Data Visualization with R

Alexandra L Emmons Ph.D. & Joe Wu Ph.D.

BTEP/GAU/CCR/NCI/NIH - email [email protected]
Bioinformatics Training and Education Program
Table of Contents

Course Overview

• Course Overview 10

• Welcome to the Data Visualization with R Series 10

• A series of lessons designed to introduce learners to the R package ggplot2 10

• Course objectives 10

• What this course is not! 10

• Course Expectations 10

• Lesson 1: Introduction to plot types 10

• Lesson 2: Getting Started with ggplot2 11

• Lesson 3: Scatter plots and Non-data elements of ggplot2 customization 11

• Lesson 4: Visualizing summary statistics with ggplot2 11

• Lesson 5: Visualizing clusters with heatmaps 11

• Lesson 6: Combining multiple plots to create a figure panel 11

• Required Course Materials 11

Lesson 1: Course Introduction

• Lesson 1: Course Introduction 12

Lesson 2: The Basics of GGplot2

• Introduction to ggplot2 13

• Objectives 13
 • Going beyond Excel 13

• Why ggplot2? 16

• Getting started with ggplot2 16

• Getting help 17

• The ggplot2 template 17

• Geom functions 20

• Mapping and aesthetics (aes()) 21

• R objects can also store figures 24

• Colors 24

• Facets 30

• A better example of facet_grid() using data("Titanic"). 33

• Building upon our template 34

• Using multiple geoms per plot 35

• Labels, legends, scales, and themes 36

• Saving plots (ggsave()) 37

• Resource list 38

• Acknowledgements 38

Lesson 3: Scatter plots and ggplot2 customization

• Scatter plots and plot customization 39

• Objectives 39

• The Data 39

• The grammar of graphics 40

• Scatter plots 41

• Basic Scatter 41

• Coordinate Systems 42
 • Perform and plot PCA data using iris. 43

• What is PCA? 43

• Perform PCA 44

• Plot PCA 45

• Add custom axes labels 47

• Add a stat to our plot with stat_ellipse(). 48

• Plot customization 49

• Themes 49

• A helpful color trick 51

• Putting it all together 53

• Creating a publication ready volcano plot 53

• Changing axes scales 56

• Modifying legends 57

• Save to an .rds file 60

• References 61

Lesson 4: Stat Transformations: Bar plots, box plots, and histograms

• Stat Transformations: Bar plots, box plots, and histograms 62

• Our grammar of graphics template 62

• Our grammar of graphics template 62

• Libraries 63

• The Data 63

• Variable Types 64

• The stat argument 65

• Bar Plot 66

• stat = count 67

• stat = identity 69
 • Using factors 71

• Adding error bars 73

• Histogram 75

• Box plot 78

Lesson5: Visualizing clusters with heatmap and dendrogram

• Visualizing clusters with heatmaps 81

• What is a heatmap? 81

• Heatmap can be used to visualize the following 81

• What is a dendrogram? 81

• Applications of dendrograms 81

• Methods available to produce a heatmap in R 84

• Load the libraries 85

• Import data 85

• Heatmap of the top 20 genes from differential expression analysis 87

• Distance, clustering, and scaling 88

• Distance calculation 88

• Cluster generation 88

• Scaling 88

• Working with color customization 92

• Adding treatment group information to samples (annotation_col, annotation_colors) 93

• Add the annotations column 94

• Modify the annotations colors 95

• Splitting heatmap into multiple columns and rows 96

• Add a title to heatmap 98

• Saving a non-ggplot 100

 • Make this plot compatible with ggplot2 using the package ggplotify. 102

• Save as an R object 102

Lesson 6: Multi-figure panel

• Objectives 105

• Why do we need to learn to combine figures? 105

• Example journals and their figure limits: 105

• Load the libraries 106

• The Data 107

• What is cowplot? 111

• Using cowplot to arrange figures 112

• Plotting labels with cowplot 113

• Other notable features of cowplot 114

• Taking advantage of ggdraw 115

• Using cowplot we can get an image such as this: 117

• What is patchwork? 118

• Combining two plots 119

• Using plot_layout() 125

• Adding a spacer 129

• Add our cowplot image 130

• Including a title 131

• Save the plot 132

• Which package to use? 133

• Acknowledgements 133
Getting the Data

Course Data 135

Practice Questions

Practice plotting using ggplot2: Lesson 2 137

• Load the data 137

• Exercise Questions 137

• Question 1 137

• Question 2 138

• Question 3 139

• Question 4 139

• Question 5 140

• Question 6 141

• Challenge question 1 142

• Challenge question 2 143

• Want more practice? 145

• Question 1 145

• Question 2 145

• Question 3 146

• Question 4 147

• Question 5 148

• Question 6 149
Practice plotting using ggplot2: Lesson 3 151

• Question 1 151

• Question 2 152

• Question 3 153

• Question 4 155

• Question 5 156

Lesson 4: Stat Transformations: Bar plots, box plots, and histograms 157

• Activate packages 157

• Load the mtcars dataset using the code below. This is a dataset that comes with R. 157

• Question 1 157

• Question 2 158

• Question 3 159

• Question 4 160

Lesson5: Visualizing clusters with heatmap and dendrogram 162

• Load necessary packages 162

• Question 1 162

• Question 2 163

• Question 3 163

• Question 4 164

• Question 5 165

Additional Resources

For Further Reading 167

• Getting started with R 167

• General help with ggplot2 167

• Troubleshooting error messages 167

 • Other Resources 167

DNAnexus (Class Registrants Only)

Navigating DNAnexus 169

• Instruction for using DNAnexus for the Intro to R class 169

DNAnexus Outside of Class 173

• Setting up the R-Studio environment outside of Class hours 173

• Log into DNAnexus 173

• To Start the Application 174

• Open the R-Studio Web interface 176

• Proceed with running content from the R Class 178

10 Course Overview

Course Overview

Welcome to the Data Visualization with R Series

A series of lessons designed to introduce learners to the R package
ggplot2
This course will include a series of lessons for scientists with beginner level experience in R.
The primary purpose of this course is to introduce learners to data visualization in R with a
primary focus on ggplot2 and related packages.

Course objectives
1. Learn how to generate basic plot types in ggplot2
2. Understand how basic plot types can be customized to generate more complex plots

What this course is not!

