Experimental Design and the Analysis of Variance Comparing t > 2 Groups - Numeric Responses • Extension of Methods used to Compare 2 Groups • Independent Samples and Paired Data Designs • Normal and non-normal data distributions Data Design Normal Nonnormal Independent Samples (CRD) Paired Data (RBD) F-Test 1-Way ANOVA F-Test 2-Way ANOVA KruskalWallis Test Friedman’s Test Completely Randomized Design (CRD) • Controlled Experiments - Subjects assigned at random to one of the t treatments to be compared • Observational Studies - Subjects are sampled from t existing groups • Statistical model yij is measurement from the jth subject from group i: yij i ij i ij where is the overall mean, i is the effect of treatment i , ij is a random error, and i is the population mean for group i 1-Way ANOVA for Normal Data (CRD) • For each group obtain the mean, standard deviation, and sample size: y i. yij j ni si 2 ( y y ) ij i. j ni 1 • Obtain the overall mean and sample size N n1 ... nt n1 y1. ... nt y t . i j yij y .. N N Analysis of Variance - Sums of Squares • Total Variation TSS i 1 j 1 ( yij y.. ) 2 k ni dfTotal N 1 • Between Group (Sample) Variation SST i 1 ji1 ( y i. y .. ) 2 i 1 ni ( y i. y.. ) 2 t n t dfT t 1 • Within Group (Sample) Variation SSE i 1 j 1 ( yij y i. ) i 1 (ni 1) si2 t ni TSS SST SSE 2 t dfTotal dfT df E df E N t Analysis of Variance Table and F-Test Source of Variation Treatments Error Total Sum of Squares SST SSE TSS Degrres of Freedom t-1 N-t N-1 Mean Square MST=SST/(t-1) MSE=SSE/(N-t) F F=MST/MSE • Assumption: All distributions normal with common variance •H0: No differences among Group Means (1 t =0) • HA: Group means are not all equal (Not all i are 0) MST T .S . : Fobs MSE R.R. : Fobs F ,t 1, N t P val : P( F Fobs ) (Table 9) Expected Mean Squares • Model: yij = +i + ij with ij ~ N(0,s2), Si = 0: E ( MSE ) s 2 t E ( MST ) s 2 2 n i i i 1 t 1 t s 2 E ( MST ) E ( MSE ) 2 n i i t i 1 t 1 s2 1 2 n i i i 1 2 s (t 1) E ( MST ) When H 0 : 1 t 0 is true, 1 E ( MSE ) E ( MST ) otherwise ( H a is true), 1 E ( MSE ) Expected Mean Squares • 3 Factors effect magnitude of F-statistic (for fixed t) – True group effects (1,…,t) – Group sample sizes (n1,…,nt) – Within group variance (s2) • Fobs = MST/MSE • When H0 is true (1=…=t=0), E(MST)/E(MSE)=1 • Marginal Effects of each factor (all other factors fixed) – As spread in (1,…,t) E(MST)/E(MSE) – As (n1,…,nt) E(MST)/E(MSE) (when H0 false) – As s2 E(MST)/E(MSE) (when H0 false) 0.09 0.09 0.08 0.08 0.07 0.07 0.06 0.06 0.05 0.05 0.04 0.04 0.03 E ( MST ) E ( MSE ) 0.02 0.01 0 0 20 40 60 80 100 120 140 160 180 200 A) =100, t1=-20, t2=0, t3=20, s = 20 n 4 8 12 20 0.09 0.08 0.07 A 9 17 25 41 B 129 257 385 641 C 1.5 2 2.5 3.5 0.03 0.02 0.01 0 0 20 40 60 80 100 120 140 160 180 200 B) =100, t1=-20, t2=0, t3=20, s = 5 D 9 17 25 41 0.09 0.08 0.06 0.07 0.05 0.06 0.04 0.05 0.03 0.04 0.02 0.03 0.01 0.02 0 0.01 0 20 40 60 80 100 120 140 160 180 0 0 C) =100, t1=-5, t2=0, t3=5, s = 20 20 40 60 80 100 120 140 160 180 D) =100, t1=-5, t2=0, t3=5, s = 5 200 Example - Seasonal Diet Patterns in Ravens • “Treatments” - t = 4 seasons of year (3 “replicates” each) – – – – Winter: November, December, January Spring: February, March, April Summer: May, June, July Fall: August, September, October • Response (Y) - Vegetation (percent of total pellet weight) • Transformation (For approximate normality): Y Y ' arcsin 100 Source: K.A. Engel and L.S. Young (1989). “Spatial and Temporal Patterns in the Diet of Common Ravens in Southwestern Idaho,” The Condor, 91:372-378 Seasonal Diet Patterns in Ravens - Data/Means Y j=1 j=2 j=3 Winter(i=1) 94.3 90.3 83.0 Fall(i=2) 80.7 90.5 91.8 Summer(i=3) 80.5 74.3 32.4 Fall (i=4) 67.8 91.8 89.3 Y' j=1 j=2 j=3 Winter(i=1) 1.329721 1.254080 1.145808 Fall(i=2) 1.115957 1.257474 1.280374 Summer(i=3) 1.113428 1.039152 0.605545 Fall (i=4) 0.967390 1.280374 1.237554 1.329721 1.254080 1.145808 y 1. 1.24203 3 1.115957 1.257474 1.280374 y 2. 1.217935 3 1.113428 1.039152 0.605545 y 3. 0.919375 3 0.967390 1.280374 1.237554 y 4. 1.16773 3 1.329721 ... 1.237554 y .. 