Data Analysis for Graduate Research – Inference between multiple means

Analysis of Variance (ANOVA) Model

Response data from observation belonging to group
The random error terms are independently and identically distributed (iid) as normal with mean zero and common variance.
Look familiar? T-test is a special case of ANOVA.

Hypothesis specification

The null hypothesis is that there is no difference in the mean of the quantitative variable across groups (categorical variable), while the alternative is that there is a difference.

: The mean outcome is the same across all groups.
: At least one mean is different.

Multiple Comparison

Why not multiple T-tests between all pairs of groups?

Each time you conduct a test, you risk coming to the wrong conclusion.

Repeated tests compound that chance of being wrong.

Img Ref: https://xkcd.com/882

Proof

Set a Type 1 error rate of (significant results due to randomness)
Conduct 10 analyses on the same data, but on different subsets.
Chance of making an error on any one test: .05
Chance of NOT making an error on any one test: .95
Chance of NOT making an error on ALL 10 tests:
–> 40% chance of making a Type 1 error on at least one test.

Visual Comparison

Compare groups I, II, and III. Can you visually determine if the differences in the group centers is due to chance or not? What about groups IV, V, and VI?

Side-by-side dot plot for the outcomes for six groups.

Analysis of Variance

The total amount of variation in our quantitative outcome can be broken into two parts:

Total Variation = Between Group Variation (model) + Within Group Variation (residual)

The portion of the variance in the outcome that is explained by the groups (model)
The portion that’s leftover due to unexplained randomness (residual)

By looking at a ratio of these variance portions, we can determine if the variation observed is due to the groups, or random chance.

Sum of Squares

Variation is measured using the Sum of Squares (SS): The sum of the squares within a group (SSE), the sum of squares between groups (SSG), and the total sum of squares (SST).

SST (Total): Measures the variation of the data points around the overall mean.

Sum of Squares

SSG (Between groups): Measures the variation of the group means around the overall mean.

SSE (Within group): Measures the variation of each observation around its group mean.

Analysis of Variance Table

The results are typically summarized in an ANOVA table.

Source	SS	MS
Groups	SSG	MSG =
Error	SSE	MSE =
Total	SST

The value in the F column is the test statistic, and has a F distribution with degrees of freedom (df) dependent on the number of groups (I-1), and the number of observations (N-I).

The F-distribution

The -value is the area to the right of the F statistic density curve. This is always to the right because the F-distribution is truncated at 0 and skewed right. This is true regardless of the .

Show the code

df1 <- c(3, 5, 8)
df2 <- c(4, 6, 10)

plot(NULL, xlim = c(0, 5), ylim = c(0, 1), xlab = expression(F), ylab="", main = "F Distribution", axes=FALSE)
axis(2, labels = FALSE, tick = FALSE)
axis(1); box()
for (i in 1:3) {
  curve(df(x, df1[i], df2[i]), from = 0, to = 5, col = i, add = TRUE, lty = i, lwd=2)
}
legend("topright", legend = paste("df1 =", df1, ", df2 =", df2), col = 1:3, lty = 1:3)

Assumptions

Generally we must check three conditions on the data before performing ANOVA:

The observations are independent within and across groups
The data within each group are nearly normal
The variability across the groups is about equal.

Example: March of the Penguins

Show the code

pen <- palmerpenguins::penguins

1. Identify response and explanatory variables

Species = categorical explanatory variable
Flipper length = quantitative response variable

2. Visualize and summarise

Show the code

library(ggdist) # for the "half-violin" plot (stat_slab)
ggplot(pen, aes(x=flipper_length_mm, y=species, fill=species)) + 
      stat_slab(alpha=.5, justification = 0) + 
      geom_boxplot(width = .2,  outlier.shape = NA) + 
      geom_jitter(alpha = 0.5, height = 0.05) +
      stat_summary(fun="mean", geom="point", col="red", size=4, pch=17) + 
      theme_bw() + 
      labs(x="Flipper Length (mm)", y = "Species") + 
      theme(legend.position = "none")

Show the code

pen %>% group_by(species) %>% 
  summarise(mean=mean(flipper_length_mm, na.rm=TRUE), 
            sd = sd(flipper_length_mm, na.rm=TRUE), 
            IQR = IQR(flipper_length_mm, na.rm=TRUE)) %>%
  kable(digits = 1)

species	mean	sd	IQR
Adelie	190.0	6.5	9
Chinstrap	195.8	7.1	10
Gentoo	217.2	6.5	9

The distribution of flipper length varies across the species, with Gentoo having the largest flippers on average at 217.2mm compared to Adelie (190mm) and Chinstrap (195.8mm). The distributions are normally distributed with very similar spreads, Chinstrap has the most variable flipper length with a SD of 7.1 compared to 6.5 for the other two species.

A blog post about raincloud plots vs violins

3. Write the null and research hypothesis in words and symbols.

Null Hypothesis: There is no association between flipper length and species.
Alternate Hypothesis: There is an association between flipper length and species.

