---
title: "Using bubbleHeatmap to Plot Nightingale Metabolomics Data"
output: rmarkdown::html_vignette
vignette: >
  %\VignetteIndexEntry{"Using bubbleHeatmap to Plot Nightingale Metabolomics Data"}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
---

```{r, include = FALSE}
knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>",
  bubbleLegends.device.warning = FALSE
)
```

```{r setup}
library(bubbleHeatmap)
```

# Abstract

We present bubbleHeatmap, an R plotting package based on the grid system, which 
combines elements of a bubble plot and heatmap to conveniently display two 
numerical variables, (represented by color and size) grouped by categorical 
variables on the x and y axes. This is a useful alternative to a forest plot 
when the data can be grouped in two dimensions, such as predictors x outcomes.
We also demonstrate the particular advantage of this plot type over a 
traditional forest plot for visualising the 225 metabolic measures produced by 
the automated, high-throughput NMR-based platform developed by Nightingale 
Health. Nightingale metabolomic profiles are already available for several large 
biobanks and global cohorts (including recently released data on 100K 
individuals in UK Biobank) and have been used in over 300 peer-reviewed 
publications. We therefore expect the figure template included in this package 
to be of ongoing general interest. 

# The bubbleHeatmap Plotting Function

The bubbleHeatmap function provided in this package returns a graphical object 
(grob) representing a plot consisting of a rectangular grid with squares 
containing colored “bubbles”. The color and size of the bubbles can be scaled 
according to the values of different variables which are input to the function 
as two numeric matrices with identical dimensions. The row and column names of 
the matrices can be added to the plot to label the grid. There are also various 
options to add plot and axis titles, legends, label a row or column of plots in 
a multi-plot figure, and to change the color scale and grid/bubble sizes. 
Legends can be built into the plot or drawn separately using bubbleHMLegends(). 
Additional edits to individual elements (including graphical parameters) can 
easily be made before the plot is drawn. 

All plots are drawn in a viewport with a 5 x 5 layout grid and a given object is 
always positioned in the same cell. This allows multiple plots to be easily 
aligned for combining in a single figure. The positions of the elements in the 
layout grid (column, row) are: PlotTitle (3, 1), XTitle(3, 2), 
LeftLabelsTitle(2, 3), TopLabels(3:4, 3), YTitle(1, 4), LeftLabels(2, 4), 
PlotGrid(3, 4), RowBracket/RowTitle(4, 4), Legends (5, 4), 
ColBracket/ColTitle(3, 5).

```{r bubbleHeatmap}
#Simulate data
names <- list(paste0("leftLabels", 1:6), paste0("topLabels", 1:10))
colorMat <- matrix(rnorm(60), nrow=6, ncol=10, dimnames = names)
sizeMat <- matrix(abs(rnorm(60)), nrow=6, ncol=10, dimnames = names)

#Create sample plot tree containing all elements
tree <-  bubbleHeatmap(colorMat, sizeMat, treeName = "example",
             leftLabelsTitle = "leftLabelsTitle", showRowBracket = T,
             rowTitle = "rowTitle", showColBracket = T, colTitle="colTitle",
             plotTitle="plotTitle", xTitle="xTitle", yTitle="yTitle",
             legendTitles = c("legendTitles[1]", "legendTitles[2]"))
```
```{r draw-plot1, dev = "png", dev.args = list(type = "cairo-png"), fig.width = 6.5, fig.height=3.5, out.width="100%"}
#Draw plot
grid.newpage()
grid.draw(tree)
```


# The Nightingale Plot Templates
## Nightingale Metabolomics Platform
Nightingale Health is a Finnish company which has developed a fully automated 
platform for deriving over 200 quantitative metabolomics measures from a single 
blood serum sample using NMR technology, at a comparable cost to standard lipid 
clinical chemistry. These measures include concentration and composition of 14 
lipoprotein subclasses, apolipoproteins, fatty acids, amino acids and glycolysis 
related metabolites. Over 1M samples have already been processed, including 
biobank participants, population cohorts and clinical studies, with capacity to 
process an additional 250K samples per year. Over 100K profiles of UK Biobank 
participants have recently been released, with data on the remaining individuals 
to follow. The wide availability of standardised Nightingale datasets in global 
cohorts facilitates collaborative research and is contributing to the 
identification of new biomarkers across a range of diseases. 

