README.Rmd

---
title: "PCAtools: everything Principal Component Analysis"
author: "Kevin Blighe, Aaron Lun"
date: "`r Sys.Date()`"
package: "`r packageVersion('PCAtools')`"
output:
  github_document:
    toc: true
    toc_depth: 3
fig_width: 7
bibliography: library.bib
vignette: >
    %\VignetteIndexEntry{PCAtools: everything Principal Component Analysis}
    %\VignetteEngine{knitr::rmarkdown}
    %\VignetteEncoding{UTF-8}
    %\usepackage[utf8]{inputenc}
---

# Introduction

Principal Component Analysis (PCA) is a very powerful technique that has wide applicability in data science, bioinformatics, and further afield. It was initially developed to analyse large volumes of data in order to tease out the differences/relationships between the logical entities being analysed. It extracts the fundamental structure of the data without the need to build any model to represent it. This 'summary' of the data is arrived at through a process of reduction that can transform the large number of variables into a lesser number that are uncorrelated (i.e. the ‘principal components'), while at the same time being capable of easy interpretation on the original data [@PCAtools] [@BligheK].

*PCAtools* provides functions for data exploration via PCA, and allows the user to generate publication-ready figures. PCA is performed via *BiocSingular* [@Lun] - users can also identify optimal number of principal components via different metrics, such as elbow method and Horn's parallel analysis [@Horn] [@Buja], which has relevance for data reduction in single-cell RNA-seq (scRNA-seq) and high dimensional mass cytometry data.

```{r VROOM_CONNECTION_SIZE, eval = TRUE, echo = FALSE}

  Sys.setenv(VROOM_CONNECTION_SIZE='512000')

```

# Installation

## 1. Download the package from Bioconductor

```{r getPackage, eval = FALSE}

  if (!requireNamespace('BiocManager', quietly = TRUE))
    install.packages('BiocManager')

  BiocManager::install('PCAtools')

```

Note: to install development version direct from GitHub:

```{r getPackageDevel, eval = FALSE}

  if (!requireNamespace('remotes', quietly = TRUE))
    install.packages('remotes')

  remotes::install_github('kevinblighe/PCAtools')

```

## 2. Load the package into R session

```{r Load, message = FALSE}

  library(PCAtools)

```

# Quick start: *DESeq2*

For this example, we will follow the tutorial (from Section 3.1) of [RNA-seq workflow: gene-level
exploratory analysis and differential expression](http://master.bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html). Specifically, we will load the 'airway' data, where different airway smooth muscle cells were treated with dexamethasone.

```{r message = FALSE}

  library(airway)
  library(magrittr)

  data('airway')
  airway$dex %<>% relevel('untrt')

```

Annotate the Ensembl gene IDs to gene symbols:

```{r message = FALSE}

  ens <- rownames(airway)

  library(org.Hs.eg.db)
  symbols <- mapIds(org.Hs.eg.db, keys = ens,
    column = c('SYMBOL'), keytype = 'ENSEMBL')
  symbols <- symbols[!is.na(symbols)]
  symbols <- symbols[match(rownames(airway), names(symbols))]
  rownames(airway) <- symbols
  keep <- !is.na(rownames(airway))
  airway <- airway[keep,]

```

Normalise the data and transform the normalised counts to variance-stabilised expression levels:

```{r message = FALSE, warning = FALSE}

  library('DESeq2')

  dds <- DESeqDataSet(airway, design = ~ cell + dex)
  dds <- DESeq(dds)
  vst <- assay(vst(dds))

```

## Conduct principal component analysis (PCA):

```{r}

  p <- pca(vst, metadata = colData(airway), removeVar = 0.1)

```

## A scree plot

```{r ex1, warning = FALSE, fig.height = 7, fig.width = 6, fig.cap = 'Figure 1: A scree plot'}

  screeplot(p, axisLabSize = 18, titleLabSize = 22)

```

## A bi-plot

Different interpretations of the biplot exist. In the OMICs era, for most
general users, a biplot is a simple representation of samples in a
2-dimensional space, usually focusing on just the first two PCs:

```{r ex2a, eval = FALSE}

  biplot(p)

