# Analysis of Variance - ANOVA 30/09/09

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1 Analysis of Variance - ANOVA Eleisa Heron 30/09/09

2 Introduction Analysis of variance (ANOVA) is a method for testing the hypothesis that there is no difference between two or more population means (usually at least three) Often used for testing the hypothesis that there is no difference between a number of treatments 2

3 Independent Two Sample t-test Recall the independent two sample t-test which is used to test the null hypothesis that the population means of two groups are the same Let and be the sample means of the two groups, then the test statistic for the independent t-test is given by: with and, sample sizes,, standard deviations The test statistic is compared with the t-distribution with degrees of freedom (df) 3

4 Why Not Use t-test Repeatedly? The t-test, which is based on the standard error of the difference between two means, can only be used to test differences between two means With more than two means, could compare each mean with each other mean using t- tests Conducting multiple t-tests can lead to severe inflation of the Type I error rate (false positives) and is NOT RECOMMENDED ANOVA is used to test for differences among several means without increasing the Type I error rate The ANOVA uses data from all groups to estimate standard errors, which can increase the power of the analysis 4

5 Why Look at Variance When Interested in Means? Three groups tightly spread about their respective means, the variability within each group is relatively small Easy to see that there is a difference between the means of the three groups 5

6 Why Look at Variance When Interested in Means? Three groups have the same means as in previous figure but the variability within each group is much larger Not so easy to see that there is a difference between the means of the three groups 6

7 Why Look at Variance When Interested in Means? To distinguish between the groups, the variability between (or among) the groups must be greater than the variability of, or within, the groups If the within-groups variability is large compared with the between-groups variability, any difference between the groups is difficult to detect To determine whether or not the group means are significantly different, the variability between groups and the variability within groups are compared 7

8 One-Way ANOVA and Assumptions One-Way ANOVA - When there is only one qualitative variable which denotes the groups and only one measurement variable (quantitative), a one-way ANOVA is carried out - For a one-way ANOVA the observations are divided into mutually exclusive categories, giving the one-way classification ASSUMPTIONS - Each of the populations is Normally distributed with the same variance (homogeneity of variance) - The observations are sampled independently, the groups under consideration are independent ANOVA is robust to moderate violations of its assumptions, meaning that the probability values (P-values) computed in an ANOVA are sufficiently accurate even if the assumptions are violated 8

9 Simulated Data Example 46 observations 15 AA observations mean IQ for AA = AG observations mean IQ for AG = GG observations mean IQ for GG =

10 Introduction of Notation Consider groups, whose means we want to compare Let be the sample size of group For the simulated verbal IQ and genotype data,, representing the three possible genotypes at the particular locus of interest. Each person in this data set, as well as having a genotype, also has a verbal IQ score Want to examine if the mean verbal IQ score is the same across the 3 genotype groups - Null hypothesis is that the mean verbal IQ is the same in the three genotype groups 10

11 Within-Groups Variance Remember assumption that the population variances of the three groups is the same Under this assumption, the three variances of the three groups all estimate this common value - True population variance = - Within-groups variance = within-groups mean square = error mean square = For groups with equal sample size this is given by the average of the variances of the groups For unequal sample sizes, the variances are weighted by their degrees of freedom 11

12 Within-Groups Variance In our example data Since the population variances are assumed to be equal, the estimate of the population variance, derived from the separate within-group estimates, is valid whether or not the null hypothesis is true 12

13 Between-Groups Variance If the null hypothesis is true, the three groups can be considered as random samples from the same population (assumed equal variances, because the null hypothesis is true, then the population means are equal) The three means are three observations from the same sampling distribution of the mean The sampling distribution of the mean has variance This gives a second method of obtaining an estimate of the population variance The observed variance of the treatment means is an estimate of and is given by 13

14 Between-Groups Variance For equal sample sizes, the between-groups variance is then given by: For unequal sample sizes, the between-groups variance is given by: 14

15 Testing the Null Hypothesis, F-test If the null hypothesis is true then the between-groups variance and the within-groups variance are both estimates of the population variance If the null hypothesis is not true, the population means are not all equal, then will be greater then the population variance, it will be increased by the treatment differences To test the null hypothesis we compare the ratio of and to 1 using an F-test F statistic is given by: with and degrees of freedom 15

17 F Distribution and F-test The F distribution is the continuous distribution of the ratio of two estimates of variance The F distribution has two parameters: degrees of freedom numerator (top) and degrees of freedom denominator (bottom) The F-test is used to test the hypothesis that two variances are equal The validity of the F-test is based on the requirement that the populations from which the variances were taken are Normal In the ANOVA, a one-sided F-test is used, why not two-sided in this case? 17