This course will not:

1. Make anyone an R expert

2. Make anyone a ggplot2 expert

Note

While this course may be useful to learners with intermediate R experience who would like to learn more regarding
ggplot2, the pace of the course will be set assuming a beginner level of understanding.

Course Expectations
This course will include a series of six, 1-1.25 hour lessons over a period of three weeks. Each
lesson will be followed by a 45 minute help session in which students can ask questions and /
or get individual help with their data.

Lesson 1: Introduction to plot types

This lesson will answer that burning question: Why R for data visualization? In addition, we will
introduce the various plot types that will be generated throughout the course and will showcase
related plots that you will be able to create in the future using the foundational skills gained over
the next 3 weeks. This will not be a hands-on lesson so no coding yet. The hands-on portion of

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11 Course Overview

this series will start with lesson 2. In the help session afterwards we will help those having
trouble with DNAnexus accounts.

Lesson 2: Getting Started with ggplot2

Lesson 2 will focus on the basics of ggplot2, including the grammar of graphics philosophy and
its application. This lesson will provide a hands on introduction to the ggplot2 syntax, geom
functions, mapping and aesthetics, and plot layering.

Lesson 3: Scatter plots and Non-data elements of ggplot2

customization
Lesson 3 will continue the discussion on the grammar of graphics, with a focus on ggplot2 plot
customization including axes labels, coordinate systems, axes scales, and themes. This hands
on lesson will showcase these features of plot building through the generation of increasingly
complex scatter plots using data included with a base R installation as well as RNASeq data.

Lesson 4: Visualizing summary statistics with ggplot2

It is common to obtain summary statistics for a dataset to understand parameters like mean,
standard deviation, and distribution. In this lesson, we will learn to generate plots that will help
with visualization of summary statistics including bar plot with error bars, histogram, as well as
the box and whiskers plot.

Lesson 5: Visualizing clusters with heatmaps

Lesson 5 will introduce the heatmap and dendrogram as tools for visualizing clusters in data.
This lesson will primarily use the R package pheatmap.

Lesson 6: Combining multiple plots to create a figure panel

Scientific journals almost always have limits on the number of figures that can be included in a
publication. Don't fret, in lesson 6, we will focus on generating sub plots and multi plot figure
panels using ggplot2 associated packages.

Required Course Materials

To participate in this class you will need your government-issued computer and a reliable
internet connection. You do not need to download or install any software to participate in the
class.

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Lesson 1: Course Introduction

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Introduction to ggplot2

Objectives
1. Learn how to import spreadsheet data.

2. Learn the ggplot2 syntax.

3. Build a ggplot2 general template.

By the end of the course, students should be able to create simple, pretty, and effective figures.

Going beyond Excel

Excel is a great program for visualizing and manipulating small data sets. However, it isn't great
for working with "big data", and resulting plots are generally not publishable. Learning R and
associated plotting packages is a great way to generate publishable figures in a reproducible
fashion. Using R will not only keep you from accidentally editing your data, but it will also allow
you to generate scripts that can be viewed later or reused to generate the same plot using
different data. This will keep you from having to rely on your memory when wondering what data
was used or how a plot was generated.

Let's read in and look at some excel data.

#If the package readxl is not installed,

#you will need to install the package and load the library
#install.packages("readxl")
#library(readxl)

#data import from excel

data<-readxl::read_xlsx(
"./data/RNASeq_totalcounts_vs_totaltrans.xlsx",1)

#View data
data

## # A tibble: 8 × 4
## `Sample Name` Treatment `Number of Transcripts` `Total Counts`
## <chr> <chr> <dbl> <dbl>
## 1 GSM1275863 Dexamethasone 10768 18783120
## 2 GSM1275867 Dexamethasone 10051 15144524

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## 3 GSM1275871 Dexamethasone 11658 30776089

## 4 GSM1275875 Dexamethasone 10900 21135511
## 5 GSM1275862 None 11177 20608402
## 6 GSM1275866 None 11526 25311320
## 7 GSM1275870 None 11425 24411867
## 8 GSM1275874 None 11000 19094104

These data include total transcript read counts summed by sample and the total number of
transcripts recovered by sample that had at least 100 reads. These data derive from a bulk
RNAseq experiment described by Himes et al. (2014) (https://pubmed.ncbi.nlm.nih.gov/
24926665/). In the experiment, the authors "characterized transcriptomic changes in four
primary human ASM cell lines that were treated with dexamethasone," a common therapy for
asthma. Each cell line included a treated and untreated negative control resulting in a total
sample size of 8.

Notice those column names.

#Notice those column names

colnames(data)

## [1] "Sample Name" "Treatment" "Number of Transcripts"

## [4] "Total Counts"

Spaces cause problems for data wrangling in R, but we can change our load parameters to
repair our column names.

#data import from excel

data<-readxl::read_xlsx("./data/RNASeq_totalcounts_vs_totaltrans.xlsx",
1,.name_repair = "universal")

## New names:
## • `Sample Name` -> `Sample.Name`
## • `Number of Transcripts` -> `Number.of.Transcripts`
## • `Total Counts` -> `Total.Counts`

Readxl’s default is .name_repair = "unique", which ensures each column has a

unique name. If that is already true of the column names, readxl won’t touch them.
The value .name_repair = "universal" goes further and makes column names
syntactic, i.e. makes sure they don’t contain any forbidden characters or reserved
words. This makes life easier if you use packages like ggplot2 and dplyr
downstream, because the column names will “just work” everywhere and won’t

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require protection via backtick quotes --- readxl.tidyverse.org (https://

readxl.tidyverse.org/articles/column-names.html).

We could plot this data in excel. If we did, we would get something like this:

This isn't too bad, but it took an unnecessary amount of time, and there weren't a lot of options
for customization.

RECOMMENDATION

You should save metadata or other tabular data as either comma separated files (.csv) or tab-delimited files (.txt,
.tsv). Using these file extensions will make it easier to use the data with bioinformatic programs. There are multiple
functions available to read in delimited data in R. We will see a few of these over the next few weeks.