```

However, the original definition of a biplot by Gabriel KR [@Gabriel] is a
plot that plots both variables and observations (samples) in the same space.
The variables are indicated by arrows drawn from the origin, which indicate
their 'weight' in different directions. We touch on this later via the
*plotLoadings* function.

```{r ex2b, fig.height = 7, fig.width = 7, fig.cap = 'Figure 2: A bi-plot'}

  biplot(p, showLoadings = TRUE,
    labSize = 5, pointSize = 5, sizeLoadingsNames = 5)

```



# Quick start: Gene Expression Omnibus (GEO)

Here, we will instead start with data from [Gene Expression Omnibus](https://www.ncbi.nlm.nih.gov/geo/).
We will load breast cancer gene expression data with recurrence free survival (RFS) from
[Gene Expression Profiling in Breast Cancer: Understanding the Molecular Basis of Histologic Grade To Improve Prognosis](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2990).

First, let's read in and prepare the data:

```{r, message = FALSE}

  library(Biobase)
  library(GEOquery)

  # load series and platform data from GEO
    gset <- getGEO('GSE2990', GSEMatrix = TRUE, getGPL = FALSE)
    mat <- exprs(gset[[1]])

  # remove Affymetrix control probes
    mat <- mat[-grep('^AFFX', rownames(mat)),]

  # extract information of interest from the phenotype data (pdata)
   idx <- which(colnames(pData(gset[[1]])) %in%
      c('relation', 'age:ch1', 'distant rfs:ch1', 'er:ch1',
        'ggi:ch1', 'grade:ch1', 'size:ch1',
        'time rfs:ch1'))
    metadata <- data.frame(pData(gset[[1]])[,idx],
      row.names = rownames(pData(gset[[1]])))

  # tidy column names
    colnames(metadata) <- c('Study', 'Age', 'Distant.RFS', 'ER', 'GGI', 'Grade',
      'Size', 'Time.RFS')

  # prepare certain phenotypes of interest
    metadata$Study <- gsub('Reanalyzed by: ', '', as.character(metadata$Study))
    metadata$Age <- as.numeric(gsub('^KJ', NA, as.character(metadata$Age)))
    metadata$Distant.RFS <- factor(metadata$Distant.RFS,
      levels = c(0,1))
    metadata$ER <- factor(gsub('\\?', NA, as.character(metadata$ER)),
      levels = c(0,1))
    metadata$ER <- factor(ifelse(metadata$ER == 1, 'ER+', 'ER-'),
      levels = c('ER-', 'ER+'))
    metadata$GGI <- as.numeric(as.character(metadata$GGI))
    metadata$Grade <- factor(gsub('\\?', NA, as.character(metadata$Grade)),
      levels = c(1,2,3))
    metadata$Grade <- gsub(1, 'Grade 1', gsub(2, 'Grade 2', gsub(3, 'Grade 3', metadata$Grade)))
    metadata$Grade <- factor(metadata$Grade, levels = c('Grade 1', 'Grade 2', 'Grade 3'))
    metadata$Size <- as.numeric(as.character(metadata$Size))
    metadata$Time.RFS <- as.numeric(gsub('^KJX|^KJ', NA, metadata$Time.RFS))

  # remove samples from the pdata that have any NA value
    discard <- apply(metadata, 1, function(x) any(is.na(x)))
    metadata <- metadata[!discard,]

  # filter the expression data to match the samples in our pdata
    mat <- mat[,which(colnames(mat) %in% rownames(metadata))]

  # check that sample names match exactly between pdata and expression data 
    all(colnames(mat) == rownames(metadata))

```

Conduct principal component analysis (PCA):

```{r}

  p <- pca(mat, metadata = metadata, removeVar = 0.1)

```

## A bi-plot

```{r ex3a, eval = FALSE}

  biplot(p)

```

```{r ex3b, fig.height = 6, fig.width = 7, fig.cap = 'Figure 3: A bi-plot'}

  biplot(p, showLoadings = TRUE, lab = NULL)

```

One of the probes pointing downward is *205225_at*, which targets the *ESR1*
gene. This is already a useful validation, as the oestrogen receptor, which
is in part encoded by *ESR1*, is strongly represented by PC2 (y-axis), with
negative-to-positive receptor status going from top-to-bottom.

