1 Dataset and model presentation

This data set comes from the study of the modulation of immunity in rainbow trout (Oncorhynchus mykiss) by exposure to cadmium (Cd) combined with polyunsaturated fatty acids (PUFAs) enriched diets [Cornet et al., 2018].

The responses were quantified by measuring the modification of the expression of 15 immune-related genes (m = 15) by RT-qPCR (reverse transcription quantitative polymerase chain reaction). The experiment was carried out on 72 trouts and 3 factors were considered in the experimental design:

  • Day: Measurements on trouts were collected on days 28, 70 and 72
  • Treatment : Four polyunsaturated fatty acid diets: alpha-linolenic acid (ALA), linoleic acid (LA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)
  • Exposure: Trouts were exposed (level = 2) or not (level = 0) to high cadmium concentrations.

This gives a 3 × 4 × 2 factorial design. Each of the 24 trials corresponds to a different aquarium. Three fishes were analysed (3 replicates) for each condition, giving a total of 72 observations.

In this limpca vignette the data are first explored in order to prepare an appropriate data set for ASCA/APCA analysis. Data are first represented by PCA and outliers removed. The remaining observations are then log transformed. Next the data of each aquarium are mean aggregated in order to avoid the inclusion of an aquarium random factor in the statistical model because limpca is not yet able to handle mixed linear models. The data are finally centered and scaled by column.

The estimated model in then a (general) linear model for fixed factors including main effects and all two way interactions. The three way interaction is not included because the aggregated design has no replicate.

A detailed presentation and analysis of this dataset is also available in [Benaiche, 2022].

2 Data import and exploration

2.1 Data import and design visualization

The trout data set is list of three objects : the model outcomes, the design and the model formula.

data("trout")
# print number of and response names
cat("\n Nb of Responses :  ", ncol(trout$outcomes), "\n ")

 Nb of Responses :   15 
 
cat("\nResponses :\n", colnames(trout$outcomes), "\n ")

Responses :
 IL.1b IL6 IL8 Lysozyme IgM MCSFR.a MPO TGF.b TLR3 TLR9 MyD88 SOD Elov5 C3 Cox2 
 
# Order responses by alphabetic order
trout$outcomes <- trout$outcomes[, order(dimnames(trout$outcomes)[[2]])]
cat("\n Ordered responses :\n  ", colnames(trout$outcomes), "\n ")

 Ordered responses :
   C3 Cox2 Elov5 IL.1b IL6 IL8 IgM Lysozyme MCSFR.a MPO MyD88 SOD TGF.b TLR3 TLR9 
 
# print factor names
cat("\nDesign factors :  ", colnames(trout$design), "\n ")

Design factors :   Day Treatment Exposure Aquarium 
 
# plot the design with plotDesign function
limpca::plotDesign(
    design = trout$design, x = "Treatment",
    y = "Day", cols = "Exposure",
    title = "Initial design of the trout dataset"
)

This graph confirms that the design is balanced.

2.2 Principal Component Analysis of row data

Functions pcaBySvd, pcaScreePlot and pcaScorePlot are used to take a first look at the data by PCA.

This PCA shows that the data should be log transformed.

resPCA <- limpca::pcaBySvd(trout$outcomes)
limpca::pcaScreePlot(resPCA, nPC = 8)

limpca::pcaScorePlot(
    resPcaBySvd = resPCA, axes = c(1, 2),
    title = "Score plot of original data ",
    design = trout$design, color = "Aquarium",
    points_labs_rn = TRUE
)
Warning: ggrepel: 64 unlabeled data points (too many overlaps). Consider
increasing max.overlaps

2.3 Log10 transformation of the data and new PCA

Data are log transfomed and a new PCA is applied.

The score plot show clearly two outliers (i.e. fishes D72EPA2.1 and D28EPA2.2). They will removed of the analysis for the next steps.

# Log Transformation
trout_log <- trout
trout_log$outcomes <- as.matrix(log10(trout$outcomes))

# new PCA
resPCA1 <- limpca::pcaBySvd(trout_log$outcomes)
limpca::pcaScreePlot(resPCA1, nPC = 8)

limpca::pcaScorePlot(
    resPcaBySvd = resPCA1, axes = c(1, 2),
    title = "Score plot of Log10 data ",
    design = trout_log$design, color = "Aquarium",
    drawShapes = "polygon", points_labs_rn = TRUE
)
Warning: ggrepel: 71 unlabeled data points (too many overlaps). Consider
increasing max.overlaps

2.4 New PCA without the outliers

New data are created without the 2 outliers and a new PCA performed. The option polygon of pcaScorePlot function allows to group data by aquarium. In some aquariums, the 3 fishes shows very similar results and in other not.