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Example of .csv structure:

Why ggplot2?
Outside of base R plotting, one of the most popular packages used to generate graphics in R is
ggplot2, which is associated with a family of packages collectively known as the tidyverse.
GGplot2 allows the user to create informative plots quickly by using a 'grammar of graphics'
implementation, which is described as "a coherent system for describing and building graphs"
(R4DS). We will see this in action shortly. The power of this package is that plots are built in
layers and few changes to the code result in very different outcomes. This makes it easy to
reuse parts of the code for very different figures. GGplot2 is incredibly versatile and can create
most types of plots, especially when you consider the numerous packages (https://
exts.ggplot2.tidyverse.org/gallery/) that further extend its capabilities.

Being a part of the tidyverse collection, ggplot2 works best with data organized so that
individual observations are in rows and variables are in columns ("tidy data (https://
r4ds.had.co.nz/tidy-data.html)").

Getting started with ggplot2

To begin plotting, we need to load our ggplot2 library. Package libraries must be loaded every
time you open and use R. If you haven't yet installed the ggplot2 package on your local
machine, you will need to do that using install.packages("ggplot2").

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#load the ggplot2 library

library(ggplot2)

Getting help
The R community is extensive and getting help is now easier than ever with a simple web
search. If you can't figure out how to plot something, give a quick web search a try. Great
resources include internet tutorials, R bookdowns, and stackoverflow. You should also use the
help features within RStudio to get help on specific functions or to find vignettes. Try entering
ggplot2 in the help search bar in the lower right panel under the Help tab.

Note

You may also find Microsoft's Bing GPT-4 chatbot (https://www.bing.com/new?

toWww=1&redig=7AA1192E6A3E4A04938B4E1AB3C98A19) useful for creating code examples that can be
modified to fit your data. Currently, Bing is the only GPT-4 browser that is authorized for use by NCI.

Though it was created for ChatGPT, you may find this resource from Datacamp (https://www.datacamp.com/cheat-
sheet/chatgpt-cheat-sheet-data-science) useful for prompting appropriate responses.

The ggplot2 template

The following represents the basic ggplot2 template.

ggplot(data = <DATA>) +
<GEOM_FUNCTION>(mapping = aes(<MAPPINGS>))

The main components include the data we want to plot, geom function(s), and mapping
aesthetics. Notice the + symbol following the ggplot() function. This symbol will precede
each additional layer of code for the plot, and it is important that it is placed at the end of the
line. More on geom functions and mapping aesthetics to come.

Let's see this template in practice.

What is the relationship between total transcript sums per sample and the number of recovered
transcripts per sample?

#let's plot our data

ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts))

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We can easily see that there is a relationship between the number of transcripts per sample and
the total transcripts recovered per sample. ggplot2 default parameters are great for
exploratory data analysis. But, with only a few tweaks, we can make some beautiful, publishable
figures.

Let's take a closer look at the above code

The first step in creating this plot was initializing the ggplot object using the function ggplot().
Remember, we can look further for help using ?ggplot(). The function ggplot() takes data,
mapping, and further arguments. However, none of this needs to actually be provided at the
initialization phase, which creates the coordinate system from which we build our plot. But,
typically, you should at least call the data at this point.

The data we called was from the data frame data, which we created above. Next, we provided
a geom function (geom_point()), which created a scatter plot. This scatter plot required
mapping information, which we provided for the x and y axes. More on this in a moment.

Let's break down the individual components of the code.

#What does running ggplot() do?

ggplot(data=data)

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#What about just running a geom function?

geom_point(data=data,aes(x=Number.of.Transcripts, y = Total.Counts))

## mapping: x = ~Number.of.Transcripts, y = ~Total.Counts

## geom_point: na.rm = FALSE
## stat_identity: na.rm = FALSE
## position_identity

#what about this

ggplot() +
geom_point(data=data,aes(x=Number.of.Transcripts, y = Total.Counts))

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Geom functions
A geom is the geometrical object that a plot uses to represent data. People often
describe plots by the type of geom that the plot uses. --- R4DS (https://
r4ds.had.co.nz/data-visualisation.html#geometric-objects)

There are multiple geom functions that change the basic plot type or the plot representation. We
can create scatter plots (geom_point()), line plots (geom_line(),geom_path()), bar plots
(geom_bar(), geom_col()), line modeled to fitted data (geom_smooth()), heat maps
(geom_tile()), geographic maps (geom_polygon()), etc.

ggplot2 provides over 40 geoms, and extension packages provide even more (see
https://exts.ggplot2.tidyverse.org/gallery/ (https://exts.ggplot2.tidyverse.org/
gallery/) for a sampling). The best way to get a comprehensive overview is the
ggplot2 cheatsheet, which you can find at http://rstudio.com/resources/cheatsheets
(http://rstudio.com/resources/cheatsheets). --- R4DS (https://r4ds.had.co.nz/data-
visualisation.html)

You can also see a number of options pop up when you type geom into the console, or you can
look up the ggplot2 documentation in the help tab.

We can see how easy it is to change the way the data is plotted. Let's plot the same data using
geom_line().

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ggplot(data=data) +
geom_line(aes(x=Number.of.Transcripts, y = Total.Counts))

Mapping and aesthetics (aes())

The geom functions require a mapping argument. The mapping argument includes the aes()
function, which "describes how variables in the data are mapped to visual properties
(aesthetics) of geoms" (ggplot2 R Documentation). If not included it will be inherited from the
ggplot() function.

An aesthetic is a visual property of the objects in your plot.---R4DS (https://

r4ds.had.co.nz/data-visualisation.html)

Mapping aesthetics include some of the following:

1. the x and y data arguments
2. shapes
3. color
4. fill
5. size
6. linetype
7. alpha

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This is not an all encompassing list.

Let's return to our plot above. Is there a relationship between treatment ("dex") and the number
of transcripts or total counts?

#adding the color argument to our mapping aesthetic

ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,
color=Treatment))

There is potentially a relationship. ASM cells treated with dexamethasone in general have lower
total numbers of transcripts and lower total counts.

Notice how we changed the color of our points to represent a variable, in this case. To do this,
we set color equal to 'Treatment' within the aes() function. This mapped our aesthetic, color, to
a variable we were interested in exploring. Aesthetics that are not mapped to our variables are
placed outside of the aes() function. These aesthetics are manually assigned and do not
undergo the same scaling process as those within aes().