1.135572 12 Seasonal Diet Patterns in Ravens - Data/Means Plot of Transformed Data by Season 1.500000 1.400000 1.300000 Transformed % Vegetation 1.200000 1.100000 1.000000 0.900000 0.800000 0.700000 0.600000 0.500000 0 1 2 3 Season 4 5 Seasonal Diet Patterns in Ravens - ANOVA Total Variation : (dfTotal 12 - 1 11) TSS (1.329721 1.135572) 2 ... (1.27554 1.135572) 2 0.438425 Between Group Variation : (dfT 4 - 1 3) SST 3 (1.24203 1.135572) 2 ... (1.161773 1.135572) 2 0.197387 Within Group Variation : (dfE 12 - 4 8) SSE (1.329721 1.243203) 2 ... (1.237554 1.161773) 2 0.241038 ANOVA Source of Variation Between Groups Within Groups SS 0.197387 0.241038 df 3 8 Total 0.438425 11 MS F P-value 0.065796 2.183752 0.167768 0.03013 F crit 4.06618 Do not conclude that seasons differ with respect to vegetation intake Seasonal Diet Patterns in Ravens - Spreadsheet Month NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT Season 1 1 1 2 2 2 3 3 3 4 4 4 Y' 1.329721 1.254080 1.145808 1.115957 1.257474 1.280374 1.113428 1.039152 0.605545 0.967390 1.280374 1.237554 Season MeanOverall Mean 1.243203 1.135572 1.243203 1.135572 1.243203 1.135572 1.217935 1.135572 1.217935 1.135572 1.217935 1.135572 0.919375 1.135572 0.919375 1.135572 0.919375 1.135572 1.161773 1.135572 1.161773 1.135572 1.161773 1.135572 Sum TSS 0.037694 0.014044 0.000105 0.000385 0.014860 0.020968 0.000490 0.009297 0.280928 0.028285 0.020968 0.010400 0.438425 Total SS Between Season SS (Y’-Overall Mean)2 (Group Mean-Overall Mean)2 SST 0.011584 0.011584 0.011584 0.006784 0.006784 0.006784 0.046741 0.046741 0.046741 0.000687 0.000687 0.000687 0.197387 SSE 0.007485 0.000118 0.009486 0.010400 0.001563 0.003899 0.037657 0.014346 0.098489 0.037785 0.014066 0.005743 0.241038 Within Season SS (Y’-Group Mean)2 CRD with Non-Normal Data Kruskal-Wallis Test • Extension of Wilcoxon Rank-Sum Test to k > 2 Groups • Procedure: – Rank the observations across groups from smallest (1) to largest ( N = n1+...+nk ), adjusting for ties – Compute the rank sums for each group: T1,...,Tk . Note that T1+...+Tk = N(N+1)/2 Kruskal-Wallis Test •H0: The k population distributions are identical (1=...=k) •HA: Not all k distributions are identical (Not all i are equal) 2 12 k Ti T .S . : H 3 ( N 1 ) N ( N 1) i 1 ni R.R. : H ,k 1 2 P val : P( H ) 2 An adjustment to H is suggested when there are many ties in the data. Formula is given on page 344 of O&L. Example - Seasonal Diet Patterns in Ravens Month NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT Season 1 1 1 2 2 2 3 3 3 4 4 4 Y' 1.329721 1.254080 1.145808 1.115957 1.257474 1.280374 1.113428 1.039152 0.605545 0.967390 1.280374 1.237554 H 0 : No seasonal difference Rank 12 8 6 5 9 10.5 4 3 1 2 10.5 7 • T1 = 12+8+6 = 26 • T2 = 5+9+10.5 = 24.5 • T3 = 4+3+1 = 8 • T4 = 2+10.5+7 = 19.5 H a : Seasonal Difference s (26) 2 (24.5) 2 (8) 2 (19.5) 2 12 T .S . : H 3(12 1) 44.12 39 5.12 12(12 1) 3 3 3 3 R.R.( 0.05) : H .205, 41 7.815 P value : P( 2 H 5.12) .1632 Post-hoc Comparisons of Treatments • If differences in group means are determined from the Ftest, researchers want to compare pairs of groups. Three popular methods include: – Fisher’s LSD - Upon rejecting the null hypothesis of no differences in group means, LSD method is equivalent to doing pairwise comparisons among all pairs of groups as in Chapter 6. – Tukey’s Method - Specifically compares all t(t-1)/2 pairs of groups. Utilizes a special table (Table 11, p. 701). – Bonferroni’s Method - Adjusts individual comparison error rates so that all conclusions will be correct at desired confidence/significance level. Any number of comparisons can be made. Very general approach can be applied to any inferential problem Fisher’s Least Significant Difference Procedure • Protected Version is to only apply method after significant result in overall F-test • For each pair of groups, compute the least significant difference (LSD) that the sample means need to differ by to conclude the population means are not equal LSDij t / 2 1 1 MSE n n j i with df N t Conclude i j if y i. y j . LSDij Fisher' s Confidence Interval : y i. y j . LSDij Tukey’s W Procedure • More conservative than Fisher’s LSD (minimum significant difference and confidence interval width are higher). • Derived so that the probability that at least one false difference is detected is (experimentwise error rate) Wij q (t , ) MSE n q given in Table 11, p. 701 with N-t Conclude i j if y i. y j . Wij Tukey' s Confidence Interval : y i. y j . Wij When the sample sizes are unequal, use n t 1 1 n1 nt Bonferroni’s Method (Most General) • Wish to make C comparisons of pairs of groups with simultaneous confidence intervals or 2-sided tests •When all pair of treatments are to be compared, C = t(t-1)/2 • Want the overall confidence level for all intervals to be “correct” to be 95% or the overall type I error rate for all tests to be 0.05 • For confidence intervals, construct (1-(0.05/C))100% CIs for the difference in each pair of group means (wider than 95% CIs) • Conduct each