The data are now clean for further analysis

# Remove outliers and create new dataset
trout_clean <- trout_log
outliers <- match(
    c("D72EPA2.1", "D28EPA2.2"),
    rownames(trout_log$outcomes)
)
trout_clean$outcomes <- trout_log$outcomes[-outliers, ]
trout_clean$design <- trout_log$design[-outliers, ]
# PCA
resPCA2 <- limpca::pcaBySvd(trout_clean$outcomes)
limpca::pcaScreePlot(resPCA2, nPC = 8)

# Score plot Components 1 and 2
limpca::pcaScorePlot(
    resPcaBySvd = resPCA2, axes = c(1, 2),
    title = "Score plot of Log10 data without outliers (PC 1&2)",
    design = trout_clean$design, color = "Aquarium",
    drawShapes = "polygon",
    points_labs_rn = FALSE
)

# Score plot Components 3 and 4
limpca::pcaScorePlot(
    resPcaBySvd = resPCA2, axes = c(3, 4),
    title = "Score plot of Log10 data without outliers (PC 3&4)",
    design = trout_clean$design, color = "Aquarium",
    drawShapes = "polygon", points_labs_rn = FALSE
)

The option polygon of pcaScorePlot function allows to group data by aquarium. In some aquariums, the 3 fishes shows very similar results and in other not.

2.5 Mean agregation by aquarium and scaling

Data are now mean aggregated by aquarium. This will remove the hierarchy in the design and allow to apply a classical fixed effect general linear model to the data.

Data are next centered and scaled by column. This will give the same importance to each response in the analysis.

# Mean aggregation
mean_outcomes <- matrix(0, nrow = 24, ncol = 15)
mean_design <- matrix(0, nrow = 24, ncol = 3)
y <- list(
    trout_clean$design[["Day"]],
    trout_clean$design[["Treatment"]],
    trout_clean$design[["Exposure"]]
)
for (i in 1:15) {
    mean_outcomes[, i] <- aggregate(trout_clean$outcomes[, i], by = y, mean)[, 4]
}
mean_design <- aggregate(trout_clean$outcomes[, 1], by = y, mean)[, c(1:3)]

# Set row and col names
colnames(mean_outcomes) <- colnames(trout_clean$outcomes)
colnames(mean_design) <- colnames(trout_clean$design)[1:3]
trout_mean_names <- apply(mean_design, 1, paste, collapse = "")
rownames(mean_outcomes) <- trout_mean_names
rownames(mean_design) <- trout_mean_names
# Outcomes centering and Scaling
mean_outcomes <- scale(mean_outcomes, center = TRUE, scale = TRUE)
# New data object creation
trout_mean <- list(
    "outcomes" = mean_outcomes,
    "design" = mean_design,
    "formula" = trout$formula
)
# Clean objects
rm(
    resPCA, resPCA1, resPCA2, y, mean_design, mean_outcomes,
    trout_mean_names
)

3 Exploration of aggregated data

Aggregated data are now explored.

3.1 Design

Note that there is no replicate in this new design. There is only one observation (i.e. one aquarium) for each of the 24 factor combinations.

pander(head(trout_mean$design))
  Day Treatment Exposure
D28ALA0 D28 ALA 0
D70ALA0 D70 ALA 0
D72ALA0 D72 ALA 0
D28DHA0 D28 DHA 0
D70DHA0 D70 DHA 0
D72DHA0 D72 DHA 0 
limpca::plotDesign(
    design = trout_mean$design,
    title = "Design of mean aggregated trout dataset"
)

3.2 Example of lineplot of the responses for two observations

limpca::plotLine(trout_mean$outcomes,
    rows = c(1, 24),
    xaxis_type = "character", type = "s"
) +
    ggplot2::theme(axis.text.x = element_text(angle = 45, hjust = 1))

3.3 PCA aggregated data

resPCA_mean <- limpca::pcaBySvd(trout_mean$outcomes)
pcaScreePlot(resPCA_mean, nPC = 6)