For example

#map the shape aesthetic to the variable "dex"

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#use the color purple across all points (NOT mapped to a variable)
ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,
shape=Treatment), color="purple")

We can also see from this that 'Treatment' could be mapped to other aesthetics. In the above
example, we see it mapped to shape rather than color. By default, ggplot2 will only map six
shapes at a time, and if your number of categories goes beyond 6, the remaining groups will go
unmapped. This is by design because it is hard to discriminate between more than six shapes
at any given moment. This is a clue from ggplot2 that you should choose a different aesthetic to
map to your variable. However, if you choose to ignore this functionality, you can manually
assign more than six shapes (https://r-graphics.org/recipe-scatter-shapes).

We could have just as easily mapped it to alpha, which adds a gradient to the point visibility by
category, or we could map it to size. There are multiple options, so feel free to explore a little
with your plots.

Other things to note

The assignment of color, shape, or alpha to our variable was automatic, with a unique aesthetic level representing
each category (i.e., 'Dexamethasone', 'none') within our variable. You will also notice that ggplot2 automatically
created a legend to explain the levels of the aesthetic mapped. We can change aesthetic parameters - what colors
are used, for example - by adding additional layers to the plot.

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R objects can also store figures

As we have discussed, R objects are used to store things created in R to memory. This includes
plots.

scatter_plot<-ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,
color=Treatment))

scatter_plot

We can add additional layers directly to our object. We will see how this works by defining some
colors for our 'dex' variable.

Colors

ggplot2 will automatically assign colors to the categories in our data. Colors are assigned to
the fill and color aesthetics in aes(). We can change the default colors by providing an
additional layer to our figure. To change the color, we use the scale_color functions:
scale_color_manual(), scale_color_brewer(), scale_color_grey(), etc. We can
also change the name of the color labels in the legend using the labels argument of these
functions

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scatter_plot +
scale_color_manual(values=c("red","black"),
labels=c('treated','untreated'))

scatter_plot +
scale_color_grey()

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scatter_plot +
scale_color_brewer(palette = "Paired")

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Similarly, if we want to change the fill, we would use the scale_fill options. To apply
scale_fill to shape, we will have to alter the shapes, as only some shapes take a fill
argument.

Image from https://ggplot2.tidyverse.org/articles/ggplot2-specs.html (https://

ggplot2.tidyverse.org/articles/ggplot2-specs.html):

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ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,fill=Treatment),
shape=21,size=2) + #increase size and change points
scale_fill_manual(values=c("purple", "yellow"))

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There are a number of ways to specify the color argument including by name, number, and hex
code. Here (https://www.r-graph-gallery.com/ggplot2-color.html) is a great resource from the R
Graph Gallery (https://www.r-graph-gallery.com/index.html) for assigning colors in R.

There are also a number of complementary packages in R that expand our color options. One
of my favorites is viridis, which provides colorblind friendly palettes. randomcoloR is a
great package if you need a large number of unique colors.

library(viridis) #Remember to load installed packages before use

## Loading required package: viridisLite

scatter_plot + scale_color_viridis(discrete=TRUE, option="viridis")

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Paletteer contains a comprehensive set of color palettes, if you want to load the palettes
from multiple packages all at once. See the Github page (https://github.com/EmilHvitfeldt/
paletteer) for details.

Facets
A way to add variables to a plot beyond mapping them to an aesthetic is to use facets or
subplots. There are two primary functions to add facets, facet_wrap() and facet_grid().
If faceting by a single variable, use facet_wrap(). If multiple variables, use facet_grid().
The first argument of either function is a formula, with variables separated by a ~ (See below).
Variables must be discrete (not continuous).

#plot
ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,fill=Sample.Name),
shape=21,size=2) + #increase size and change points
scale_fill_viridis(discrete=TRUE, option="viridis") +
facet_wrap(~Treatment)

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Note the help options with ?facet_wrap(). How would we make our plot facets vertical rather
than horizontal?

ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,fill=Sample.Name),
shape=21,size=2) + #increase size and change points
scale_fill_viridis(discrete=TRUE, option="viridis") +
facet_wrap(~Treatment, ncol=1)

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Be sure to take a look at facet_grid(). Facet_grid would allow us to map even more
variables in our data

ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,fill=Sample.Name),
shape=21,size=2) + #increase size and change points
scale_fill_viridis(discrete=TRUE, option="viridis") +
facet_grid(Sample.Name~Treatment)

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This is a silly example, but hopefully it gets across the point.

A better example of facet_grid() using data("Titanic").

This data set provides information on the fate of passengers on the fatal maiden
voyage of the ocean liner ‘Titanic’, summarized according to economic status
(class), sex, age and survival. --- R Documentation ?Titanic

data("Titanic")
Titanic <- as.data.frame(Titanic)
head(Titanic)

## Class Sex Age Survived Freq

## 1 1st Male Child No 0
## 2 2nd Male Child No 0
## 3 3rd Male Child No 35
## 4 Crew Male Child No 0
## 5 1st Female Child No 0
## 6 2nd Female Child No 0

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ggplot() + geom_col(data = Titanic, aes(x = Class, y = Freq, fill = Survived),

facet_grid(Sex ~ Age)

Building upon our template

This is the grammar of graphics. Adding layers to create unique figures.

ggplot(data = <DATA>) +
<GEOM_FUNCTION>(
mapping = aes(<MAPPINGS>),
) +
<FACET_FUNCTION>

Note that there are a lot of invisible (default) layers that often go into each ggplot2, and there
are ways to customize these layers. See this chapter (https://r4ds.had.co.nz/data-
visualisation.html#the-layered-grammar-of-graphics) from R for Data Science for more
information on the grammar of graphics.

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Using multiple geoms per plot

Because we build plots using layers in ggplot2. We can add multiple geoms to a plot to
represent the data in unique ways.

#We can combine geoms; here we combine a scatter plot with a

#with a linear model regression line using geom_smooth
ggplot(data=data) +
geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,
color=Treatment)) +
geom_smooth(method='lm', aes(x=Number.of.Transcripts,
y = Total.Counts, color= Treatment))

## `geom_smooth()` using formula = 'y ~ x'

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#to make our code more effective, we can put shared aesthetics in the
#ggplot function
ggplot(data=data, aes(x=Number.of.Transcripts,
y = Total.Counts, color= Treatment)) +
geom_point() +
geom_smooth(method='lm')

## `geom_smooth()` using formula = 'y ~ x'

Labels, legends, scales, and themes

How do we ultimately get our figures to a publishable state? The bread and butter of pretty plots
really falls to the additional non-data layers of our ggplot2 code. These layers will include code
to label the axes, scale the axes, and customize the legends and theme (https://
ggplot2.tidyverse.org/reference/theme.html). We will be working with these additional plot
features in the weeks to come, so stay tuned.