test at =0.05/C significance level (rejection region cut-offs more extreme than when =0.05) • Critical t-values are given in table on class website, we will use notation: t/2,C, where C=#Comparisons, = df Bonferroni’s Method (Most General) 1 1 Bij t / 2,C ,v MSE n n j i (t given on class website with v N-t ) Conclude i j if y i. y j . Bij Bonferroni ' s Confidence Interval : y i. y j . Bij Example - Seasonal Diet Patterns in Ravens Note: No differences were found, these calculations are only for demonstration purposes MSE 0.03013 ni 3 t.025,8 2.306 q.05,t 4,df E 8 4.53 t.025,C 6,df E 8 3.479 1 1 LSDij 2.306 (0.03013) 0.3268 3 3 1 Wij 4.53 (0.03013) 0.4540 3 1 1 Bij 3.479 (0.03013) 0.4930 3 3 Comparison(i vs j) Group i Mean Group j MeanDifference 1 vs 2 1.243203 1.217935 0.025267 1 vs 3 1.243203 0.919375 0.323828 1 vs 4 1.243203 1.161773 0.081430 2 vs 3 1.217935 0.919375 0.298560 2 vs 4 1.217935 1.161773 0.056162 3 vs 4 0.919375 1.161773 -0.242398 Randomized Block Design (RBD) • t > 2 Treatments (groups) to be compared • b Blocks of homogeneous units are sampled. Blocks can be individual subjects. Blocks are made up of t subunits • Subunits within a block receive one treatment. When subjects are blocks, receive treatments in random order. • Outcome when Treatment i is assigned to Block j is labeled Yij • Effect of Trt i is labeled i • Effect of Block j is labeled bj • Random error term is labeled ij • Efficiency gain from removing block-to-block variability from experimental error Randomized Complete Block Designs • Model: Yij i b j ij i b j ij t i 0 E ( ij ) 0 i 1 V ( ij ) s 2 • Test for differences among treatment effects: • H0: 1 ... t 0 • HA: Not all i = 0 (1 ... t ) (Not all i are equal) Typically not interested in measuring block effects (although sometimes wish to estimate their variance in the population of blocks). Using Block designs increases efficiency in making inferences on treatment effects RBD - ANOVA F-Test (Normal Data) • Data Structure: (t Treatments, b Subjects) • Mean for Treatment i: y i. • Mean for Subject (Block) j: • Overall Mean: y. j y .. • Overall sample size: N = bt • ANOVA:Treatment, Block, and Error Sums of Squares TSS i 1 j 1 yij y .. t b SSB t y SSE y 2 df Total bt 1 y SST bi 1 y i . y .. 2 df T t 1 b 2 df B b 1 t j 1 .j .. 2 ij y i. y . j y .. TSS SST SSB df E (b 1)(t 1) RBD - ANOVA F-Test (Normal Data) • ANOVA Table: Source Treatments Blocks Error Total SS SST SSB SSE TSS df t-1 b-1 (b-1)(t-1) bt-1 MS MST = SST/(t-1) MSB = SSB/(b-1) MSE = SSE/[(b-1)(t-1)] •H0: 1 ... t 0 (1 ... t ) • HA: Not all i = 0 T .S . : Fobs R.R. : Fobs (Not all i are equal) MST MSE F ,t 1,( b 1)( t 1) P val : P ( F Fobs ) F F = MST/MSE Pairwise Comparison of Treatment Means • Tukey’s Method- q in Studentized Range Table with = (b-1)(t-1) MSE Wij q (t , v) b Conclude i j if y i. y j . Wij Tukey' s Confidence Interval : y i. y j . Wij • Bonferroni’s Method - t-values from table on class website with = (b-1)(t-1) and C=t(t-1)/2 Bij t / 2,C ,v 2 MSE b Conclude i j if y i. y j . Bij Bonferroni ' s Confidence Interval : y i. y j . Bij Expected Mean Squares / Relative Efficiency • Expected Mean Squares: As with CRD, the Expected Mean Squares for Treatment and Error are functions of the sample sizes (b, the number of blocks), the true treatment effects (1,…,t) and the variance of the random error terms (s2) • By assigning all treatments to units within blocks, error variance is (much) smaller for RBD than CRD (which combines block variation&random error into error term) • Relative Efficiency of RBD to CRD (how many times as many replicates would be needed for CRD to have as precise of estimates of treatment means as RBD does): MSECR (b 1) MSB b(t 1) MSE RE ( RCB , CR) MSE RCB (bt 1) MSE Example - Caffeine and Endurance • • • • Treatments: t=4 Doses of Caffeine: 0, 5, 9, 13 mg Blocks: b=9 Well-conditioned cyclists Response: yij=Minutes to exhaustion for cyclist j @ dose i Data: Dose \ Subject 0 5 9 13 1 36.05 42.47 51.50 37.55 2 52.47 85.15 65.00 59.30 3 56.55 63.20 73.10 79.12 4 45.20 52.10 64.40 58.33 5 35.25 66.20 57.45 70.54 6 66.38 73.25 76.49 69.47 7 40.57 44.50 40.55 46.48 8 57.15 57.17 66.47 66.35 9 28.34 35.05 33.17 36.20 Plot of Y versus Subject by Dose 90.00 80.00 70.00 Time to Exhaustion 60.00 50.00 0 mg 5 mg 9mg 40.00 13 mg 30.00 20.00 10.00 0.00 0 1 2 3 4 5 Cyclist 6 7 8 9 10 Example - Caffeine and Endurance Subject\Dose 1 2 3 4 5 6 7 8 9 Dose Mean Dose Dev Squared Dev TSS 0mg 36.05 52.47 56.55 45.20 35.25 66.38 40.57 57.15 28.34 46.44 -8.80 77.38 5mg 42.47 