation to the planned sample size \( n \) in each of two groups is

\[ n \approx 16\left(\frac{D}{\sigma}\right)^{-2}, \]

where \( D = |\mu_{1}-\mu_{2}| \) is the effect size.

Pwr package in R

library(pwr)
function power calculations for
pwr.2p.test two proportions (equal n)
pwr.2p2n.test two proportions (unequal n)
pwr.anova.test balanced one way ANOVA
pwr.chisq.test chi-square test
pwr.f2.test general linear model
pwr.p.test proportion (one sample)
pwr.r.test correlation
pwr.t.test t-tests (one sample, 2 sample, paired)
pwr.t2n.test t-test (two samples with unequal n)

Two-sample t-test example

Two-sample \( t \)-test with significance level 0.05, 80% power, and relative effect size \( d = \frac{|\mu_{1}-\mu_{2}|}{\sigma} = 0.3 \).

pwr.t.test(d=0.3, power=0.8, type="two.sample")

     Two-sample t test power calculation 

              n = 175.3847
              d = 0.3
      sig.level = 0.05
          power = 0.8
    alternative = two.sided

NOTE: n is number in *each* group

Two-sample t-test example

plot of chunk unnamed-chunk-4

Additional Reading

  • Whitlock & Schluter, Interleaf 2: Pseudoreplication (pp. 115-116)
  • Whitlock & Schluter, Chapter 14: Designing experiments