Here's a teaser.

ggplot(data=data) +

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geom_point(aes(x=Number.of.Transcripts, y = Total.Counts,
fill=Treatment),
shape=21,size=2) +
scale_fill_manual(values=c("purple", "yellow"),
labels=c('treated','untreated'))+
#can change labels of fill levels along with colors
xlab("Recovered transcripts per sample") + #add x label
ylab("Total sequences per sample") +#add y label
guides(fill = guide_legend(title="Treatment")) + #label the legend
scale_y_continuous(trans="log10") + #log transform the y axis
theme_bw()

Saving plots (ggsave())

Finally, we have a quality plot ready to publish. The next step is to save our plot to a file. The
easiest way to do this with ggplot2 is ggsave(). This function will save the last plot that you
displayed by default. Look at the function parameters using ?ggsave().

ggsave("Plot1.png",width=5.5,height=3.5,units="in",dpi=300)

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Check out this article (https://www.tidyverse.org/blog/2020/08/taking-control-of-plot-scaling/) for

recommendations on effectively scaling plots.

Resource list
1. ggplot2 cheatsheet
2. The R Graph Gallery (https://www.r-graph-gallery.com/)
3. The R Graphics Cookbook (https://r-graphics.org/recipe-quick-bar)
4. Ggplot2 extensions (https://exts.ggplot2.tidyverse.org/gallery/)

Acknowledgements
Material from this lesson was adapted from Chapter 3 of R for Data Science (https://
r4ds.had.co.nz/data-visualisation.html) and from a 2021 workshop entitled Introduction to Tidy
Transciptomics (https://stemangiola.github.io/bioc2021_tidytranscriptomics/articles/
tidytranscriptomics.html) by Maria Doyle and Stefano Mangiola.

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Scatter plots and plot customization

Objectives
1. Learn to customize your ggplot with labels, axes, text annotations, and themes.

2. Learn how to make and modify scatter plots to make fairly different overall plot
representations.

3. Load a different data set using new load functions

The primary purpose of this lesson is to learn how to customize our ggplot2 plots. We will do
this by focusing on different types of scatter plots.

The Data
In this lesson we will use two different sets of data. First, we will use data available with your
base R installation, the iris data set, which is stored in object iris. These data include
measurements from the petals and sepals of different Iris species including Iris setosa,
versicolor, and virginica. See ?iris for more information about these data.

The Irises: image from https://www.datacamp.com/community/tutorials/machine-learning-in-r

Second, we will use some more complicated bioinformatics data related to the RNAseq project
introduced in Lesson 2.

First, let's load our libraries using the library function, library():

library(ggrepel) #Needed for label repel

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## Loading required package: ggplot2

library(ggplot2)
library(dplyr)

##
## Attaching package: 'dplyr'

## The following objects are masked from 'package:stats':

##
## filter, lag

## The following objects are masked from 'package:base':

##
## intersect, setdiff, setequal, union

Now, let's load the RNA data that we will use toward the end of this lesson. We will use the
function read.delim() to load tab delimited RNASeq data and the function readLines() to
load the list of top genes.

dexp_sigtrnsc<-read.delim("../data/sig_dexp_results.txt",
as.is=TRUE)
topgenes<-readLines("../data/topgenes.txt")

The grammar of graphics

We are returning to the core grammar of graphics concept introduced in Lesson 2. Remember,
to create a plot all you you need are data, geom_function(s), and mapping arguments.

Here is the basic template we left off with:

ggplot(data = <DATA>) +
<GEOM_FUNCTION>(
mapping = aes(<MAPPINGS>),
) +
<FACET_FUNCTION>

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However, there are additional components, highlighted in bold, that can be added to our core
components to enable us to generate even more diverse plot types.

Our grammar of graphics:

• one or more datasets,

• one or more geometric objects that serve as the visual representations of the
data, – for instance, points, lines, rectangles, contours,

• descriptions of how the variables in the data are mapped to visual properties
(aesthetics) of the geometric objects, and an associated scale (e. g., linear,
logarithmic, rank),

• a facet specification, i.e. the use of multiple similar subplots to look at

subsets of the same data,

• one or more coordinate systems,

• optional parameters that affect the layout and rendering, such text size, font
and alignment, legend positions.

• statistical summarization rules, [See LESSON 4]

---(Holmes and Huber, 2021 (https://web.stanford.edu/class/bios221/book/03-

chap.html))

We will extend our basic template throughout this lesson as we make a variety of scatter plots
and in lesson 4.

Scatter plots
Scatterplots are useful for visualizing treatment–response comparisons,
associations between variables, or paired data (e.g., a disease biomarker in several
patients before and after treatment).Holmes and Huber, 2021 (https://
web.stanford.edu/class/bios221/book/03-chap.html)

Because scatter plots involve mapping each data point, the geom function used is
geom_point(). We saw a fairly basic implementation of this in Lesson 2.

Basic Scatter
Let's take another look at a simple scatter plot using the iris data. We can look at the
relationship between petal length and petal width (i.e., variable association) for the various Iris
species.

ggplot(data = iris) + #include our data

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geom_point(aes(x = Petal.Length, y = Petal.Width,

color = Species, shape = Species)) +
#geom and mappings
coord_fixed(xlim = c(0,7),
ylim=c(0,3),expand=FALSE) # NEW CORE COMPONENT

This code should look fairly familiar, with the exception of a new function, coord_fixed(). This
is a modification of the ggplot2 coordinate system.

