Experimental design

Overview

Teaching: XX min
Exercises: XX min

Questions

How do we design experiments optimally?

How do we interpret a given design?

Objectives

Explain the formula notation and design matrices.

Explore different designs and learn how to interpret coefficients.

Contribute!

This episode is intended to discuss experimental design - what it means, why it is important, how you would translate your metadata into a suitable design matrix, how coefficients are to be interpreted.

suppressPackageStartupMessages({
    library(SummarizedExperiment)
    library(ExploreModelMatrix)
    library(dplyr)
})

meta <- read.csv("data/GSE96870_coldata_all.csv", row.names = 1)
meta

                     title geo_accession     organism     age    sex   infection  strain time     tissue mouse
GSM2545336 CNS_RNA-seq_10C    GSM2545336 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day8 Cerebellum    14
GSM2545337 CNS_RNA-seq_11C    GSM2545337 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     9
GSM2545338 CNS_RNA-seq_12C    GSM2545338 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum    10
GSM2545339 CNS_RNA-seq_13C    GSM2545339 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    15
GSM2545340 CNS_RNA-seq_14C    GSM2545340 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Cerebellum    18
GSM2545341 CNS_RNA-seq_17C    GSM2545341 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day8 Cerebellum     6
GSM2545342  CNS_RNA-seq_1C    GSM2545342 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day8 Cerebellum     5
GSM2545343 CNS_RNA-seq_20C    GSM2545343 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum    11
GSM2545344 CNS_RNA-seq_21C    GSM2545344 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    22
GSM2545345 CNS_RNA-seq_22C    GSM2545345 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Cerebellum    13
GSM2545346 CNS_RNA-seq_25C    GSM2545346 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day8 Cerebellum    23
GSM2545347 CNS_RNA-seq_26C    GSM2545347 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day8 Cerebellum    24
GSM2545348 CNS_RNA-seq_27C    GSM2545348 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     8
GSM2545349 CNS_RNA-seq_28C    GSM2545349 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum     7
GSM2545350 CNS_RNA-seq_29C    GSM2545350 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Cerebellum     1
GSM2545351  CNS_RNA-seq_2C    GSM2545351 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day8 Cerebellum    16
GSM2545352 CNS_RNA-seq_30C    GSM2545352 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    21
GSM2545353  CNS_RNA-seq_3C    GSM2545353 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     4
GSM2545354  CNS_RNA-seq_4C    GSM2545354 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum     2
GSM2545355 CNS_RNA-seq_571    GSM2545355 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Spinalcord     1
GSM2545356 CNS_RNA-seq_574    GSM2545356 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     2
GSM2545357 CNS_RNA-seq_575    GSM2545357 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     3
GSM2545358 CNS_RNA-seq_583    GSM2545358 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     4
GSM2545359 CNS_RNA-seq_585    GSM2545359 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day8 Spinalcord     5
GSM2545360 CNS_RNA-seq_589    GSM2545360 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day8 Spinalcord     6
GSM2545361 CNS_RNA-seq_590    GSM2545361 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     7
GSM2545362  CNS_RNA-seq_5C    GSM2545362 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    20
GSM2545363  CNS_RNA-seq_6C    GSM2545363 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Cerebellum    12
GSM2545364 CNS_RNA-seq_709    GSM2545364 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     8
GSM2545365 CNS_RNA-seq_710    GSM2545365 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     9
GSM2545366 CNS_RNA-seq_711    GSM2545366 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord    10
GSM2545367 CNS_RNA-seq_713    GSM2545367 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord    11
GSM2545368 CNS_RNA-seq_728    GSM2545368 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Spinalcord    12
GSM2545369 CNS_RNA-seq_729    GSM2545369 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Spinalcord    13
GSM2545370 CNS_RNA-seq_730    GSM2545370 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day8 Spinalcord    14
GSM2545371 CNS_RNA-seq_731    GSM2545371 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    15
GSM2545372 CNS_RNA-seq_733    GSM2545372 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day8 Spinalcord    17
GSM2545373 CNS_RNA-seq_735    GSM2545373 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day4 Spinalcord    18
GSM2545374 CNS_RNA-seq_736    GSM2545374 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day8 Spinalcord    19
GSM2545375 CNS_RNA-seq_738    GSM2545375 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    20
GSM2545376 CNS_RNA-seq_740    GSM2545376 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    21
GSM2545377 CNS_RNA-seq_741    GSM2545377 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    22
GSM2545378 CNS_RNA-seq_742    GSM2545378 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day8 Spinalcord    23
GSM2545379 CNS_RNA-seq_743    GSM2545379 Mus musculus 8 weeks   Male  InfluenzaA C57BL/6 Day8 Spinalcord    24
GSM2545380  CNS_RNA-seq_9C    GSM2545380 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day8 Cerebellum    19

vd <- VisualizeDesign(sampleData = meta, 
                      designFormula = ~ tissue + time + sex)
vd$cooccurrenceplots