85.15 63.20 52.10 66.20 73.25 44.50 57.17 35.05 57.68 2.44 5.95 9mg 51.50 65.00 73.10 64.40 57.45 76.49 40.55 66.47 33.17 58.68 3.44 11.86 13mg 37.55 59.30 79.12 58.33 70.54 69.47 46.48 66.35 36.20 58.15 2.91 8.48 Subj MeanSubj Dev Sqr Dev 41.89 -13.34 178.07 65.48 10.24 104.93 67.99 12.76 162.71 55.01 -0.23 0.05 57.36 2.12 4.51 71.40 16.16 261.17 43.03 -12.21 149.12 61.79 6.55 42.88 33.19 -22.05 486.06 55.24 1389.50 103.68 7752.773 TSS (36.05 55.24) 2 (36.20 55.24) 2 7752.773 dfTotal 4(9) 1 35 SSB 4(41.89 55.24) (33.19 55.24) 4(1389.50) 5558.00 SST 9 (46.44 55.24) 2 (58.15 55.24) 2 9(103.68) 933.12 dfT 4 1 3 2 2 df B 9 1 8 SSE (36.05 41.89 46.44 55.24) 2 (36.20 33.19 58.15 55.24) 2 TSS SST SSB 7752.773 933.12 5558 1261.653 df E (4 1)(9 1) 24 Example - Caffeine and Endurance Source Dose Cyclist Error Total df 3 8 24 35 SS 933.12 5558.00 1261.65 7752.77 MS 311.04 694.75 52.57 H 0 : No Caffeine Dose Effect (1 4 0) H A : Difference s Exist Among Doses MST 311.04 T .S . : Fobs 5.92 MSE 52.57 R.R.( 0.05) : Fobs F.05,3, 24 3.01 P value : P( F 5.92) .0036 (From EXCEL) Conclude that true means are not all equal F 5.92 Example - Caffeine and Endurance Tukey' s W : q.05, 4, 24 1 3.90 W 3.90 52.57 9.43 9 Bonferroni ' s B : t.05 / 2, 6, 24 Doses 5mg vs 0mg 9mg vs 0mg 13mg vs 0mg 9mg vs 5mg 13mg vs 5mg 13mg vs 9mg 2 2.875 B 2.875 52.57 9.83 9 High Mean 57.6767 58.6811 58.1489 58.6811 58.1489 58.1489 Low Mean Difference Conclude 46.4400 11.2367 50 46.4400 12.2411 90 46.4400 11.7089 130 57.6767 1.0044 NSD 57.6767 0.4722 NSD 58.6811 -0.5322 NSD Example - Caffeine and Endurance Relative Efficiency of Randomized Block to Completely Randomized Design : t 4 b 9 MSB 694.75 MSE 52.57 (b 1) MSB b(t 1) MSE 8(694.75) 9(3)(52.57) 6977.39 RE ( RCB , CR) 3.79 (bt 1) MSE (9(4) 1)(52.57) 1839.95 Would have needed 3.79 times as many cyclists per dose to have the same precision on the estimates of mean endurance time. • 9(3.79) 35 cyclists per dose • 4(35) = 140 total cyclists RBD -- Non-Normal Data Friedman’s Test • When data are non-normal, test is based on ranks • Procedure to obtain test statistic: – Rank the k treatments within each block (1=smallest, k=largest) adjusting for ties – Compute rank sums for treatments (Ti) across blocks – H0: The k populations are identical (1=...=k) – HA: Differences exist among the k group means 12 k 2 T .S . : Fr T 3b(k 1) i 1 i bk (k 1) R.R. : Fr 2 ,k 1 P val : P( 2 Fr ) Example - Caffeine and Endurance Subject\Dose 1 2 3 4 5 6 7 8 9 0mg 36.05 52.47 56.55 45.2 35.25 66.38 40.57 57.15 28.34 5mg 42.47 85.15 63.2 52.1 66.2 73.25 44.5 57.17 35.05 9mg 51.5 65 73.1 64.4 57.45 76.49 40.55 66.47 33.17 13mg 37.55 59.3 79.12 58.33 70.54 69.47 46.48 66.35 36.2 Ranks Total 0mg 1 1 1 1 1 1 2 1 1 10 5mg 3 4 2 2 3 3 3 2 3 25 9mg 4 3 3 4 2 4 1 4 2 27 13mg 2 2 4 3 4 2 4 3 4 28 H 0 : No Dose Difference s H a : Dose Difference s Exist 12 26856 2 2 T .S . : Fr (10) (28) 3(9)( 4 1) 135 14.2 9(4)( 4 1) 180 R.R.( 0.05) : Fr .205, 41 7.815 P - value : P( 2 14.2) .0026 (From EXCEL) Conclude Means (Medians) are not all equal Latin Square Design • Design used to compare t treatments when there are two sources of extraneous variation (types of blocks), each observed at t levels • Best suited for analyses when t 10 • Classic Example: Car Tire Comparison – Treatments: 4 Brands of tires (A,B,C,D) – Extraneous Source 1: Car (1,2,3,4) – Extraneous Source 2: Position (Driver Front, Passenger Front, Driver Rear, Passenger Rear) Car\Position 1 2 3 4 DF A B C D PF B C D A DR C D A B PR D A B C Latin Square Design - Model • Model (t treatments, rows, columns, N=t2) : yijk k b i k ijk Overall Mean ^ y ... ^ k Effect of Treatment k k y .. k y ... bi j Effect due to row i ^ b i y i.. y ... ^ Effect due to Column j j y . j . y ... ijk Random Error Term Latin Square Design - ANOVA & F-Test t t Total Sum of Squares : TSS yijk y ... 2 df t 2 1 i 1 j 1 t Treatment Sum of Squares SST t y .. k y ... k 1 t Row Sum of Squares SSR t y i.. y ... Column Sum of Squares SSC t y . j . y ... j 1 dfT t 1 2 i 1 t 2 df R t 1 2 df C t 1 Error Sum of Squares SSE TSS SST SSR SSC • H0: 1 = … = t = 0 df E (t 2 1) 3(t 1) (t 1)(t 2) Ha: Not all k = 0 • TS: Fobs = MST/MSE = (SST/(t-1))/(SSE/((t-1)(t-2))) • RR: Fobs F, t-1, (t-1)(t-2) Pairwise Comparison of Treatment Means • Tukey’s Method- q in Studentized Range Table with = (t-1)(t-2) MSE Wij q (t , v) t Conclude i j if y i. y j . Wij Tukey' s Confidence Interval : y i. y j . Wij • Bonferroni’s Method - t-values from table on class website with = (t-1)(t-2) and C=t(t-1)/2 Bij t / 