Coordinate Systems
Coordinate systems are probably the most complicated part of ggplot2. The default
coordinate system is the Cartesian coordinate system where the x and y positions
act independently to determine the location of each point. --- R4DS (https://
r4ds.had.co.nz/data-visualisation.html#coordinate-systems)

coord_fixed() with the default argument ratio=1 ensures that the units are represented
equally in physical space on the plot. Because the x and y measurements were both taken in
centimeters, it is good practice to make sure that the "same mapping of data space to physical
space is used." --- Holmes and Huber, 2021 (https://web.stanford.edu/class/bios221/book/03-
chap.html)

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You will not need to worry about the coordinate system of your plot in most cases, but it is likely
you will need to mess with the coordinate system at some point in the future. Another commonly
used coordinate function is coord_flip(), which allows you to flip the representation of the
plot, for example, by switching bars in a bar plot from vertical to horizontal. See ?
coord_flip() for more information.

Our new template:

ggplot(data = <DATA>) +
<GEOM_FUNCTION>(
mapping = aes(<MAPPINGS>),
) +
<FACET_FUNCTION> +
<COORDINATE SYSTEM>

Perform and plot PCA data using iris.

Many complex plots (e.g., PCA ordinations) are in their basic form a scatter plot. Here we are
going to apply PCA to the iris data and generate a plot using ggplot2.

What is PCA?
Principal component analysis (PCA) is a linear dimension reduction method applied to highly
dimensional data. The goal of PCA is to reduce the dimensionality of the data by transforming
the data in a way that maximizes the variance explained. Read more here (https://
towardsdatascience.com/principal-component-analysis-pca-79d228eb9d24) and here (https://
www.huber.embl.de/msmb/Chap-Multivariate.html).

Key points:

• New variables are defined by a linear combination of original variables

• Each subsequent new variable contains less information

• Applications: dimensionality reduction, clustering, outlier identification ---

Shawhin Talebi, 2021 (https://towardsdatascience.com/principal-component-
analysis-pca-79d228eb9d24)

Note

PCAs are used frequently in -omics fields. However, often than not, there will be package specific functions for PCA
and plotting PCA for different -omics analyses. Because of this, we will show the main features here using a simpler
data set, iris.

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Perform PCA

We can use the function prcomp() to run PCA on the first four columns of the iris data. The
function takes numeric data.

colnames(iris)[1:4]

## [1] "Sepal.Length" "Sepal.Width" "Petal.Length" "Petal.Width"

pca <- prcomp(iris[,1:4], scale = TRUE)

pca

## Standard deviations (1, .., p=4):

## [1] 1.7083611 0.9560494 0.3830886 0.1439265
##
## Rotation (n x k) = (4 x 4):
## PC1 PC2 PC3 PC4
## Sepal.Length 0.5210659 -0.37741762 0.7195664 0.2612863
## Sepal.Width -0.2693474 -0.92329566 -0.2443818 -0.1235096
## Petal.Length 0.5804131 -0.02449161 -0.1421264 -0.8014492
## Petal.Width 0.5648565 -0.06694199 -0.6342727 0.5235971

#get structure of df
str(pca)

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## List of 5
## $ sdev : num [1:4] 1.708 0.956 0.383 0.144
## $ rotation: num [1:4, 1:4] 0.521 -0.269 0.58 0.565 -0.377 ...
## ..- attr(*, "dimnames")=List of 2
## .. ..$ : chr [1:4] "Sepal.Length" "Sepal.Width" "Petal.Length" "Petal.Widt
## .. ..$ : chr [1:4] "PC1" "PC2" "PC3" "PC4"
## $ center : Named num [1:4] 5.84 3.06 3.76 1.2
## ..- attr(*, "names")= chr [1:4] "Sepal.Length" "Sepal.Width" "Petal.Length
## $ scale : Named num [1:4] 0.828 0.436 1.765 0.762
## ..- attr(*, "names")= chr [1:4] "Sepal.Length" "Sepal.Width" "Petal.Length
## $ x : num [1:150, 1:4] -2.26 -2.07 -2.36 -2.29 -2.38 ...
## ..- attr(*, "dimnames")=List of 2
## .. ..$ : NULL
## .. ..$ : chr [1:4] "PC1" "PC2" "PC3" "PC4"
## - attr(*, "class")= chr "prcomp"

The object pca is a list of 5: the standard deviations of the principal components, a matrix of
variable loadings, the scaling used, and the data projected on the principal components.

Plot PCA
To plot the first two axes of variation along with species information, we will need to make a data
frame with this information. The axes are in pca$x.

#Build a data frame

pcaData <- as.data.frame(pca$x[, 1:2]) # extract first two PCs
pcaData <- cbind(pcaData, iris$Species) # add species to df
colnames(pcaData) <- c("PC1", "PC2", "Species") # change column names

#Plot
ggplot(pcaData) +
aes(PC1, PC2, color = Species, shape = Species) + # define plot area
geom_point(size = 2) + # adding data points
coord_fixed() # fixing coordinates

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Info

Tip 6, here (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586259/), discusses the importance of aspect ratio and

using coord_fixed() more in detail.

This is a decent plot showing us how the species relate based on characteristics of their sepals
and petals. From this plot, we see that Iris virginica and Iris versicolor are more similar than Iris
setosa.

But, the axes are missing the % explained variance. Let's add custom axes. We can do this with
the xlab() and ylab() functions or the functions labs(). But first we need to grab some
information from our PCA analysis. Let's use summary(pca). This function provides a summary
of results for a variety of model fitting functions and methods.

#Extract % Variance Explained

summary(pca)

## Importance of components:
## PC1 PC2 PC3 PC4
## Standard deviation 1.7084 0.9560 0.38309 0.14393

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## Proportion of Variance 0.7296 0.2285 0.03669 0.00518

## Cumulative Proportion 0.7296 0.9581 0.99482 1.00000

PC1 and PC2 combined account for 96% of variance in the data. We can add this information
directly to our plot using custom axes labels.

Add custom axes labels

#Plot
ggplot(pcaData) +
aes(PC1, PC2, color = Species, shape = Species) +
geom_point(size = 2) +
coord_fixed() +
xlab("PC1: 73%")+ #x axis label text
ylab("PC2: 23%") # y axis label text

Automating % Variance in axis labels 

If you want to automate the "Proportion of Variance", you should call it directly in the code. For example,

ggplot(pcaData) +
aes(PC1, PC2, color = Species, shape = Species) +

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geom_point(size = 2) +
coord_fixed() +
labs(x=paste0("PC1: ",summary(pca)$importance[2,1]*100,"%"),
y=paste0("PC2: ",summary(pca)$importance[2,2]*100,"%"))

Add a stat to our plot with stat_ellipse().