More on this later in this vignette.


## A pairs plot

```{r ex4, message = FALSE, fig.height = 10, fig.width = 10, fig.cap = 'Figure 4: A pairs plot'}

  pairsplot(p)

```

## A loadings plot

If the biplot was previously generated with *showLoadings = TRUE*, check how
this loadings plot corresponds to the biplot loadings - they should match up
for the top hits.

```{r ex5, fig.height = 6, fig.width = 8, fig.cap = 'Figure 5: A loadings plot'}

  plotloadings(p, labSize = 3)

```


## An eigencor plot

```{r ex6, warning = FALSE, fig.height = 4, fig.width = 8, fig.cap = 'Figure 6: An eigencor plot'}

  eigencorplot(p,
    metavars = c('Study','Age','Distant.RFS','ER',
      'GGI','Grade','Size','Time.RFS'))

```

## Access the internal data

The rotated data that represents the observations / samples is stored in *rotated*,
while the variable loadings are stored in *loadings*

```{r}

  p$rotated[1:5,1:5]

  p$loadings[1:5,1:5]

```


# Advanced features

All functions in *PCAtools* are highly configurable and should cover virtually
all basic and advanced user requirements. The following sections take a look
at some of these advanced features, and form a somewhat practical example of
how one can use *PCAtools* to make a clinical interpretation of data.

First, let's sort out the gene annotation by mapping the probe IDs to gene
symbols. The array used for this study was the Affymetrix U133a, so let's use
the *hgu133a.db* Bioconductor package:

```{r, messages = FALSE}

  suppressMessages(require(hgu133a.db))
  newnames <- mapIds(hgu133a.db,
    keys = rownames(p$loadings),
    column = c('SYMBOL'),
    keytype = 'PROBEID')

  # tidy up for NULL mappings and duplicated gene symbols
  newnames <- ifelse(is.na(newnames) | duplicated(newnames),
    names(newnames), newnames)
  rownames(p$loadings) <- newnames
    
```


## Determine optimum number of PCs to retain

A scree plot on its own just shows the accumulative proportion of explained
variation, but how can we determine the optimum number of PCs to retain?

*PCAtools* provides four metrics for this purpose:

* Elbow method (`findElbowPoint()`)
* Horn's parallel analysis [@Horn] [@Buja] (`parallelPCA()`)
* Marchenko-Pastur limit (`chooseMarchenkoPastur()`)
* Gavish-Donoho method (`chooseGavishDonoho()`)

Let's perform Horn's parallel analysis first:

```{r, warning = FALSE}

  horn <- parallelPCA(mat)
  horn$n

```

Now the elbow method:

```{r}

  elbow <- findElbowPoint(p$variance)
  elbow

```

In most cases, the identified values will disagree. This is because finding the
correct number of PCs is a difficult task and is akin to finding the 'correct'
number of clusters in a dataset - there is no correct answer.

Taking these values, we can produce a new scree plot and mark these:

```{r ex7, fig.height = 7, fig.width = 9, fig.cap = 'Figure 7: Advanced scree plot illustrating optimum number of PCs'}

  library(ggplot2)

  screeplot(p,
    components = getComponents(p, 1:20),
    vline = c(horn$n, elbow)) +

    geom_label(aes(x = horn$n + 1, y = 50,
      label = 'Horn\'s', vjust = -1, size = 8)) +
    geom_label(aes(x = elbow + 1, y = 50,
      label = 'Elbow method', vjust = -1, size = 8))

```

If all else fails, one can simply take the number of PCs that contributes to
a pre-selected total of explained variation, e.g., in this case, 27 PCs account
for >80% explained variation.

```{r}

  which(cumsum(p$variance) > 80)[1]

```


## Modify bi-plots

The bi-plot comparing PC1 versus PC2 is the most characteristic plot of PCA.
However, PCA is much more than the bi-plot and much more than PC1 and PC2. This
said, PC1 and PC2, by the very nature of PCA, are indeed usually the most
important parts of a PCA analysis.