2,C ,v 2 MSE t Conclude i j if y i. y j . Bij Bonferroni ' s Confidence Interval : y i. y j . Bij Expected Mean Squares / Relative Efficiency • Expected Mean Squares: As with CRD, the Expected Mean Squares for Treatment and Error are functions of the sample sizes (t, the number of blocks), the true treatment effects (1,…,t) and the variance of the random error terms (s2) • By assigning all treatments to units within blocks, error variance is (much) smaller for LS than CRD (which combines block variation&random error into error term) • Relative Efficiency of LS to CRD (how many times as many replicates would be needed for CRD to have as precise of estimates of treatment means as LS does): MSECR MSR MSC (t 1) MSE RE ( LS , CR) MSE LS (t 1) MSE 2-Way ANOVA • 2 nominal or ordinal factors are believed to be related to a quantitative response • Additive Effects: The effects of the levels of each factor do not depend on the levels of the other factor. • Interaction: The effects of levels of each factor depend on the levels of the other factor • Notation: ij is the mean response when factor A is at level i and Factor B at j 2-Way ANOVA - Model yijk i b j bij ijk i 1,..., a j 1,..., b k 1,..., r yijk Measuremen t on k th unit receiving Factors A at level i, B at level j Overall Mean i Effect of i th level of factor A b j Effect of j th level of factor B bij Interactio n effect whe n i th level of A and j th level of B are combined ijk Random Error Terms •Model depends on whether all levels of interest for a factor are included in experiment: • Fixed Effects: All levels of factors A and B included • Random Effects: Subset of levels included for factors A and B • Mixed Effects: One factor has all levels, other factor a subset Fixed Effects Model • Factor A: Effects are fixed constants and sum to 0 • Factor B: Effects are fixed constants and sum to 0 • Interaction: Effects are fixed constants and sum to 0 over all levels of factor B, for each level of factor A, and vice versa • Error Terms: Random Variables that are assumed to be independent and normally distributed with mean 0, variance s2 a i 0, i 1 b bj 0 j 1 a bij 0 j i 1 2 b 0 i ~ N 0 , s ij ijk b j 1 Example - Thalidomide for AIDS • • • • Response: 28-day weight gain in AIDS patients Factor A: Drug: Thalidomide/Placebo Factor B: TB Status of Patient: TB+/TBSubjects: 32 patients (16 TB+ and 16 TB-). Random assignment of 8 from each group to each drug). Data: – – – – Thalidomide/TB+: 9,6,4.5,2,2.5,3,1,1.5 Thalidomide/TB-: 2.5,3.5,4,1,0.5,4,1.5,2 Placebo/TB+: 0,1,-1,-2,-3,-3,0.5,-2.5 Placebo/TB-: -0.5,0,2.5,0.5,-1.5,0,1,3.5 ANOVA Approach • Total Variation (TSS) is partitioned into 4 components: – Factor A: Variation in means among levels of A – Factor B: Variation in means among levels of B – Interaction: Variation in means among combinations of levels of A and B that are not due to A or B alone – Error: Variation among subjects within the same combinations of levels of A and B (Within SS) Analysis of Variance a b r Total Variation : TSS yijk y ... 2 dfTotal abr 1 i 1 j 1 k 1 a Factor A Sum of Squares : SSA br y i.. y ... 2 df A a 1 i 1 b Factor B Sum of Squares : SSB ar y . j . y ... 2 j 1 a b df B b 1 Interactio n Sum of Squares : SSAB r y ij. y i.. y . j . y ... 2 i 1 j 1 a b r Error Sum of Squares : SSE yijk y ij. i 1 j 1 k 1 • TSS = SSA + SSB + SSAB + SSE • dfTotal = dfA + dfB + dfAB + dfE 2 df E ab(r 1) df AB (a 1)(b 1) ANOVA Approach - Fixed Effects Source Factor A Factor B Interaction Error Total df a-1 b-1 (a-1)(b-1) ab(r-1) abr-1 SS SSA SSB SSAB SSE TSS MS MSA=SSA/(a-1) MSB=SSB/(b-1) MSAB=SSAB/[(a-1)(b-1)] MSE=SSE/[ab(r-1)] F FA=MSA/MSE FB=MSB/MSE FAB=MSAB/MSE • Procedure: • First test for interaction effects • If interaction test not significant, test for Factor A and B effects Test for Interactio n : Test for Factor A Test for Factor B H 0 : b11 ... bab 0 H 0 : 1 ... a 0 H 0 : b1 ... b b 0 H a : Not all bij 0 H a : Not all i 0 H a : Not all b j 0 MSAB TS : FAB MSE RR : FAB F ,( a 1)( b 1),ab( r 1) MSA MSB TS : FA TS : FB MSE MSE RR : FA F ,( a 1),ab( r 1) RR : FB F ( b 1),ab( r 1) Example - Thalidomide for AIDS Individual Patients Group Means tb 7.5 Negative Positive 3.000 wtgain 5.0 2.5 0.0 -2.5 meanwg 2.000 1.000 0.000 -1.000 Placebo Placebo Thalidomide Thalidomide drug drug Report WTGAIN GROUP TB+/Thalidomide TB-/Thalidomide TB+/Placebo TB-/Placebo Total Mean 3.688 2.375 -1.250 .688 1.375 N 8 8 8 8 32 Std. Deviation 2.6984 1.3562 1.6036 1.6243 2.6027 Example - Thalidomide for AIDS Tests of Between-Subjects Effects