In scatter plots, the raw data is the focus of the plot, but for many other plots, this is not the
case. We will discuss statistical transformation more in lesson 4 and how they apply. However,
you may wish to overlay a stat on your PCA. For example, ellipses are often added to PCA
ordinations to emphasize group clustering with confidence intervals. By default,
stat_ellipse() uses the bivariate t distribution, but this can be modified. Let's add ellipses
with 95% confidence intervals to our plot.

ggplot(pcaData) +
aes(PC1, PC2, color = Species, shape = Species) +
geom_point(size = 2) +
coord_fixed() +
xlab("PC1: 73%")+
ylab("PC2: 23%") +
stat_ellipse(geom="polygon", level=0.95, alpha=0.2) #adding a stat

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Plot customization
Themes
The plot above is looking pretty good, but there are many more features that can be customized
to make this publishable or fit a desired style. Changing non-data elements (related to axes,
titles subtitles, gridlines, legends, etc.) of our plot can be done with theme(). GGplot2 has a
definitive default style that falls under one of their precooked themes, theme_gray().
theme_gray() is one of eight complete themes provided by ggplot2.

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We can also specify and build a theme within our plot code or develop a custom theme to be
reused across multiple plots. The theme function is the bread and butter of plot customization.
Check out ?ggplot2::theme() for a list of available parameters. There are many.

Let's see how this works by changing the fonts and text sizes and dropping minor grid lines:

ggplot(pcaData) +
aes(PC1, PC2, color = Species, shape = Species) + # define plot area
geom_point(size = 2) + # adding data points
coord_fixed() +
xlab("PC1: 73%")+
ylab("PC2: 23%") +
stat_ellipse(geom="polygon", level=0.95, alpha=0.2) +
theme_bw() + #start with a custom theme

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theme(axis.text=element_text(size=12,family="Times New Roman"),

axis.title = element_text(size=12,family="Times New Roman"),
legend.text = element_text(size=12,family="Times New Roman"),
legend.title = element_text(size=12,family="Times New Roman"),
panel.grid.minor = element_blank())

You may want to establish a custom theme for reuse with a number of plots. See this great
tutorial (https://rpubs.com/mclaire19/ggplot2-custom-themes) by Madeline Pickens for steps on
how to do that.

A helpful color trick

When you have a lot of colors and you want to keep these colors consistent, you can use the
following convenient functions to set a name attribute for a vector of colors.

Let's do this for our iris species.

#defining colors
iriscolors<-setNames(c("blue","black","orange"),levels(iris$Species))

#Now plot
ggplot(pcaData) +
aes(PC1, PC2, color = Species, shape = Species) +
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geom_point(size = 2) +
scale_color_manual(values=iriscolors)+ #Adding the color argument
coord_fixed() +
xlab("PC1: 73%")+
ylab("PC2: 23%") +
stat_ellipse(geom="polygon", level=0.95, alpha=0.2,aes(fill=Species)) +
scale_fill_manual(values=iriscolors)+ #Fill ellipses
theme_bw() +
theme(axis.text=element_text(size=12,family="Times New Roman"),
axis.title = element_text(size=12,family="Times New Roman"),
legend.text = element_text(size=12,family="Times New Roman"),
legend.title = element_text(size=12,family="Times New Roman"),
panel.grid.minor = element_blank())

We can use this color palette for all plots of these three species to keep our figures consistent
throughout a presentation or publication.

Using ggfortify to make a PCA 

ggfortify is an excellent package to consider for easily generating PCA plots. ggfortify provides "unified
plotting tools for statistics commonly used, such as GLM, time series, PCA families, clustering and survival analysis.
The package offers a single plotting interface for these analysis results and plots in a unified style using 'ggplot2'."
(https://cran.r-project.org/web/packages/ggfortify/)

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library(ggfortify)
autoplot(pca, data = iris, colour = 'Species',size=2)

Since this is a ggplot2 object, this can easily be customized by adding ggplot2 customization layers.

Putting it all together

Let's update our ggplot2 grammar of graphics template:

ggplot(data = <DATA>) +
<GEOM_FUNCTION>(
mapping = aes(<MAPPINGS>),
stat = <STAT>
) +
<FACET_FUNCTION> +
<COORDINATE SYSTEM> +
<THEME>

Creating a publication ready volcano plot

We now know enough to put our new skills to use to make a volcano plot from RNASeq data.

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A volcano plot is a type of scatterplot that shows statistical significance (P value)

versus magnitude of change (fold change). It enables quick visual identification of
genes with large fold changes that are also statistically significant. These may be
the most biologically significant genes. --- Maria Doyle, 2021 (https://
training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/rna-seq-
viz-with-volcanoplot/tutorial.html)

Introducing EnhancedVolcano 

There is a dedicated package for creating volcano plots available on Bioconductor, EnhancedVolcano (https://
bioconductor.org/packages/release/bioc/html/EnhancedVolcano.html). Plots created using this package can be
customized using ggplot2 functions and syntax.

Using EnhancedVolcano:

#The default cut-off for log2FC is >|2|

#the default cut-off for P value is 10e-6
library(EnhancedVolcano)
EnhancedVolcano(dexp_sigtrnsc,
title = "Enhanced Volcano with Airways",
lab = dexp_sigtrnsc$transcript,
x = 'logFC',
y = 'FDR')

Let's take a quick look at the data we loaded at the beginning of the lesson:

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head(dexp_sigtrnsc)

## feature albut transcript ref_genome .abundant logFC logCPM

## 1 ENSG00000109906 untrt ZBTB16 hg38 TRUE 7.146970 4.149689
## 2 ENSG00000165995 untrt CACNB2 hg38 TRUE 3.280645 4.508931
## 3 ENSG00000106976 untrt DNM1 hg38 TRUE -1.764478 5.381597
## 4 ENSG00000120129 untrt DUSP1 hg38 TRUE 2.938116 7.313059
## 5 ENSG00000146250 untrt PRSS35 hg38 TRUE -2.761556 3.908222
## 6 ENSG00000152583 untrt SPARCL1 hg38 TRUE 4.555765 5.534393
## F PValue FDR Significant
## 1 1429.3427 5.107615e-11 4.067194e-07 TRUE
## 2 1575.2829 3.342498e-11 4.067194e-07 TRUE
## 3 645.7452 1.616082e-09 2.573772e-06 FALSE
## 4 694.4165 1.179551e-09 2.573772e-06 TRUE
## 5 806.5147 6.162483e-10 2.573772e-06 TRUE
## 6 721.3794 9.999692e-10 2.573772e-06 TRUE

topgenes

## [1] "ZBTB16" "CACNB2" "DNM1" "DUSP1" "PRSS35" "SPARCL1"

Significant differential expression was assigned based on an absolute log fold change greater
than or equal to 2 and an FDR corrected p-value less than 0.05.