In a bi-plot, we can shade the points by different groups and add many more features.

### Colour by a metadata factor, use a custom label, add lines through origin, and add legend

```{r ex8, fig.height = 7, fig.width = 7.5, fig.cap = 'Figure 8: Colour by a metadata factor, use a custom label, add lines through origin, and add legend'}

  biplot(p,
    lab = paste0(p$metadata$Age, ' años'),
    colby = 'ER',
    hline = 0, vline = 0,
    legendPosition = 'right')

```


### Supply custom colours and encircle variables by group

The encircle functionality literally draws a polygon around
each group specified by *colby*. It says nothing about any statistic
pertaining to each group.

```{r ex9, message = FALSE, fig.height = 7.5, fig.width = 7, fig.cap = 'Figure 9: Supply custom colours and encircle variables by group'}

  biplot(p,
    colby = 'ER', colkey = c('ER+' = 'forestgreen', 'ER-' = 'purple'),
    colLegendTitle = 'ER-\nstatus',
    # encircle config
      encircle = TRUE,
      encircleFill = TRUE,
    hline = 0, vline = c(-25, 0, 25),
    legendPosition = 'top', legendLabSize = 16, legendIconSize = 8.0)

  biplot(p,
    colby = 'ER', colkey = c('ER+' = 'forestgreen', 'ER-' = 'purple'),
    colLegendTitle = 'ER-\nstatus',
    # encircle config
      encircle = TRUE, encircleFill = FALSE,
      encircleAlpha = 1, encircleLineSize = 5,
    hline = 0, vline = c(-25, 0, 25),
    legendPosition = 'top', legendLabSize = 16, legendIconSize = 8.0)

```


### Stat ellipses

Stat ellipses are also drawn around each group but have a greater statistical
meaning and can be used, for example, as a strict determination of outlier
samples. Here, we draw ellipses around each group at the 95% confidence level:

```{r ex10, message = FALSE, fig.height = 7.5, fig.width = 7, fig.cap = 'Figure 10: Stat ellipses'}

  biplot(p,
    colby = 'ER', colkey = c('ER+' = 'forestgreen', 'ER-' = 'purple'),
    # ellipse config
      ellipse = TRUE,
      ellipseType = 't',
      ellipseLevel = 0.95,
      ellipseFill = TRUE,
      ellipseAlpha = 1/4,
      ellipseLineSize = 1.0,
    xlim = c(-125,125), ylim = c(-50, 80),
    hline = 0, vline = c(-25, 0, 25),
    legendPosition = 'top', legendLabSize = 16, legendIconSize = 8.0)

  biplot(p,
    colby = 'ER', colkey = c('ER+' = 'forestgreen', 'ER-' = 'purple'),
    # ellipse config
      ellipse = TRUE,
      ellipseType = 't',
      ellipseLevel = 0.95,
      ellipseFill = TRUE,
      ellipseAlpha = 1/4,
      ellipseLineSize = 0,
      ellipseFillKey = c('ER+' = 'yellow', 'ER-' = 'pink'),
    xlim = c(-125,125), ylim = c(-50, 80),
    hline = 0, vline = c(-25, 0, 25),
    legendPosition = 'top', legendLabSize = 16, legendIconSize = 8.0)

```


### Change shape based on tumour grade, remove connectors, and add titles

```{r ex11, message = FALSE, eval = FALSE}

  biplot(p,
    colby = 'ER', colkey = c('ER+' = 'forestgreen', 'ER-' = 'purple'),
    hline = c(-25, 0, 25), vline = c(-25, 0, 25),
    legendPosition = 'top', legendLabSize = 13, legendIconSize = 8.0,
    shape = 'Grade', shapekey = c('Grade 1' = 15, 'Grade 2' = 17, 'Grade 3' = 8),
    drawConnectors = FALSE,
    title = 'PCA bi-plot',
    subtitle = 'PC1 versus PC2',
    caption = '27 PCs ≈ 80%')