Dependent Variable: WTGAIN Type III Sum Source of Squares df Mean Square a Corrected Model 109.688 3 36.563 Intercept 60.500 1 60.500 DRUG 87.781 1 87.781 TB .781 1 .781 DRUG * TB 21.125 1 21.125 Error 100.313 28 3.583 Total 270.500 32 Corrected Total 210.000 31 a. R Squared = .522 (Adjusted R Squared = .471) F 10.206 16.887 24.502 .218 5.897 Sig. .000 .000 .000 .644 .022 • There is a significant Drug*TB interaction (FDT=5.897, P=.022) • The Drug effect depends on TB status (and vice versa) Comparing Main Effects (No Interaction) • Tukey’s Method- q in Studentized Range Table with = ab(r-1) MSE Wij q (a, v) br A MSE Wij q (b, v) ar B Conclude : i j if y i.. y j .. WijA b i b j if y .i. y . j . W B Tukey' s CI : ( i j ) : y i.. y j .. WijA ij ( b i b j ) : y .i. y . j . WijB • Bonferroni’s Method - t-values in Bonferroni table with =ab (r-1) Bij t / 2,a ( a 1) / 2,v A 2MSE br Bij t / 2,b (b 1) / 2,v B Conclude : i j if y i.. y j .. BijA 2MSE ar b i b j if y .i. y . j . B B Bonferroni ' s CI : (αi -α j ) : y i.. y j .. B ijA ij ( b i -b j ) : y .i. y . j . B ijB Comparing Main Effects (Interaction) • Tukey’s Method- q in Studentized Range Table with = ab(r-1) MSE Wij q (a, v) r A Within k th level of Factor B, Conclude : i j if y ik. y j . WijA Tukey' s CI : ( i j ) : y ik. y jk. WijA Similar for Factor B in A • Bonferroni’s Method - t-values in Bonferroni table with =ab (r-1) Bij t / 2,a ( a 1) / 2,v A 2MSE r Within k th level of B, Conclude : i j if y ik. y jk. BijA Bonferroni ' s CI : (αi -α j ) : y ik. y jk. BijA Miscellaneous Topics • 2-Factor ANOVA can be conducted in a Randomized Block Design, where each block is made up of ab experimental units. Analysis is direct extension of RBD with 1-factor ANOVA • Factorial Experiments can be conducted with any number of factors. Higher order interactions can be formed (for instance, the AB interaction effects may differ for various levels of factor C). • When experiments are not balanced, calculations are immensely messier and you must use statistical software packages for calculations Mixed Effects Models • Assume: – Factor A Fixed (All levels of interest in study) 1 2 … 0 – Factor B Random (Sample of levels used in study) bj ~ N(0,sb2) (Independent) • AB Interaction terms Random b)ij ~ N(0,sab2 (Independent) • Analysis of Variance is computed exactly as in Fixed Effects case (Sums of Squares, df’s, MS’s) • Error terms for tests change (See next slide). ANOVA Approach – Mixed Effects Source Factor A Factor B Interaction Error Total df a-1 b-1 (a-1)(b-1) ab(r-1) abr-1 SS SSA SSB SSAB SSE TSS MS MSA=SSA/(a-1) MSB=SSB/(b-1) MSAB=SSAB/[(a-1)(b-1)] MSE=SSE/[ab(r-1)] F FA=MSA/MSAB FB=MSB/MSAB FAB=MSAB/MSE • Procedure: • First test for interaction effects • If interaction test not significant, test for Factor A and B effects Test for Interactio n : Test for Factor A Test for Factor B 2 H 0 : s ab 0 H 0 : 1 ... a 0 H 0 : s b2 0 2 H a : s ab 0 H a : Not all i 0 H a : s b2 0 MSAB TS : FAB MSE RR : FAB F ,( a 1)( b 1),ab( r 1) MSA MSB TS : FA TS : FB MSAB MSAB RR : FA F ,( a 1),( a 1)( b 1) RR : FB F (b 1),( a 1)( b 1) Comparing Main Effects for A (No Interaction) • Tukey’s Method- q in Studentized Range Table with = (a-1)(b-1) MSAB Wij q (a, v) br A Conclude : i j if y i.. y j .. WijA Tukey' s CI : ( i j ) : y i.. y j .. WijA • Bonferroni’s Method - t-values in Bonferroni table with = (a-1)(b-1) Bij t / 2,a ( a 1) / 2,v A 2MSAB br Conclude : i j if y i.. y j .. BijA Bonferroni ' s CI : (αi -α j ) : y i.. y j .. BijA Random Effects Models • Assume: – Factor A Random (Sample of levels used in study) i ~ N(0,sa2) (Independent) – Factor B Random (Sample of levels used in study) bj ~ N(0,sb2) (Independent) • AB Interaction terms Random b)ij ~ N(0,sab2 (Independent) • Analysis of Variance is computed exactly as in Fixed Effects case (Sums of Squares, df’s, MS’s) • Error terms for tests change (See next slide). ANOVA Approach – Mixed Effects Source Factor A Factor B Interaction Error Total df a-1 b-1 (a-1)(b-1) ab(n-1) abn-1 SS SSA SSB SSAB SSE TSS MS MSA=SSA/(a-1) MSB=SSB/(b-1) MSAB=SSAB/[(a-1)(b-1)] MSE=SSE/[ab(n-1)] F FA=MSA/MSAB FB=MSB/MSAB FAB=MSAB/MSE • Procedure: • First test for interaction effects • If interaction test not significant, test for Factor A and B effects Test for Interactio n : 2 H 0 : s ab 0 2 H a : s ab 0 MSAB TS : FAB MSE RR : FAB F ,( a 1)( b 1),ab( r 1) Test for Factor A Test for Factor B H 0 : s a2 0 H 0 : s b2 0 H a : s a2 0 H a : s b2 0 MSA MSB TS : FA TS : FB MSAB MSAB RR : FA F ,( a 1),( a 1)( b 1) RR : FB