3.3.1 Score plots

The three score plots below show clearly that the Day is the more important effect.

limpca::pcaScorePlot(
    resPcaBySvd = resPCA_mean, axes = c(1, 2),
    title = "PCA score plot by Exposure and Day",
    design = trout_mean$design,
    shape = "Exposure", color = "Day",
    points_labs_rn = FALSE
)

limpca::pcaScorePlot(
    resPcaBySvd = resPCA_mean, axes = c(1, 2),
    title = "PCA scores plot by Treatment and Day",
    design = trout_mean$design,
    shape = "Day", color = "Treatment",
    points_labs_rn = FALSE
)

limpca::pcaScorePlot(
    resPcaBySvd = resPCA_mean, axes = c(1, 2),
    title = "PCA scores plot by Exposure and Treatment",
    design = trout_mean$design,
    shape = "Treatment", color = "Exposure",
    points_labs_rn = FALSE
)

3.3.2 1D Loading plots

limpca::pcaLoading1dPlot(
    resPcaBySvd = resPCA_mean, axes = c(1, 2),
    title = "PCA loadings plot trout", xlab = " ",
    ylab = "Expression", xaxis_type = "character", type = "s"
) +
    ggplot2::theme(axis.text.x = element_text(angle = 45, hjust = 1))

3.3.3 2D Loading plots

This 2D loading plot allows already to observe that most responses are correlated with each others. Igm, SOD and TLR9 behave quite differently.

limpca::pcaLoading2dPlot(
    resPcaBySvd = resPCA_mean, axes = c(1, 2),
    title = "PCA loadings plot trout", addRownames = TRUE
)

3.4 Scatterplot matrix of all 15 responses

The plotScatterM function allows to visualize the 2 by 2 relation between all (or some of) the responses simultaneously and choose different markers and colors above and below the diagonal according to factor levels. Strong relations between expressions are confirmed here for most genes.

limpca::plotScatterM(
    Y = trout_mean$outcomes, cols = c(1:15),
    design = trout_mean$design,
    varname.colorup = "Day",
    vec.colorup = c("CadetBlue4", "pink", "orange"),
    varname.colordown = "Day",
    vec.colordown = c("CadetBlue4", "pink", "orange"),
    varname.pchup = "Treatment",
    varname.pchdown = "Exposure"
)

4 GLM decomposition

The estimated model is the following :

outcomes ~ Day + Treatment + Exposure + Day:Treatment + Day:Exposure + Treatment:Exposure

Since the design has only one replicate, the three way interaction has been removed because it is confounded with residuals.

4.1 Model matrix X generation

resLmpModelMatrix <- limpca::lmpModelMatrix(trout_mean)
pander::pander(head(resLmpModelMatrix$modelMatrix))
Table continues below
  (Intercept) Day1 Day2 Treatment1 Treatment2 Treatment3
D28ALA0 1 1 0 1 0 0
D70ALA0 1 0 1 1 0 0
D72ALA0 1 -1 -1 1 0 0
D28DHA0 1 1 0 0 1 0
D70DHA0 1 0 1 0 1 0
D72DHA0 1 -1 -1 0 1 0
Table continues below
  Exposure1 Day1:Treatment1 Day2:Treatment1 Day1:Treatment2
D28ALA0 1 1 0 0
D70ALA0 1 0 1 0
D72ALA0 1 -1 -1 0
D28DHA0 1 0 0 1
D70DHA0 1 0 0 0
D72DHA0 1 0 0 -1
Table continues below
  Day2:Treatment2 Day1:Treatment3 Day2:Treatment3
D28ALA0 0 0 0
D70ALA0 0 0 0
D72ALA0 0 0 0
D28DHA0 0 0 0
D70DHA0 1 0 0
D72DHA0 -1 0 0
Table continues below
  Day1:Exposure1 Day2:Exposure1 Treatment1:Exposure1
D28ALA0 1 0 1
D70ALA0 0 1 1
D72ALA0 -1 -1 1
D28DHA0 1 0 0
D70DHA0 0 1 0
D72DHA0 -1 -1 0
  Treatment2:Exposure1 Treatment3:Exposure1
D28ALA0 0 0
D70ALA0 0 0
D72ALA0 0 0
D28DHA0 1 0
D70DHA0 1 0
D72DHA0 1 0