```

### Modify line types, remove gridlines, and increase point size

```{r ex12a}

  biplot(p,
    lab = NULL,
    colby = 'ER', colkey = c('ER+'='royalblue', 'ER-'='red3'),
    hline = c(-25, 0, 25), vline = c(-25, 0, 25),
    vlineType = c('dotdash', 'solid', 'dashed'),
    gridlines.major = FALSE, gridlines.minor = FALSE,
    pointSize = 5,
    legendPosition = 'left', legendLabSize = 14, legendIconSize = 8.0,
    shape = 'Grade', shapekey = c('Grade 1'=15, 'Grade 2'=17, 'Grade 3'=8),
    drawConnectors = FALSE,
    title = 'PCA bi-plot',
    subtitle = 'PC1 versus PC2',
    caption = '27 PCs ≈ 80%')

```

Let's plot the same as above but with loadings:

```{r ex12b, message = FALSE, fig.height = 5.5, fig.width = 11, fig.cap = 'Figure 11: Modify line types, remove gridlines, and increase point size'}

  biplot(p,
    # loadings parameters
      showLoadings = TRUE,
      lengthLoadingsArrowsFactor = 1.5,
      sizeLoadingsNames = 4,
      colLoadingsNames = 'red4',
    # other parameters
      lab = NULL,
      colby = 'ER', colkey = c('ER+'='royalblue', 'ER-'='red3'),
      hline = 0, vline = c(-25, 0, 25),
      vlineType = c('dotdash', 'solid', 'dashed'),
      gridlines.major = FALSE, gridlines.minor = FALSE,
      pointSize = 5,
      legendPosition = 'left', legendLabSize = 14, legendIconSize = 8.0,
      shape = 'Grade', shapekey = c('Grade 1'=15, 'Grade 2'=17, 'Grade 3'=8),
      drawConnectors = FALSE,
      title = 'PCA bi-plot',
      subtitle = 'PC1 versus PC2',
      caption = '27 PCs ≈ 80%')

```

### Colour by a continuous variable and plot other PCs

There are two ways to colour by a continuous variable. In the first way, we simply
'add on' a continuous colour scale via *scale_colour_gradient*:

```{r ex13a, fig.height = 5.5, fig.width = 6, fig.cap = 'Figure 12: Colour by a continuous variable and plot other PCs'}

  # add ESR1 gene expression to the metadata
  p$metadata$ESR1 <- mat['205225_at',]

  biplot(p,
    x = 'PC2', y = 'PC3',
    lab = NULL,
    colby = 'ESR1',
    shape = 'ER',
    hline = 0, vline = 0,
    legendPosition = 'right') +

  scale_colour_gradient(low = 'gold', high = 'red2')

```

We can also just permit that the internal *ggplot2* engine picks the colour
scheme - here, we also plot PC10 versus PC50:

```{r ex13b, eval = FALSE}

  # was always eval = FALSE
  biplot(p, x = 'PC10', y = 'PC50',
    lab = NULL,
    colby = 'Age',
    hline = 0, vline = 0,
    hlineWidth = 1.0, vlineWidth = 1.0,
    gridlines.major = FALSE, gridlines.minor = TRUE,
    pointSize = 5,
    legendPosition = 'left', legendLabSize = 16, legendIconSize = 8.0,
    shape = 'Grade', shapekey = c('Grade 1'=15, 'Grade 2'=17, 'Grade 3'=8),
    drawConnectors = FALSE,
    title = 'PCA bi-plot',
    subtitle = 'PC10 versus PC50',
    caption = '27 PCs ≈ 80%')

```

## Quickly explore potentially informative PCs via a pairs plot

The pairs plot in PCA unfortunately suffers from a lack of use; however, for
those who love exploring data and squeezing every last ounce of information out
of data, a pairs plot provides for a relatively quick way to explore useful
leads for other downstream analyses.