$`tissue = Cerebellum`

plot of chunk unnamed-chunk-5


$`tissue = Spinalcord`

plot of chunk unnamed-chunk-5

Compare males and females, non-infected spinal cord

meta_noninf_spc <- meta %>% filter(time == "Day0" & 
                                       tissue == "Spinalcord")
meta_noninf_spc

                     title geo_accession     organism     age    sex   infection  strain time     tissue mouse
GSM2545356 CNS_RNA-seq_574    GSM2545356 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     2
GSM2545357 CNS_RNA-seq_575    GSM2545357 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     3
GSM2545358 CNS_RNA-seq_583    GSM2545358 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     4
GSM2545361 CNS_RNA-seq_590    GSM2545361 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     7
GSM2545364 CNS_RNA-seq_709    GSM2545364 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     8
GSM2545365 CNS_RNA-seq_710    GSM2545365 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     9
GSM2545366 CNS_RNA-seq_711    GSM2545366 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord    10
GSM2545367 CNS_RNA-seq_713    GSM2545367 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord    11

vd <- VisualizeDesign(sampleData = meta_noninf_spc, 
                      designFormula = ~ sex)
vd$designmatrix

           (Intercept) sexMale
GSM2545356           1       1
GSM2545357           1       1
GSM2545358           1       0
GSM2545361           1       1
GSM2545364           1       0
GSM2545365           1       0
GSM2545366           1       0
GSM2545367           1       1

vd$plotlist

[[1]]

plot of chunk unnamed-chunk-6

Challenge: Can you do it?

Set up the design formula to compare the three time points (Day0, Day4, Day8) in the male spinal cord samples, and visualize it using ExploreModelMatrix.

Solution

meta_male_spc <- meta %>% filter(sex == "Male" & tissue == "Spinalcord")
meta_male_spc

                     title geo_accession     organism     age  sex   infection  strain time     tissue mouse
GSM2545355 CNS_RNA-seq_571    GSM2545355 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day4 Spinalcord     1
GSM2545356 CNS_RNA-seq_574    GSM2545356 Mus musculus 8 weeks Male NonInfected C57BL/6 Day0 Spinalcord     2
GSM2545357 CNS_RNA-seq_575    GSM2545357 Mus musculus 8 weeks Male NonInfected C57BL/6 Day0 Spinalcord     3
GSM2545360 CNS_RNA-seq_589    GSM2545360 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day8 Spinalcord     6
GSM2545361 CNS_RNA-seq_590    GSM2545361 Mus musculus 8 weeks Male NonInfected C57BL/6 Day0 Spinalcord     7
GSM2545367 CNS_RNA-seq_713    GSM2545367 Mus musculus 8 weeks Male NonInfected C57BL/6 Day0 Spinalcord    11
GSM2545368 CNS_RNA-seq_728    GSM2545368 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day4 Spinalcord    12
GSM2545369 CNS_RNA-seq_729    GSM2545369 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day4 Spinalcord    13
GSM2545372 CNS_RNA-seq_733    GSM2545372 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day8 Spinalcord    17
GSM2545373 CNS_RNA-seq_735    GSM2545373 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day4 Spinalcord    18
GSM2545378 CNS_RNA-seq_742    GSM2545378 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day8 Spinalcord    23
GSM2545379 CNS_RNA-seq_743    GSM2545379 Mus musculus 8 weeks Male  InfluenzaA C57BL/6 Day8 Spinalcord    24

vd <- VisualizeDesign(sampleData = meta_male_spc, designFormula = ~ time)
vd$designmatrix

           (Intercept) timeDay4 timeDay8
GSM2545355           1        1        0
GSM2545356           1        0        0
GSM2545357           1        0        0
GSM2545360           1        0        1
GSM2545361           1        0        0
GSM2545367           1        0        0
GSM2545368           1        1        0
GSM2545369           1        1        0
GSM2545372           1        0        1
GSM2545373           1        1        0
GSM2545378           1        0        1
GSM2545379           1        0        1

vd$plotlist

[[1]]