4.2 Computation of effect matrices and importances

As observed before, the more important effect in the model is the Day main effect.

resLmpEffectMatrices <- lmpEffectMatrices(resLmpModelMatrix)
resLmpEffectMatrices$varPercentagesPlot

4.3 Bootstrap test of effect significance

The bootstrap test shows that, in addition to the Day effect, the Treatment effect is also significant (p<0.05) and the DxT effect is nearly significant (p close to 0.1). The corresponding effect matrices will be studied more deeply by PCA in the next sections.

resLmpBootstrapTests <- lmpBootstrapTests(
    resLmpEffectMatrices = resLmpEffectMatrices,
    nboot = 1000
)

# Print p-values
pander::pander(t(resLmpBootstrapTests$resultsTable))
Table continues below
  Day Treatment Exposure Day:Treatment
% of variance (T III) 71.74 8.43 0.67 8.85
Bootstrap p-values < 0.001 0.022 0.594 0.107
  Day:Exposure Treatment:Exposure Residuals
% of variance (T III) 2.07 2.74 5.49
Bootstrap p-values 0.381 0.488 -

5 ASCA and APCA

Visualization of single or combined effect matrices using ASCA and APCA. ASCA-E is also provided in limpca but not shown here.

5.1 ASCA

5.1.1 PCA decomposition of effect matrices

In addition to single model effects, a combined effect matrix Day+Treatment+Day:Treatment also is computed in order to visualize the combined effect of these two most important factors.

resASCA <- lmpPcaEffects(
    resLmpEffectMatrices = resLmpEffectMatrices,
    method = "ASCA",
    combineEffects = list(c(
        "Day", "Treatment",
        "Day:Treatment"
    ))
)

5.1.2 Contributions

Print contributions of each model effect and of each PC for each effect matrix decomposition by PCA. This last result is given effect by effect and then reported to the global variance.

resLmpContributions <- lmpContributions(resASCA)
pander::pander(resLmpContributions$totalContribTable)
  Percentage of Variance
Day 71.74
Treatment 8.43
Exposure 0.67
Day:Treatment 8.85
Day:Exposure 2.07
Treatment:Exposure 2.74
Residuals 5.49 
pander::pander(resLmpContributions$effectTable)
  PC1 PC2 PC3 PC4 PC5 Sum
Day 88.66 11.34 0 0 0 100
Treatment 72.02 23.61 4.36 0 0 99.99
Exposure 100 0 0 0 0 100
Day:Treatment 47.35 21.52 17.42 8.97 2.8 98.06
Day:Exposure 81.01 18.99 0 0 0 100
Treatment:Exposure 78.74 15.81 5.46 0 0 100
Residuals 39.25 28.02 17.55 8.76 4.49 98.07 
pander::pander(resLmpContributions$contribTable)
  PC1 PC2 PC3 PC4 PC5 Contrib
Day 63.61 8.14 0 0 0 71.74
Treatment 6.07 1.99 0.37 0 0 8.43
Exposure 0.67 0 0 0 0 0.67
Day:Treatment 4.19 1.9 1.54 0.79 0.25 8.85
Day:Exposure 1.68 0.39 0 0 0 2.07
Treatment:Exposure 2.16 0.43 0.15 0 0 2.74
Residuals 2.16 1.54 0.96 0.48 0.25 5.49 
pander::pander(resLmpContributions$combinedEffectTable)
  PC1 PC2 PC3 PC4 PC5 Sum
Day+Treatment+Day:Treatment 75.51 10.08 6.53 3.62 1.91 97.65
Residuals 39.25 28.02 17.55 8.76 4.49 98.07 
## Visualize the more important contributions
resLmpContributions$plotContrib

5.1.3 Scores and loadings plots

2D Score plots of the most important effects and of the residual matrix are given below with their related loading plots. These allow to see for which response each effect is or is not important

5.1.3.1 Day effect

A <- lmpScorePlot(resASCA,
    effectNames = "Day",
    color = "Day", shape = "Day"
)
B <- lmpLoading2dPlot(resASCA,
    effectNames = "Day",
    points_labs = colnames(trout$outcomes)
)
grid.arrange(A, B, ncol = 2)