F (b 1),( a 1)( b 1) Nested Designs • Designs where levels of one factor are nested (as opposed to crossed) wrt other factor • Examples Include: – – – – – Classrooms nested within schools Litters nested within Feed Varieties Hair swatches nested within shampoo types Swamps of varying sizes (e.g. large, medium, small) Restaurants nested within national chains Nested Design - Model Yijk i b j (i ) ijk i 1,...a j 1,..., bi k 1,..., r where : Yijk Response for k th rep of Factor A at i th level, B at jth level within A Overall Mean i Effect of i th level of A (Fixed or Random) b j (i ) Effect of jth level of B within i th level of A (Fixed or Random) ijk Random error term for k th rep when A is at i, B is at j(i) Nested Design - ANOVA Total Variation : bi a r TSS Yijk Y ... a dfTotal r bi 1 2 i 1 j 1 k 1 i 1 Factor A : a SSA r bi Y i.. Y ... 2 df A a 1 i 1 Factor B Nested Within A bi a SSB( A) r Y ij. Y i.. i 1 j 1 2 a df B ( A) bi a i 1 Error : a bi r SSE Yijk Y ij. i 1 j 1 k 1 2 a df E (r 1) bi i 1 Factors A and B Fixed a i 0 i 1 2 b 0 i 1 ,..., a ~ N 0 , s j (i ) ijk bi j 1 Tests for Difference s Among Factor A Effects H 0 : 1 ... a 0 H A : Not all i 0 MSA Test Statistic : FA P - value : PF FA MSE Rejection Region : FA F ,a 1,( r 1) b i Tests for Difference s Among Factor B Effects H 0 : b j ( i ) 0 i, j H A : Not all b j (i ) 0 MSB ( A) P - value : PF FB ( A) MSE Rejection Region : FB ( A) F , b a ,( r 1) b i i Test Statistic : FB ( A) Comparing Main Effects for A • Tukey’s Method- q in Studentized Range Table with = (r-1)Sbi MSE 1 1 Wij q (a, v) 2 rbi rb j A Conclude : i j if y i.. y j .. WijA Tukey' s CI : ( i j ) : y i.. y j .. WijA • Bonferroni’s Method - t-values in Bonferroni table with = (r-1)Sbi Bij t / 2,a ( a 1) / 2,v A 1 1 MSE rb rb j i Conclude : i j if y i.. y j .. B ijA Bonferroni ' s CI : (αi -α j ) : y i.. y j .. B ijA Comparing Effects for Factor B Within A • Tukey’s Method- q in Studentized Range Table with = (r-1)Sbi B Wij ( k ) MSE q (bk , v) r Conclude : b i ( k ) b j ( k ) if y ki. y kj . WijB( k ) Tukey' s CI : ( b i ( k ) b j ( k ) ) : y ki. y kj . WijB( k ) • Bonferroni’s Method - t-values in Bonferroni table with = (r-1)Sbi Bij ( k ) t / 2,bk (bk 1) / 2,v B 2 MSE r Conclude : b i ( k ) b j ( k ) if y ki. y kj . BijB( k ) Bonferroni ' s CI : ( b i ( k ) -b j ( k ) ) : y ki. y kj . B ijB( k ) Factor A Fixed and B Random a i 1 i 0 b j ( i ) ~ N 0, s b2 ijk ~ N 0, s 2 Tests for Difference s Among Factor A Effects H 0 : 1 ... a 0 H A : Not all i 0 MSA Test Statistic : FA P - value : PF FA MSB ( A) Rejection Region : FA F ,a 1, b a i Tests for Difference s Among Factor B Effects H 0 : s b2 0 i, j H A : s b2 0 MSB ( A) P - value : PF FB ( A) MSE Rejection Region : FB ( A) F , b a ,( r 1) b i i Test Statistic : FB ( A) Comparing Main Effects for A (B Random) • Tukey’s Method- q in Studentized Range Table with = Sbi-a MSAB 1 1 Wij q (a, v) 2 rbi rb j A Conclude : i j if y i.. y j .. WijA Tukey' s CI : ( i j ) : y i.. y j .. WijA • Bonferroni’s Method - t-values in Bonferroni table with = Sbi-a Bij t / 2,a ( a 1) / 2,v A 1 1 MSAB rb rb j i Conclude : i j if y i.. y j .. B ijA Bonferroni ' s CI : (αi -α j ) : y i.. y j .. B ijA Factors A and B Random i ~ N 0, s a2 b j (i ) ~ N 0, s b2 ijk ~ N 0, s 2 Tests for Difference s Among Factor A Effects H 0 : s a2 0 H A : s a2 0 MSA Test Statistic : FA P - value : PF FA MSB ( A) Rejection Region : FA F ,a 1, b a i Tests for Difference s Among Factor B Effects H 0 : s b2 0 i, j H A : s b2 0 MSB ( A) P - value : P F FB ( A) MSE Rejection Region : FB ( A) F , b a ,( r 1) b i i Test Statistic : FB ( A) Elements of Split-Plot Designs • Split-Plot Experiment: Factorial design with at least 2 factors, where experimental units wrt factors differ in “size” or “observational points”. • Whole plot: Largest experimental unit • Whole Plot Factor: Factor that has levels assigned to whole plots. Can be extended to 2 or more factors • Subplot: Experimental units that the whole plot is split into (where observations are made) • Subplot Factor: Factor that has levels assigned to subplots • Blocks: Aggregates of whole plots that receive all levels of whole plot factor Split Plot Design Block 1 A=1, B=1 A=1, B=2 A=1, B=3 A=1, B=4 A=2, B=1 A=2, B=2 A=2, B=3 A=2, B=4 A=3, B=1 A=3, B=2 A=3, B=3 A=3, B=4 Block 2 A=1, B=1 A=1, B=2 A=1, B=3 A=1, B=4 A=2, B=1 A=2, B=2 A=2, B=3 A=2, B=4 A=3, B=1 A=3, B=2 A=3, B=3 A=3, B=4 Block 3 A=1, B=1 A=1, B=2 A=1, B=3 A=1, B=4 A=2, B=1 A=2, B=2 A=2, B=3 A=2, B=4 A=3, B=1 A=3, B=2 A=3, B=3 A=3, B=4 Block 