plot of chunk unnamed-chunk-7

Factorial design without interactions

meta_noninf <- meta %>% filter(time == "Day0")
meta_noninf

                     title geo_accession     organism     age    sex   infection  strain time     tissue mouse
GSM2545337 CNS_RNA-seq_11C    GSM2545337 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     9
GSM2545338 CNS_RNA-seq_12C    GSM2545338 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum    10
GSM2545343 CNS_RNA-seq_20C    GSM2545343 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum    11
GSM2545348 CNS_RNA-seq_27C    GSM2545348 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     8
GSM2545349 CNS_RNA-seq_28C    GSM2545349 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum     7
GSM2545353  CNS_RNA-seq_3C    GSM2545353 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     4
GSM2545354  CNS_RNA-seq_4C    GSM2545354 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum     2
GSM2545356 CNS_RNA-seq_574    GSM2545356 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     2
GSM2545357 CNS_RNA-seq_575    GSM2545357 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     3
GSM2545358 CNS_RNA-seq_583    GSM2545358 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     4
GSM2545361 CNS_RNA-seq_590    GSM2545361 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     7
GSM2545364 CNS_RNA-seq_709    GSM2545364 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     8
GSM2545365 CNS_RNA-seq_710    GSM2545365 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     9
GSM2545366 CNS_RNA-seq_711    GSM2545366 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord    10
GSM2545367 CNS_RNA-seq_713    GSM2545367 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord    11

vd <- VisualizeDesign(sampleData = meta_noninf, 
                      designFormula = ~ sex + tissue)
vd$designmatrix

           (Intercept) sexMale tissueSpinalcord
GSM2545337           1       0                0
GSM2545338           1       0                0
GSM2545343           1       1                0
GSM2545348           1       0                0
GSM2545349           1       1                0
GSM2545353           1       0                0
GSM2545354           1       1                0
GSM2545356           1       1                1
GSM2545357           1       1                1
GSM2545358           1       0                1
GSM2545361           1       1                1
GSM2545364           1       0                1
GSM2545365           1       0                1
GSM2545366           1       0                1
GSM2545367           1       1                1

vd$plotlist

[[1]]

plot of chunk unnamed-chunk-8

Factorial design with interactions

meta_noninf <- meta %>% filter(time == "Day0")
meta_noninf

                     title geo_accession     organism     age    sex   infection  strain time     tissue mouse
GSM2545337 CNS_RNA-seq_11C    GSM2545337 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     9
GSM2545338 CNS_RNA-seq_12C    GSM2545338 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum    10
GSM2545343 CNS_RNA-seq_20C    GSM2545343 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum    11
GSM2545348 CNS_RNA-seq_27C    GSM2545348 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     8
GSM2545349 CNS_RNA-seq_28C    GSM2545349 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum     7
GSM2545353  CNS_RNA-seq_3C    GSM2545353 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     4
GSM2545354  CNS_RNA-seq_4C    GSM2545354 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Cerebellum     2
GSM2545356 CNS_RNA-seq_574    GSM2545356 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     2
GSM2545357 CNS_RNA-seq_575    GSM2545357 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     3
GSM2545358 CNS_RNA-seq_583    GSM2545358 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     4
GSM2545361 CNS_RNA-seq_590    GSM2545361 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord     7
GSM2545364 CNS_RNA-seq_709    GSM2545364 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     8
GSM2545365 CNS_RNA-seq_710    GSM2545365 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     9
GSM2545366 CNS_RNA-seq_711    GSM2545366 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord    10
GSM2545367 CNS_RNA-seq_713    GSM2545367 Mus musculus 8 weeks   Male NonInfected C57BL/6 Day0 Spinalcord    11

vd <- VisualizeDesign(sampleData = meta_noninf, 
                      designFormula = ~ sex * tissue)
vd$designmatrix

           (Intercept) sexMale tissueSpinalcord sexMale:tissueSpinalcord
GSM2545337           1       0                0                        0
GSM2545338           1       0                0                        0
GSM2545343           1       1                0                        0
GSM2545348           1       0                0                        0
GSM2545349           1       1                0                        0
GSM2545353           1       0                0                        0
GSM2545354           1       1                0                        0
GSM2545356           1       1                1                        1
GSM2545357           1       1                1                        1
GSM2545358           1       0                1                        0
GSM2545361           1       1                1                        1
GSM2545364           1       0                1                        0
GSM2545365           1       0                1                        0
GSM2545366           1       0                1                        0
GSM2545367           1       1                1                        1

vd$plotlist

[[1]]

plot of chunk unnamed-chunk-9

Paired design

meta_fem_day0 <- meta %>% filter(sex == "Female" & 
                                     time == "Day0")
meta_fem_day0

                     title geo_accession     organism     age    sex   infection  strain time     tissue mouse
GSM2545337 CNS_RNA-seq_11C    GSM2545337 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     9
GSM2545338 CNS_RNA-seq_12C    GSM2545338 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum    10
GSM2545348 CNS_RNA-seq_27C    GSM2545348 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     8
GSM2545353  CNS_RNA-seq_3C    GSM2545353 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     4
GSM2545358 CNS_RNA-seq_583    GSM2545358 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     4
GSM2545364 CNS_RNA-seq_709    GSM2545364 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     8
GSM2545365 CNS_RNA-seq_710    GSM2545365 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     9
GSM2545366 CNS_RNA-seq_711    GSM2545366 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord    10

vd <- VisualizeDesign(sampleData = meta_fem_day0,
                      designFormula = ~ mouse + tissue)
vd$designmatrix