5.1.3.2 Treatment effect

A <- lmpScorePlot(resASCA,
    effectNames = "Treatment",
    color = "Treatment", shape = "Treatment"
)
B <- lmpLoading2dPlot(resASCA,
    effectNames = "Treatment",
    points_labs = colnames(trout$outcomes)
)
grid.arrange(A, B, ncol = 2)

5.1.3.3 Day:Treatment effect

A <- lmpScorePlot(resASCA,
    effectNames = "Day:Treatment",
    color = "Treatment", shape = "Day"
)
B <- lmpLoading2dPlot(resASCA,
    effectNames = "Day:Treatment",
    points_labs = colnames(trout$outcomes)
)
grid.arrange(A, B, ncol = 2)

5.1.3.4 Combined Day+Treatment+Day:Treatment effect

A <- lmpScorePlot(resASCA,
    effectNames = "Day+Treatment+Day:Treatment",
    color = "Treatment", shape = "Day"
)
B <- lmpLoading2dPlot(resASCA,
    effectNames = "Day+Treatment+Day:Treatment",
    points_labs = colnames(trout$outcomes)
)
grid.arrange(A, B, ncol = 2)

5.1.3.5 Residual matrix decomposition

The score and loading plot of the residual matrix does not show any special pattern.

A <- lmpScorePlot(resASCA,
    effectNames = "Residuals",
    color = "Treatment", shape = "Day"
)
B <- lmpLoading2dPlot(resASCA,
    effectNames = "Residuals",
    points_labs = colnames(trout$outcomes)
)
grid.arrange(A, B, ncol = 2)

5.1.4 Effect plots on scores

Interaction effects are difficult to visualize in 2D score plots. Effect plots are interesting in this context. It show the effect of one factor on a PC for different level of the other.

Below the Day:Treament interaction effect is drawn alone and then combined with the two related main effects.

The second graph shows that their is some interaction effect but small compared to the Day and even treatment main effects.

A <- lmpEffectPlot(resASCA,
    effectName = "Day:Treatment",
    x = "Day", z = "Treatment", axes = c(1, 2)
)
A$PC1 <- A$PC1 + ggtitle("PC1: Day:Treatment effect alone")
A$PC2 <- A$PC2 + ggtitle("PC2: Day:Treatment effect alone")
grid.arrange(A$PC1, A$PC2, ncol = 2)

A <- lmpEffectPlot(resASCA,
    effectName = "Day+Treatment+Day:Treatment",
    x = "Day", z = "Treatment", axes = c(1, 2)
)
A$PC1 <- A$PC1 + ggtitle("PC1: Combined D+T+D:T effects")
A$PC2 <- A$PC2 + ggtitle("PC2: Combined D+T+D:T effects")
grid.arrange(A$PC1, A$PC2, ncol = 2)

5.2 APCA

APCA allows to visualize by PCA each model effect added to model residuals. It gives an idea of the effects signal to noise ratio and significance. Be care that the significance depends also crucially of the number of observations in the experiment.

In APCA the score plots are the more interesting graphics to look at. We then only give them here for all model effects.

resAPCA <- lmpPcaEffects(
    resLmpEffectMatrices =
        resLmpEffectMatrices, method = "APCA"
)
# Day Effect
lmpScorePlot(resAPCA,
    effectNames = "Day",
    color = "Day", shape = "Day", drawShapes = "ellipse"
)

# Treatment Effect
lmpScorePlot(resAPCA,
    effectNames = "Treatment",
    color = "Treatment", shape = "Treatment", drawShapes = "ellipse"
)
Warning: Removed 30 rows containing missing values or values outside the scale range
(`geom_path()`).

# Exposure Effect
lmpScorePlot(resAPCA,
    effectNames = "Exposure",
    color = "Exposure", shape = "Exposure", drawShapes = "ellipse"
)

# Day:Treatment Effect
lmpScorePlot(resAPCA,
    effectNames = "Day:Treatment",
    color = "Treatment", shape = "Day", drawShapes = "polygon"
)

6 Univariate ANOVA

This part of the vignette links the results of the ASCA/APCA analysis to a more classical analysis applied in -omics transcriptomic data analysis (see e.g. LIMMA package).

An ANOVA model is fitted to each response separately and then, for each model effect, p-values of effect significance are corrected by FDR and ordered. These results allow to detect for which responses each effect of interest is significant. E.g. find which gene is a potential biomarker to differentiate patients with or without a given disease.