18 F Distribution 18

19 ANOVA Table Slightly more I-1 = 3-1 = 2, since complicated as the 3 genotype groups, sample sizes are AA, AG, GG not all equal ( ) = 43 For the simulated genotype, verbal IQ data: One-sided P value, statistically significant at 0.05 level Sum Sq / Df = Mean Sq 19

20 Assumption Checking Homogeneity of variance = homoscedasticity - The dependent variable (quantitative measurement) should have the same variance in each category of the independent variable (qualitative variable) - Needed since the denominator of the F-ratio is the within-group mean square, which is the average of the group variances - ANOVA is robust for small to moderate departures from homogeneity of variance, especially with equal sample sizes for the groups - Rule of thumb: the ratio of the largest to the smallest group variance should be 3:1 or less, but be careful, the more unequal the sample sizes the smaller the differences in variances which are acceptable 20

21 Assumption Checking Testing for homogeneity of variance - Levene s test of homogeneity of variance - Bartlett s test of homogeneity of variance (Chi-square test) - Examine boxplots of the data by group, will highlight visually if there is a large difference in variability between the groups - Plot residuals versus fitted values and examine scatter around zero, residuals = observations group mean, group mean = fitted value Bartlett s test: null hypothesis is that the variances in each group are the same - For the simulated genotype, IQ data: Bartlett's K-squared = , df = 2, p-value =

22 Assumption Checking Normality Assumption - The dependent variable (measurement, quantitative variable) should be Normally distributed in each category of the independent variable (qualitative variable) - Again ANOVA is robust to moderate departures from Normality Checking the Normality assumption - Boxplots of the data by group allows for the detection of skewed distributions - Quantile-Quantile plots (QQ plots) of the residuals, which should give a 45-degree line on a plot of observed versus expected values, - Usual tests for Normality may not be adequate with small sample sizes, insufficient power to detect deviations from Normality 22

23 Residuals for Simulated Genotype, IQ Data 23

24 Residuals vs Fitted for Simulated Genotype, IQ Data 24

25 Assumption Checking If data are not Normal or don t have equal variances, can consider - transforming the data, (eg. log, square root ) - non-parametric alternatives 25

26 R Code for ANOVA In R, due to the way an ANOVA is carried out the data needs to be in a specific format If the factor is numeric, R will read the analysis as if it were carrying out a regression, and not a comparison of means, (coerce the numeric into factor with as.factor()) "Genotype" " IQ" AA AA AA GG GG GG GG # Loading in the data contained in the file ANOVA_data.txt > Genotype_IQ <- read.table("anova_data.txt") # Creating a boxplot of the data > boxplot(genotype_iq[,2]~genotype_iq[,1], xlab = "Genotype", ylab = "Verbal IQ") 26

27 R Code for ANOVA # ANOVA is carried out in R using the lm linear model function > g<- lm(genotype_iq[,2]~genotype_iq[,1]) # anova function computes the ANOVA table for the fitted model object (in this case g) > anova(g) Analysis of Variance Table Response: Genotype_IQ[, 2] Df Sum Sq Mean Sq F value Pr(>F) Genotype_IQ[, 1] * Residuals Signif. codes: 0 *** ** 0.01 *

28 Summary So Far Within-Groups Mean Square - This is the average of the variances in each group - This estimates the population variance regardless of whether or not the null hypothesis is true - This is also called the error mean square Between-Groups Mean Square - This is calculated from the variance between groups - This estimates the population variance, if the null hypothesis is true Use an F-test to compare these two estimates of variance Relationship with t-test - If the one-way ANOVA is used for the comparison of two groups only, the analysis is exactly and mathematically equivalent to the use of the independent t-test, (the F statistic is exactly the square of the corresponding t statistic) 28

29 What to do with a Significant ANOVA Result (F-test) If the ANOVA is significant and the null hypothesis is rejected, the only valid inference that can be made is that at least one population mean is different from at least one other population mean The ANOVA does not reveal which population means differ from which others Some questions we might want to ask: - Is one treatment much better than all the other treatments? - Is one treatment much worse than all the other treatments? - Only think about investigating differences between individual groups when the overall comparison of groups (ANOVA) is significant, or that you had intended particular comparisons at the outset - Need to consider whether the groups are ordered or not - There are many approaches for after the ANOVA, here are some 29

30 What to do with a Significant ANOVA Result (F-test) Modified t-test: - To compare groups after the ANOVA, use a modified t-test - Modified t-test is based on the pooled estimate of variance from all the groups (within groups, residual variance in the ANOVA table), not just the pair being considered - Need to think about multiple testing corrections Least significant difference: - The least difference between two means which is significant is given by: - Arrange the treatment means in order of magnitude and the difference between any pair of means can be compared with the least significant difference 30