4 A=1, B=1 A=1, B=2 A=1, B=3 A=1, B=4 A=2, B=1 A=2, B=2 A=2, B=3 A=2, B=4 A=3, B=1 A=3, B=2 A=3, B=3 A=3, B=4 Note: Within each block we would assign at random the 3 levels of A to the whole plots and the 4 levels of B to the subplots within whole plots Examples • Agriculture: Varieties of a crop or gas may need to be grown in large areas, while varieties of fertilizer or varying growth periods may be observed in subsets of the area. • Engineering: May need long heating periods for a process and may be able to compare several formulations of a by-product within each level of the heating factor. • Behavioral Sciences: Many studies involve repeated measurements on the same subjects and are analyzed as a split-plot (See Repeated Measures lecture) Design Structure • Blocks: b groups of experimental units to be exposed to all combinations of whole plot and subplot factors • Whole plots: a experimental units to which the whole plot factor levels will be assigned to at random within blocks • Subplots: c subunits within whole plots to which the subplot factor levels will be assigned to at random. • Fully balanced experiment will have n=abc observations Data Elements (Fixed Factors, Random Blocks) • Yijk: Observation from wpt i, block j, and spt k • : Overall mean level • i : Effect of ith level of whole plot factor (Fixed) • bj: Effect of jth block (Random) • (ab )ij : Random error corresponding to whole plot elements in block j where wpt i is applied • k: Effect of kth level of subplot factor (Fixed) • )ik: Interaction btwn wpt i and spt k • (bc )jk: Interaction btwn block j and spt k (often set to 0) • ijk: Random Error= (bc )jk+ (abc )ijk • Note that if block/spt interaction is assumed to be 0, represents the block/spt within wpt interaction Model and Common Assumptions • Yijk = + i + b j + (ab )ij + k + ( )ik + ijk a i 1 i 0 b j ~ NID (0, s b2 ) 2 ( ab) ij ~ NID (0, s ab ) c k 1 k 0 a ( ) i 1 c ik ( ) ik 0 k 1 ijk ~ NID (0.s 2 ) COV (b j , ( ab) ij ) COV (b j , ijk ) COV (( ab) ij , ijk ) 0 Tests for Fixed Effects Whole Plot Trt Effects : H 0 : 1 a 0 Test Statistic : FW P MSW P MS BLOCK *W P PW P P ( F FW P | F ~ Fa 1.( a 1)( b 1) ) Subplot Trt Effects : H 0 : 1 c 0 Test Statistic : FSP MS SP MS ERROR PSP P ( F FSP | F ~ Fc 1.a ( b 1)( c 1) ) WP SP Interactio n : H 0 : ( ) ik 0 i, k Test Statistic : FW P SP MSW P SP MS ERROR PW P SP P ( F FW P SP | F ~ F( a 1)( c 1).a ( b 1)( c 1) ) Comparing Factor Levels Whole Plot Factor Levels : 95% CI for i i ' : Y i .. Y i '.. t 2 MS BLOCK W P bc Sub Plot Factor Levels : Y 2 MS ERROR ab Sub Plot Effects Within same whole plot (Interacti on) : 95% CI for ( k k ' ) : .. k Y .. k ' t 95% CI for k k ' ( )ik ( ) ik ' : Y i .k Y i .k ' t 2 MS ERROR b Whole Plot Effects within same sub plot (Interacti on) : Y i .k Y i '.k t 2MS BLOCK W P (c 1) MS ERROR bc 2 (c 1) MS ERROR MS BLOCK W P (c 1) MS ERROR 2 MS BLOCK W P 2 ^ a (b 1)(c 1) (a 1)(b 1) (df given below) Repeated Measures Designs • a Treatments/Conditions to compare • N subjects to be included in study (each subject will receive only one treatment) – n subjects receive trt i: an = N • t time periods of data will be obtained • Effects of trt, time and trtxtime interaction of primary interest. – Between Subject Factor: Treatment – Within Subject Factors: Time, TrtxTime Model Yijk i b j (i ) t k (t ) ik ijk overall mean i effect of trt i a i 1 i 0 b j (i ) effect of j th subject in trt i b j (i ) ~ NID 0, s b2 t k effect of k time period th t t k 1 k 0 (t ) ik interactio n between tr t i and time k ijk random error term ijk ~ NID 0, s 2 a (t ) i 1 t ik (t ) ik 0 k 1 Note the random error term is actually the interaction between subjects (within treatments) and time Tests for Fixed Effects Treatment Effects : H 0 : 1 a 0 Test Statistic : FTRTS MS TRTS MS SUBJECTS (TRTS ) PTRTS P ( F FTRTS | F ~ Fa 1, a ( n 1) ) Time Effects : H 0 : t 1 t t 0 Test Statistic : FTIME MS TIME MS ERROR PTIME P ( F FTIME | F ~ Ft 1, a ( n 1)( t 1) ) Treatment/ Time Interactio n : H 0 : (t ) ik 0 i, k Test Statistic : FTRT TIME MS TRT TIME MS ERROR PTRT TIME P ( F FTRT TIME | F ~ F( a 1)( t 1), a ( n 1)( t 1) ) Comparing Factor Levels Comparing Treatment Levels : 95% CI for i i ' : Y i .. Y i '.. t 2 MS SUBJECTS (TRTS ) nt Comparing Time Levels : Y t 2 MS ERROR 95% CI for t k t k ' : .. k Y .. k ' an Comparing Treatment Levels Within Time Levels : Y i .k Y i '.k t 2MS SUBJECTS (TRTS ) (t 1) MS ERROR nt with approximat e df : ^ (t 1)MS ERROR MS SUBJECT (TRT ) 2 (t 1) MS ERROR 2 MS SUBJECT (TRT ) a (n 1) a(n 1)(t 1) 2