As the number of pairwise plots increases, however, space becomes limited. We
can shut off titles and axis labeling to save space. Reducing point size and
colouring by a variable of interest can additionally help us to rapidly skim
over the data.

```{r ex14, message = FALSE, fig.height = 8, fig.width = 7, fig.cap = 'Figure 13: Quickly explore potentially informative PCs via a pairs plot'}

  pairsplot(p,
    components = getComponents(p, c(1:10)),
    triangle = TRUE, trianglelabSize = 12,
    hline = 0, vline = 0,
    pointSize = 0.4,
    gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'Grade',
    title = 'Pairs plot', plotaxes = FALSE,
    margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))

```

We can arrange these in a way that makes better use of the screen space by
setting 'triangle = FALSE'. In this case, we can further control the layout
with the 'ncol' and 'nrow' parameters, although, the function will
automatically determine these based on your input data.

```{r ex15, fig.height = 5.5, fig.width = 6, fig.cap = 'Figure 14: arranging a pairs plot horizontally'}

  pairsplot(p,
    components = getComponents(p, c(4,33,11,1)),
    triangle = FALSE,
    hline = 0, vline = 0,
    pointSize = 0.8,
    gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'ER',
    title = 'Pairs plot', titleLabSize = 22,
    axisLabSize = 14, plotaxes = TRUE,
    margingaps = unit(c(0.1, 0.1, 0.1, 0.1), 'cm'))

```

## Determine the variables that drive variation among each PC

If, on the bi-plot or pairs plot, we encounter evidence that 1 or more PCs
are segregating a factor of interest, we can explore further the genes that
are driving these differences along each PC.

For each PC of interest, 'plotloadings' determines the variables falling within
the top/bottom 5% of the loadings range, and then creates a final consensus
list of these. These variables are then plotted.

The loadings plot, like all others, is highly configurable. To modify the
cut-off for inclusion / exclusion of variables, we use *rangeRetain*, where
0.01 equates to the top/bottom 1% of the loadings range per PC.

```{r ex16, fig.height = 5.5, fig.width = 7, fig.cap = 'Figure 15: Determine the variables that drive variation among each PC'}

  plotloadings(p,
    rangeRetain = 0.01,
    labSize = 4.0,
    title = 'Loadings plot',
    subtitle = 'PC1, PC2, PC3, PC4, PC5',
    caption = 'Top 1% variables',
    shape = 24,
    col = c('limegreen', 'black', 'red3'),
    drawConnectors = TRUE)

```

At least one interesting finding is *205225_at* / *ESR1*, which is by far the gene
most responsible for variation along PC2. The previous bi-plots showed that
this PC also segregated ER+ from ER- patients. The other results could be
explored. Also, from the biplots with loadings that we have already generated,
this result is also verified in these.

With the loadings plot, in addition, we can instead plot absolute values and
modify the point sizes to be proportional to the loadings. We can also switch
off the line connectors and plot the loadings for any PCs

```{r ex17a, fig.height = 7, fig.width = 9, fig.cap = 'Figure 16: plotting absolute component loadings'}

  plotloadings(p,
    components = getComponents(p, c(4,33,11,1)),
    rangeRetain = 0.1,
    labSize = 4.0,
    absolute = FALSE,
    title = 'Loadings plot',
    subtitle = 'Misc PCs',
    caption = 'Top 10% variables',
    shape = 23, shapeSizeRange = c(1, 16),
    col = c('white', 'pink'),
    drawConnectors = FALSE)

```

We can plot just this single PC and flip the plot on its side, if we wish:

```{r ex17b, fig.height = 4.5, fig.width = 9, fig.cap = 'Figure 17: plotting absolute component loadings'}

  plotloadings(p,
    components = getComponents(p, c(2)),
    rangeRetain = 0.12, absolute = TRUE,
    col = c('black', 'pink', 'red4'),
    drawConnectors = TRUE, labSize = 4) + coord_flip()

```


## Correlate the principal components back to the clinical data

Further exploration of the PCs can come through correlations with clinical
data. This is also a mostly untapped resource in the era of 'big data' and
can help to guide an analysis down a particular path.