The code below applies such analysis and compares then responses importance based on FDR p-values to response loadings obtained by ASCA/APCA.

6.1 Parallel ANOVA modeling and FDR p-value corrections

# Creation of a matrix to store the p-values
m <- ncol(trout_mean$outcomes)
mat_pval <- matrix(nrow = m, ncol = 6)
dimnames(mat_pval) <- list(
    dimnames(trout_mean$outcomes)[[2]],
    c(
        "Day", "Treatment", "Exposure", "Day:Treatment",
        "Day:Exposure", "Treatment:Exposure"
    )
)

# Parallel ANOVA modeling
for (i in 1:m) {
    data <- cbind(y = trout_mean$outcomes[, i], trout_mean$design)
    Modl <- lm(y ~ Day + Treatment + Exposure + Day:Treatment + Day:Exposure + Treatment:Exposure,
        contrasts = list(Day = contr.sum, Treatment = contr.sum, Exposure = contr.sum),
        data = data
    )
    tabanova <- Anova(Modl, type = 3)
    mat_pval[i, ] <- tabanova[2:7, 4]
}

# FDR p-values correction
for (i in 1:6) mat_pval[, i] <- p.adjust(mat_pval[, i], method = "BH")

6.2 FDR corrected p_values (q-values)

pander(mat_pval)
Table continues below
  Day Treatment Exposure Day:Treatment Day:Exposure
C3 0.0005514 0.4772 0.9769 0.9457 0.8465
Cox2 0.0002609 0.3833 0.9183 0.9457 0.8172
Elov5 7.56e-05 0.3833 0.995 0.9457 0.8465
IL.1b 0.0002609 0.1485 0.9183 0.773 0.8172
IL6 0.0003037 0.273 0.9183 0.9457 0.8172
IL8 0.0002609 0.3833 0.9183 0.9457 0.8172
IgM 0.004411 0.1362 0.9183 0.9457 0.8465
Lysozyme 0.0002791 0.2254 0.9183 0.9457 0.8172
MCSFR.a 2.327e-05 0.1362 0.9183 0.9457 0.9502
MPO 7.251e-05 0.2254 0.9183 0.9457 0.8465
MyD88 2.327e-05 0.1362 0.995 0.7736 0.8172
SOD 0.001557 0.3833 0.9769 0.7334 0.8172
TGF.b 0.000362 0.2887 0.995 0.9457 0.9364
TLR3 0.01301 0.2254 0.9183 0.773 0.8172
TLR9 0.2085 0.2254 0.995 0.7334 0.8172
  Treatment:Exposure
C3 0.9638
Cox2 0.9625
Elov5 0.9638
IL.1b 0.6085
IL6 0.6085
IL8 0.7775
IgM 0.7775
Lysozyme 0.7775
MCSFR.a 0.6085
MPO 0.7775
MyD88 0.09138
SOD 0.7775
TGF.b 0.9625
TLR3 0.9625
TLR9 0.9638 
heatmap(-log10(mat_pval),
    Rowv = NA, Colv = "Rowv",
    cexCol = 0.8, scale = "none", main = "Heatmap of -log10(q-values)"
)

6.3 Plot ASCA loadings versus -log10(q-values)

Plot the relation between the ASCA loadings and FDR p-values for the three more important effects of the model : Day, Treatment and Day:Treatment.

FDR p-values are log transformed and loadings are summarized over the 2 first components.

Effects <- c("Day", "Treatment", "Day:Treatment")

for (i in 1:3) {
    Pval_log <- -log10(mat_pval[, Effects[i]])
    resA <- resASCA[[Effects[i]]]
    PC12Load <- as.vector(sqrt(resA$loadings[, 1:2]^2 %*% resA$singvar[1:2]^2))
    matres <- cbind(PC12Load, Pval_log)
    A[[i]] <- plotScatter(
        Y = matres, xy = c(1, 2),
        points_labs = rownames(matres),
        xlab = "PC1&2 ASCA loadings", ylab = "-log10(p-values)",
        title = paste(Effects[i], "effect")
    )
}

A[[1]]
Warning: ggrepel: 1 unlabeled data points (too many overlaps). Consider
increasing max.overlaps

A[[2]]