## CETP Dataset
The data included in this package represents associations between Nightingale 
metabolic measures and a genetic risk score (GRS) for Cholesterol Ester Transfer 
Protein (CETP) in the China Kadoorie Biobank (CKB), a prospective study of 
Chinese adults from 10 distinct areas of China. The full design of the CKB 
cohort and the methods and results of this study have been previously published. 

CETP transfers esterified cholesterol from HDL to apolipoprotein B-containing 
lipoproteins in exchange for triglycerides. This process is a key component of 
the atheroprotective reverse cholesterol transport pathway, which modulates the 
return of excess cholesterol from peripheral cells such as macrophages to the 
liver, where it can be redistributed or excreted. Reduced CETP activity results 
in higher levels of HDL cholesterol (HDL-C), which has resulted in interest in 
CETP as a drug target due to the inverse correlation between HDL-C and risk of 
atherosclerotic disease. In this analysis, five CETP SNP variants were selected 
on the basis of previously reported associations with HDL cholesterol and CETP 
activity. Genotyping data for these variants and conventional lipid biochemistry 
measures were available for a subset of 17,854 CKB participants selected for a 
CVD case-control study. This data was used to generate a CETP GRS weighted by 
HDL-C association, derived internally with 100-fold cross-validation. The 
Nightingale Health metabolomics panel consisting of 225 metabolomics measures 
was available for 4657 of these individuals, and association with the GRS was 
assessed by linear regression with adjustment for age and sex, stratified by
geographical region. In generation of the GRS and association of metabolomics,
outcomes were standardized by rank inverse normal transformation, stratified by 
region, after adjustment for age and sex. 

The sample dataset provided includes the estimate, standard error, p-value, and
-log10(p-value) for the association of the 225 metabolomics traits with the CETP
GRS, scaled to 10-mg/dL higher levels of HDL-C.

## Workflow
### Load Data
The ntngale225 and ntngale249 data frames contain the necessary 
plot groupings and row/column names to arrange a Nightingale results dataset 
into the 10 plots that make up the figure. User dataset should include a column
with either the UK Biobank or China Kadoorie Biobank variable name to enable 
the data to be matched to the template using the "merge_template" function.

```{r merge-data}
myData <- merge_template(cetp, "ckb_id")

```
### One-step Figure
The metabFigure function wraps the formatData, multiPlotInput, bubbleHeatmapList 
and metabFigurePlot functions listed below, applying default settings to build 
the standard figure in one step. Alternatively, see below for the individual 
functions. 

```{r one-step}
metabTree <- metabFigure(myData)
```

### Format Data
The formatData function splits a dataset according to the value of plotGroup, 
and reshapes each one into two matrices defined by the values of rowName and 
colName, containing the values of colorValue and sizeValue, and orders the list 
and the rows and columns of each matrix. The Nightingale sorting orders are 
built-in and will override other settings when nightingale = TRUE. 

```{r format-data}
gridData <- formatData(myData, colorValue="estimate", sizeValue = "negLog10P", 
                       nightingale = TRUE)
```

### Prepare Input for plotting function (multiPlotInput) 
multiPlotInput() returns a list of lists of the input arguments/settings for 
bubbleHeatmap(), allowing multiple plots to be generated at once. It generates 
a single set of legends based on the data across all plots. Settings 
for the Nightingale plots are built-in and are requested using nightingale 
= TRUE.

```{r tree-input}
treeInput <- multiPlotInput(colorList=gridData$colorList, 
                            sizeList=gridData$sizeList, 
                            nightingale=TRUE, legendHeight=8)
```

### BubbleHeatmapList
bubbleHeatmapList is a convenient wrapper for generating a list of plots in a 
single call. It requires a single argument of two lists, \$trees, a list of 
argument lists to create plots using bubbleHeatmap(), and \$legends, a list of 
arguments for (optionally) creating legends using bubbleHMLegends(). The output 
from multiPlotInput is formatted in this way and can be passed directly to
bubbleHeatmapList().

```{r create-trees}
treeList <- bubbleHeatmapList(treeInput)
```

### Nightingale Figure Function
The metabFigurePlot function takes a list of plot trees and a legend tree, and 
combines and arranges them to produce a single tree representing the Nightingale
plot figure style. Like the individual bubbleHeatmap trees, they can be edited,
or drawn using grid.draw()

```{r assemble-figure}
metabTree <- metabFigurePlot(treeList)
```
```{r plot2,  dev = "png", dev.args = list(type = "cairo-png"), fig.width = 8, fig.height=6, out.width="100%"}
grid.newpage()
grid.draw(metabTree)
```