We may wish, for example, to correlate all PCs that account for 80% variation in
our dataset and then explore further the PCs that have statistically significant
correlations.

'eigencorplot' is built upon another function by the *PCAtools* developers, namely
[CorLevelPlot](https://github.com/kevinblighe/CorLevelPlot). Further examples
can be found there.

**NB - for factors, ensure that these are ordered in a logical fashion prior to running this function**

```{r ex18a, warning = FALSE, fig.height = 4.25, fig.width = 12, fig.cap = 'Figure 18: Correlate the principal components back to the clinical data'}

  eigencorplot(p,
    components = getComponents(p, 1:27),
    metavars = c('Study','Age','Distant.RFS','ER',
      'GGI','Grade','Size','Time.RFS'),
    col = c('darkblue', 'blue2', 'black', 'red2', 'darkred'),
    cexCorval = 0.7,
    colCorval = 'white',
    fontCorval = 2,
    posLab = 'bottomleft',
    rotLabX = 45,
    posColKey = 'top',
    cexLabColKey = 1.5,
    scale = TRUE,
    main = 'PC1-27 clinical correlations',
    colFrame = 'white',
    plotRsquared = FALSE)

```

We can also supply different cut-offs for statistical significance, apply
p-value adjustment, plot R-squared values, and specify correlation method:

```{r ex18b, warning = FALSE, fig.height = 5, fig.width = 12, fig.cap = 'Figure 19: Correlate the principal components back to the clinical data'}

  eigencorplot(p,
    components = getComponents(p, 1:horn$n),
    metavars = c('Study','Age','Distant.RFS','ER','GGI',
      'Grade','Size','Time.RFS'),
    col = c('white', 'cornsilk1', 'gold', 'forestgreen', 'darkgreen'),
    cexCorval = 1.2,
    fontCorval = 2,
    posLab = 'all',
    rotLabX = 45,
    scale = TRUE,
    main = bquote(Principal ~ component ~ Pearson ~ r^2 ~ clinical ~ correlates),
    plotRsquared = TRUE,
    corFUN = 'pearson',
    corUSE = 'pairwise.complete.obs',
    corMultipleTestCorrection = 'BH',
    signifSymbols = c('****', '***', '**', '*', ''),
    signifCutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 1))

```

Clearly, PC2 is coming across as the most interesting PC in this experiment,
with highly statistically significant correlation (p<0.0001) to ER status,
tumour grade, and GGI (genomic Grade Index), an indicator of response.
It comes as no surprise that the gene driving most variationn along PC2 is
*ESR1*, identified from our loadings plot.

This information is, of course, not new, but shows how PCA is much more than
just a bi-plot used to identify outliers!