31 What to do with a Significant ANOVA Result (F-test) Tukey s honest significance test - The test compares the means of every group to the means of every other group - The test corrects for the multiple comparisons that are made Linear Trend - When the groups are ordered we don t want to compare each pair of groups, but rather investigate if there is a trend across the groups (linear trend) - Between groups variability can be partitioned into a component due to a linear trend and a non-linear component There are many other tests available... 31

32 Two-Way ANOVA, Factorial Designs One dependent variable (quantitative variable), two independent variables (classifying variables = factors) Advantage is that factorial designs can provide information on how the factors interact or combine in the effect that they have on the dependent variable Factor A Factor B c 1 X.X X.X X.X X.X 2 X.X X.X X.X X.X 3 X.X X.X X.X X.X : : : ; : : ; : ; ; : : ; : : r X.X X.X X.X X.X 32

33 Factorial ANOVAs Different types of effects are possible, the treatment effect: a difference in population means, can be of two types: - Main effect: a difference in population means for a factor collapsed over the levels of all other factors in the design - Interaction effect: when the effect on one factor is not the same at the levels of another Interaction: whenever the effect of one factor depends on the levels of another factor For a 2-way ANOVA, 8 possible outcomes that could have occurred: - Nothing - Main effect of factor A - Main effect of factor B - Both main effects (factor A and factor B) - AxB interaction - AxB interaction and main effect of factor A - AxB interaction and main effect of factor B - AxB interaction and both main effects (factors A and B) 33

34 Interactions in ANOVA Factor 1 Factor 2 AA AG GG F M Factor 1 Factor 2 AA AG GG F M

35 Interactions in ANOVA Factor 1 Factor 2 AA AG GG F M Factor 1 Factor 2 AA AG GG F M

36 Interactions in ANOVA Factor 1 Factor 2 AA AG GG F M Factor 1 Factor 2 AA AG GG F M

37 Repeated Measures ANOVA Repeated measures ANOVA is used when all members of a random sample are measured under a number of different conditions A standard ANOVA would treat the data as independent and fail to model the correlation between the repeated measures Time (mins) Subject X X X X 2 X X X X 3 X X X X : : : ; : : ; : ; ; : : ; : : 20 X X X 37

38 ANCOVA ANCOVA: Analysis of Covariance - Extend the ANOVA to include a qualitative independent variable (covariate) - Used to reduce the within group error variance - Used to eliminate confounders - Most useful when the covariate is linearly related to the dependent variable and is not related to the factors (independent qualitative variables) - Similar assumptions to the ANOVA 38

39 Non Parametric ANOVA Kruskal-Wallis Test ANOVA is the more general form of the t-test Kruskal-Wallis test is the more general form of the Mann-Whitney test Kruskal-Wallis test, doesn t assume normality, compares medians: - Rank the complete set of observations, 1 to (regardless of the group they belong to) - For each group, calculate the sum of the ranks - Let the sum of the ranks of the observations in group be, then the average rank in each group is given by - The statistic is defined as: average of all the ranks When the null hypothesis is true, follows a Chi squared distribution degrees of freedom Any variation among the groups will increase the test statistic (upper tail test) 30/09/09 Introduction to ANOVA 39

40 Non-Parametric Two-Way ANOVA Friedman s two way analysis of variance non-parametric hypothesis test - It s based on ranking the data in each row of the table from low to high - Each row is ranked separately - The ranks are then summed in each column (group) - The test is based on a Chi squared distribution - Just like with the ANOVA the Friedman test will only indicate a difference but won t say where the difference lies 40

41 Literature Example The MTHFR 677C-T Polymorphism and Behaviors in Children With Autism: Exploratory Genotype Phenotype Correlations Robin P. Goin-Kochel, Anne E. Porter, Sarika U. Peters, Marwan Shinawi, Trilochan Sahoo, and Arthur L. Beaudet Autism Research 2: ,

42 Literature Example No Evidence for an Effect of COMT Val158Met Genotype on Executive Function in Patients With 22q11 Deletion Syndrome Bronwyn Glaser, Martin Debbane, Christine Hinard, Michael A. Morris, Sophie P. Dahoun, Stylianos E., Antonarakis, Stephan Eliez Am J Psychiatry 2006; 163:

43 Further Reading BOOKS: Interpretation and Uses of Medical Statistics by Leslie E Daly and Geoffrey J. Bourke, Blackwell science, 2000 Practical Statistics for Medical Research by Douglas G. Altman, Chapman and Hall,

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