           (Intercept) mouse tissueSpinalcord
GSM2545337           1     9                0
GSM2545338           1    10                0
GSM2545348           1     8                0
GSM2545353           1     4                0
GSM2545358           1     4                1
GSM2545364           1     8                1
GSM2545365           1     9                1
GSM2545366           1    10                1

vd$plotlist

[[1]]

plot of chunk unnamed-chunk-10

Within- and between-subject comparisons

meta_fem_day04 <- meta %>% 
    filter(sex == "Female" & 
               time %in% c("Day0", "Day4")) %>%
    droplevels()
meta_fem_day04

                     title geo_accession     organism     age    sex   infection  strain time     tissue mouse
GSM2545337 CNS_RNA-seq_11C    GSM2545337 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     9
GSM2545338 CNS_RNA-seq_12C    GSM2545338 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum    10
GSM2545339 CNS_RNA-seq_13C    GSM2545339 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    15
GSM2545344 CNS_RNA-seq_21C    GSM2545344 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    22
GSM2545348 CNS_RNA-seq_27C    GSM2545348 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     8
GSM2545352 CNS_RNA-seq_30C    GSM2545352 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    21
GSM2545353  CNS_RNA-seq_3C    GSM2545353 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Cerebellum     4
GSM2545358 CNS_RNA-seq_583    GSM2545358 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     4
GSM2545362  CNS_RNA-seq_5C    GSM2545362 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Cerebellum    20
GSM2545364 CNS_RNA-seq_709    GSM2545364 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     8
GSM2545365 CNS_RNA-seq_710    GSM2545365 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord     9
GSM2545366 CNS_RNA-seq_711    GSM2545366 Mus musculus 8 weeks Female NonInfected C57BL/6 Day0 Spinalcord    10
GSM2545371 CNS_RNA-seq_731    GSM2545371 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    15
GSM2545375 CNS_RNA-seq_738    GSM2545375 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    20
GSM2545376 CNS_RNA-seq_740    GSM2545376 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    21
GSM2545377 CNS_RNA-seq_741    GSM2545377 Mus musculus 8 weeks Female  InfluenzaA C57BL/6 Day4 Spinalcord    22

design <- model.matrix(~ mouse, data = meta_fem_day04)
design <- cbind(design, 
                Spc.Day0 = meta_fem_day04$tissue == "Spinalcord" & 
                    meta_fem_day04$time == "Day0",
                Spc.Day4 = meta_fem_day04$tissue == "Spinalcord" & 
                    meta_fem_day04$time == "Day4")
rownames(design) <- rownames(meta_fem_day04)
design

           (Intercept) mouse Spc.Day0 Spc.Day4
GSM2545337           1     9        0        0
GSM2545338           1    10        0        0
GSM2545339           1    15        0        0
GSM2545344           1    22        0        0
GSM2545348           1     8        0        0
GSM2545352           1    21        0        0
GSM2545353           1     4        0        0
GSM2545358           1     4        1        0
GSM2545362           1    20        0        0
GSM2545364           1     8        1        0
GSM2545365           1     9        1        0
GSM2545366           1    10        1        0
GSM2545371           1    15        0        1
GSM2545375           1    20        0        1
GSM2545376           1    21        0        1
GSM2545377           1    22        0        1

vd <- VisualizeDesign(sampleData = meta_fem_day04 %>%
                          select(time, tissue, mouse),
                      designFormula = NULL, 
                      designMatrix = design, flipCoordFitted = FALSE)
vd$designmatrix

           (Intercept) mouse Spc.Day0 Spc.Day4
GSM2545337           1     9        0        0
GSM2545338           1    10        0        0
GSM2545339           1    15        0        0
GSM2545344           1    22        0        0
GSM2545348           1     8        0        0
GSM2545352           1    21        0        0
GSM2545353           1     4        0        0
GSM2545358           1     4        1        0
GSM2545362           1    20        0        0
GSM2545364           1     8        1        0
GSM2545365           1     9        1        0
GSM2545366           1    10        1        0
GSM2545371           1    15        0        1
GSM2545375           1    20        0        1
GSM2545376           1    21        0        1
GSM2545377           1    22        0        1

vd$plotlist

$`time = Day0`

plot of chunk unnamed-chunk-11


$`time = Day4`

plot of chunk unnamed-chunk-11

Key Points

Key point 1

previous episode

RNA-seq analysis with Bioconductor

next episode

Experimental design

Overview

Contribute!

Compare males and females, non-infected spinal cord

Challenge: Can you do it?

Solution

Factorial design without interactions

Factorial design with interactions

Paired design

Within- and between-subject comparisons

Key Points

previous episode

next episode