## Plot the entire project on a single panel

```{r ex19, message = FALSE, warning = FALSE, fig.height = 10, fig.width = 15, fig.cap = 'Figure 20: a merged panel of all PCAtools plots'}

  pscree <- screeplot(p, components = getComponents(p, 1:30),
    hline = 80, vline = 27, axisLabSize = 14, titleLabSize = 20,
    returnPlot = FALSE) +
    geom_label(aes(20, 80, label = '80% explained variation', vjust = -1, size = 8))

  ppairs <- pairsplot(p, components = getComponents(p, c(1:3)),
    triangle = TRUE, trianglelabSize = 12,
    hline = 0, vline = 0,
    pointSize = 0.8, gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'Grade',
    title = '', plotaxes = FALSE,
    margingaps = unit(c(0.01, 0.01, 0.01, 0.01), 'cm'),
    returnPlot = FALSE)

  pbiplot <- biplot(p,
    # loadings parameters
      showLoadings = TRUE,
      lengthLoadingsArrowsFactor = 1.5,
      sizeLoadingsNames = 4,
      colLoadingsNames = 'red4',
    # other parameters
      lab = NULL,
      colby = 'ER', colkey = c('ER+'='royalblue', 'ER-'='red3'),
      hline = 0, vline = c(-25, 0, 25),
      vlineType = c('dotdash', 'solid', 'dashed'),
      gridlines.major = FALSE, gridlines.minor = FALSE,
      pointSize = 5,
      legendPosition = 'none', legendLabSize = 16, legendIconSize = 8.0,
      shape = 'Grade', shapekey = c('Grade 1'=15, 'Grade 2'=17, 'Grade 3'=8),
      drawConnectors = FALSE,
      title = 'PCA bi-plot',
      subtitle = 'PC1 versus PC2',
      caption = '27 PCs ≈ 80%',
      returnPlot = FALSE)

  ploadings <- plotloadings(p, rangeRetain = 0.01, labSize = 4,
    title = 'Loadings plot', axisLabSize = 12,
    subtitle = 'PC1, PC2, PC3, PC4, PC5',
    caption = 'Top 1% variables',
    shape = 24, shapeSizeRange = c(4, 8),
    col = c('limegreen', 'black', 'red3'),
    legendPosition = 'none',
    drawConnectors = FALSE,
    returnPlot = FALSE)

  peigencor <- eigencorplot(p,
    components = getComponents(p, 1:10),
    metavars = c('Study','Age','Distant.RFS','ER',
      'GGI','Grade','Size','Time.RFS'),
    cexCorval = 1.0,
    fontCorval = 2,
    posLab = 'all', 
    rotLabX = 45,
    scale = TRUE,
    main = "PC clinical correlates",
    cexMain = 1.5,
    plotRsquared = FALSE,
    corFUN = 'pearson',
    corUSE = 'pairwise.complete.obs',
    signifSymbols = c('****', '***', '**', '*', ''),
    signifCutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 1),
    returnPlot = FALSE)

    library(cowplot)
    library(ggplotify)

    top_row <- plot_grid(pscree, ppairs, pbiplot,
      ncol = 3,
      labels = c('A', 'B  Pairs plot', 'C'),
      label_fontfamily = 'serif',
      label_fontface = 'bold',
      label_size = 22,
      align = 'h',
      rel_widths = c(1.10, 0.80, 1.10))

    bottom_row <- plot_grid(ploadings,
      as.grob(peigencor),
      ncol = 2,
      labels = c('D', 'E'),
      label_fontfamily = 'serif',
      label_fontface = 'bold',
      label_size = 22,
      align = 'h',
      rel_widths = c(0.8, 1.2))

    plot_grid(top_row, bottom_row, ncol = 1,
      rel_heights = c(1.1, 0.9))

```

## Make predictions on new data

It is possible to use the variable loadings as part of a matrix calculation
to 'predict' principal component eigenvectors in new data. This is elaborated
in a posting by Pandula Priyadarshana: [How to use Principal Component Analysis (PCA) to make Predictions](https://rpubs.com/PandulaP/PCA_for_Predictions). 

The *pca* class, which is created by *PCAtools*, is not configured to work with
*stats::predict*; however, trusty *prcomp* class **is** configured. We can manually
create a *prcomp* object and then use that in model prediction, as elaborated
in the following code chunk:

```{r} 

  p <- pca(mat, metadata = metadata, removeVar = 0.1)
  p.prcomp <- list(sdev = p$sdev,
    rotation = data.matrix(p$loadings),
    x = data.matrix(p$rotated),
    center = TRUE, scale = TRUE)

  class(p.prcomp) <- 'prcomp'

  # for this simple example, just use a chunk of
  # the original data for the prediction
  newdata <- t(mat[,seq(1,20)])
  predict(p.prcomp, newdata = newdata)[,1:5]

```


# Acknowledgments

The development of *PCAtools* has benefited from contributions
and suggestions from:

* Krushna Chandra Murmu
* Jinsheng
* Myles Lewis
* Anna-Leigh Brown
* Vincent Carey
* Vince Vu
* Guido Hooiveld
* pwwang
* Pandula Priyadarshana
* Barley Rose Collier Harris
* Bob Policastro

# Session info

```{r}

sessionInfo()

```

# References

@PCAtools

@BligheK

@Horn

@Buja

@Lun

@Gabriel