Let and be the average flipper length for the Adelie, Chinstrap and Gentoo species of penguins respectively.

At least one is different.

4. State and justify the analysis model. Check assumptions.

We are comparing means from multiple groups, so an ANOVA is the appropriate procedure. We need to check for independence, approximate normality and approximately equal variances across groups.

Independence: We are assuming that each penguin was sampled independently of each other, and that the species themselves are independent of each other.

Normality: The distributions of flipper length within each group are fairly normal

Equal variances: Both the standard deviation and IQR (as measures of variability) are very similar across all groups.

5. Conduct the test and make a decision about the plausibility of the alternative hypothesis.

Show the code

aov(flipper_length_mm ~ species, data=pen) |> summary()

             Df Sum Sq Mean Sq F value Pr(>F)    
species       2  52473   26237   594.8 <2e-16 ***
Residuals   339  14953      44                   
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
2 observations deleted due to missingness

The pvalue is very small, so there is evidence to support : at least one mean is different.

6. Write a conclusion in context of the problem. Include the point estimates, confidence interval for the difference and p-value.

There is sufficient evidence to believe that the average flipper length is significantly different between the Adelie, Chinstrap and Gentoo species of penguins (p<.0001).

Multiple / Post-Hoc comparisons: Which group is different?

Only appropriate if your ANOVA is significant.
The overall ANOVA can be significant and NOT have any significant differences when you look at the post hoc results. The reason is that the two analyses ask two different questions.
- The ANOVA is testing the overall pattern of the data and asking if as a whole the explanatory variable has a relationship (or lack thereof) with the response variable.
- The post hoc is asking if one level of the explanatory variable is significantly different than another for the response variable. The post hoc is not as sensitive to differences as the ANOVA.
- The family-wise error rate of is maintained
Differences in group means can be non-significant at the post hoc level, but significant at the ANOVA level.

Tukey HSD

Show the code

TukeyHSD(aov(flipper_length_mm ~ species, data=pen))

  Tukey multiple comparisons of means
    95% family-wise confidence level

Fit: aov(formula = flipper_length_mm ~ species, data = pen)

$species
                      diff       lwr       upr p adj
Chinstrap-Adelie  5.869887  3.586583  8.153191     0
Gentoo-Adelie    27.233349 25.334376 29.132323     0
Gentoo-Chinstrap 21.363462 19.000841 23.726084     0

The results of the Tukey HSD post-hoc test indicate that the average flipper length in mm is significantly different between all pairs of penguin species at the 5% significance level. Chinstrap has an average flipper length 5.87mm(3.59-8.15) larger than Adelie, whereas Gentoo has an average flipper length 27.23mm(25.33-29.13) larger than Adelie.

Coefficient of Determination

Show the code

aov(flipper_length_mm ~ species, data=pen) |> summary()

             Df Sum Sq Mean Sq F value Pr(>F)    
species       2  52473   26237   594.8 <2e-16 ***
Residuals   339  14953      44                   
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
2 observations deleted due to missingness

52473/(52473 + 14953)*100

[1] 77.82309

The coefficient of determination is interpreted as the % of the variation seen in the outcome that is due to subject level variation within each of the treatment groups. The strength of this measure can be thought of in a similar manner as the correlation coefficient , indicates a poor fit, indicates a medium fit, and indicates a good fit.

77.8% of the variation in flipper length can be explained by the species of penguin

Non-parametric tests

Many stat tests rely on assumptions that ensure the sample estimate can be modeled with a normal distribution.
What do you do if your assumptions aren’t met?
We can “relax” some of those assumptions and perform a more robust, but less powerful test.
- less power means you need more data to draw a conclusion with the same amount of confidence.
No detailed examples will be provided in these notes. You tend to learn/use these on an “as-needed” basis.

Kruskal-Wallis

The Kruskal-Wallis test is the most common non-parametric method for testing whether or not groups of observations come from the same overall distribution.
By comparing the medians instead of the means, we can remove the normality assumption on the residuals.
Null hypothesis is now that the medians of all groups are equal vs at least one population median is different.

K-W and all pairwise Wilcox Tests

Show the code

kruskal.test(flipper_length_mm ~ species, data = pen)


    Kruskal-Wallis rank sum test

data:  flipper_length_mm by species
Kruskal-Wallis chi-squared = 244.89, df = 2, p-value < 2.2e-16

Show the code

pairwise.wilcox.test(pen$flipper_length_mm, pen$species)


    Pairwise comparisons using Wilcoxon rank sum test with continuity correction 

data:  pen$flipper_length_mm and pen$species 

          Adelie Chinstrap
Chinstrap 3e-08  -        
Gentoo    <2e-16 <2e-16   

P value adjustment method: holm

In this case, the non-parametric tests come to the same conclusion as the parametric ones. If there is a discrepency, go with the non-parametric test since it has fewer assumptions.