A[[3]]
Warning: ggrepel: 6 unlabeled data points (too many overlaps). Consider
increasing max.overlaps

7 sessionInfo

sessionInfo()
R Under development (unstable) (2024-03-18 r86148)
Platform: x86_64-pc-linux-gnu
Running under: Ubuntu 22.04.4 LTS

Matrix products: default
BLAS:   /home/biocbuild/bbs-3.19-bioc/R/lib/libRblas.so 
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_GB              LC_COLLATE=C              
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

time zone: America/New_York
tzcode source: system (glibc)

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] car_3.1-2        carData_3.0-5    pander_0.6.5     gridExtra_2.3   
[5] limpca_0.99.7    ggplot2_3.5.0    BiocStyle_2.31.0

loaded via a namespace (and not attached):
 [1] SummarizedExperiment_1.33.3 gtable_0.3.4               
 [3] xfun_0.43                   bslib_0.7.0                
 [5] ggrepel_0.9.5               Biobase_2.63.1             
 [7] lattice_0.22-6              vctrs_0.6.5                
 [9] tools_4.4.0                 generics_0.1.3             
[11] stats4_4.4.0                parallel_4.4.0             
[13] tibble_3.2.1                fansi_1.0.6                
[15] highr_0.10                  pkgconfig_2.0.3            
[17] Matrix_1.7-0                tidyverse_2.0.0            
[19] S4Vectors_0.41.6            lifecycle_1.0.4            
[21] GenomeInfoDbData_1.2.12     farver_2.1.1               
[23] stringr_1.5.1               compiler_4.4.0             
[25] munsell_0.5.1               ggsci_3.0.3                
[27] codetools_0.2-20            GenomeInfoDb_1.39.13       
[29] htmltools_0.5.8.1           sass_0.4.9                 
[31] yaml_2.3.8                  tidyr_1.3.1                
[33] pillar_1.9.0                crayon_1.5.2               
[35] jquerylib_0.1.4             cachem_1.0.8               
[37] DelayedArray_0.29.9         iterators_1.0.14           
[39] abind_1.4-5                 foreach_1.5.2              
[41] tidyselect_1.2.1            digest_0.6.35              
[43] stringi_1.8.3               purrr_1.0.2                
[45] reshape2_1.4.4              dplyr_1.1.4                
[47] bookdown_0.38               labeling_0.4.3             
[49] fastmap_1.1.1               grid_4.4.0                 
[51] colorspace_2.1-0            cli_3.6.2                  
[53] SparseArray_1.3.4           magrittr_2.0.3             
[55] S4Arrays_1.3.6              utf8_1.2.4                 
[57] withr_3.0.0                 scales_1.3.0               
[59] UCSC.utils_0.99.5           rmarkdown_2.26             
[61] XVector_0.43.1              httr_1.4.7                 
[63] matrixStats_1.2.0           evaluate_0.23              
[65] knitr_1.46                  GenomicRanges_1.55.4       
[67] IRanges_2.37.1              doParallel_1.0.17          
[69] rlang_1.1.3                 Rcpp_1.0.12                
[71] glue_1.7.0                  BiocManager_1.30.22        
[73] BiocGenerics_0.49.1         jsonlite_1.8.8             
[75] plyr_1.8.9                  R6_2.5.1                   
[77] MatrixGenerics_1.15.0       zlibbioc_1.49.3

8 References

Appendix

Benaiche, N., (2022), Stabilisation of the R package LMWiRe – Linear Models for Wide Responses. UCLouvain, http://hdl.handle.net/2078.1/thesis:33996

Cornet, V., Ouaach, A., Mandiki, S., Flamion, E., Ferain, A., Van Larebeke, M., Lemaire, B., Reyes Lopez F., Tort, L., Larondelle, Y. and Kestemont, P., (2018), Environmentally-realistic concentration of cadmium combined with polyunsaturated fatty acids enriched diets modulated non-specific immunity in rainbow trout. Aquatic Toxicology, 196, 104–116. https://doi.org/10.1016/j.aquatox.2018.01.012

Thiel, M., Feraud, B. and Govaerts, B. (2017), ASCA+ and APCA+: exten- sions of ASCA and APCA in the analysis of unbalanced multifactorial designs. Journal of Chemometrics. 31 https://doi.org/10.1002/cem.2895