Let's start our plot with the <DATA>, <GEOM_FUNCTION>, and <MAPPING>. We do not need
to fix the coordinate system because we are working with two different values on the x and y
and we don't need any special coordinate system modifications. Let's plot logFC on the x axis
and the mutated column with our false discovery rate corrected p-values on the y-axis and set
the significant p-values off from the non-significant by size and color. We can also go ahead
and customize the size and color scales, since we have learned how to do that.

ggplot(data=dexp_sigtrnsc) +
geom_point(aes(x = logFC, y = log10(FDR), color = Significant,
size = Significant,
alpha = Significant)) +
scale_color_manual(values = c("black", "#e11f28")) +
scale_size_discrete(range = c(1, 2))

## Warning: Using size for a discrete variable is not advised.

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## Warning: Using alpha for a discrete variable is not advised.

This is not exactly what we want so let's keep working.

Immediately, you should notice that the figure is upside down compared to what we would
expect from a volcano plot. there are two possible ways to fix this. We could transform the FDR
corrected values by multiplying by -1 OR we could work with our axes scales. Aside from text
modifications, we haven't yet changed the scaling of the axes. Let's see how we can modify the
scale of the y-axis.

Changing axes scales

To change the y axis scale, we will need a specific function. These functions generally start with
scale_y.... In our case we want to reverse our axis so that increasingly negative is going in
the positive direction rather than the negative direction. Luckily, there is a function to reverse our
axis; see ?scale_y_reverse().

ggplot(data=dexp_sigtrnsc) +
geom_point(aes(x = logFC, y = log10(FDR), color = Significant,
size = Significant,
alpha = Significant)) +

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scale_color_manual(values = c("black", "#e11f28")) +

scale_size_discrete(range = c(1, 2)) +
scale_y_reverse(limits=c(0,-7)) #we can also set the limits

## Warning: Using size for a discrete variable is not advised.

## Warning: Using alpha for a discrete variable is not advised.

This looks pretty good, but we can tidy it up more by working with our legend guides and our
theme.

Modifying legends

We can modify many aspects of the figure legend using the function guide(). Let's see how
that works and go ahead and customize some theme arguments. Notice that the legend
position is specified in theme().

ggplot(data=dexp_sigtrnsc) +
geom_point(aes(x = logFC, y = log10(FDR), color = Significant,
size = Significant,

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alpha = Significant)) +
scale_color_manual(values = c("black", "#e11f28")) +
scale_size_discrete(range = c(1, 2)) +
scale_y_reverse(limits=c(0,-7)) + #we can also set the limits
guides(size = "none", alpha= "none",
color= guide_legend(title="Significant DE")) +
theme_bw() +
theme(
panel.border = element_blank(),
axis.line = element_line(),
panel.grid.major = element_line(size = 0.2),
panel.grid.minor = element_line(size = 0.1),
text = element_text(size = 12),
legend.position = "bottom",
axis.text.x = element_text(angle = 30, hjust = 1, vjust = 1)
)

## Warning: Using size for a discrete variable is not advised.

## Warning: The `size` argument of `element_line()` is deprecated as of ggplot2

## ℹ Please use the `linewidth` argument instead.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.

## Warning: Using alpha for a discrete variable is not advised.

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Lastly, let's layer another geom function to label our top six differentially abundant genes based
on significance. We can use geom_text_repel() from library(ggrepel), which is a
variation on geom_text().

plot_de<-ggplot(data=dexp_sigtrnsc) +
geom_point(aes(x = logFC, y = log10(FDR), color = Significant,
size = Significant,
alpha = Significant)) +
geom_text_repel(data=dexp_sigtrnsc %>%
filter(transcript %in% topgenes),
aes(x = logFC, y = log10(FDR),label=transcript),
nudge_y=0.5,hjust=0.5,direction="y",
segment.color="gray")+
scale_color_manual(values = c("black", "#e11f28")) +
scale_size_discrete(range = c(1, 2)) +
scale_y_reverse(limits=c(0,-7)) + #we can also set the limits
guides(size = "none", alpha= "none",
color= guide_legend(title="Significant DE")) +
theme_bw() +
theme(
panel.border = element_blank(),
axis.line = element_line(),
panel.grid.major = element_line(size = 0.2),

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panel.grid.minor = element_line(size = 0.1),

text = element_text(size = 12),
legend.position = "bottom",
axis.text.x = element_text(angle = 30, hjust = 1, vjust = 1)
)

## Warning: Using size for a discrete variable is not advised.

plot_de

## Warning: Using alpha for a discrete variable is not advised.

Save to an .rds file

We want to use this in a multi-panel figure in a later lesson, so let's go ahead and save it to a file
that will hold a single R object (.rds).

saveRDS(plot_de, "volcanoplot.rds")

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References
Code for PCA was adapted from Learning R through examples (https://gexijin.github.io/learnR/
index.html) by Xijin Ge, Jianli Qi, and Rong Fan, 2021. Other sources for content included R4DS
(https://r4ds.had.co.nz/) and Holmes and Huber, 2021 (https://web.stanford.edu/class/bios221/
book/).

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62 Stat Transformations: Bar plots, box plots, and histograms

Stat Transformations: Bar plots, box plots,

and histograms
Objectives

1. Review the grammar of graphics template

2. Learn about the statistical transformations inherent to geoms
3. Review data types
4. Create bar plots, box & whisker plots, and histograms.

Our grammar of graphics template

This is where we left off at the end of lesson 3.

Our grammar of graphics template

• one or more datasets,

• one or more geometric objects that serve as the visual representations of the
data, – for instance, points, lines, rectangles, contours,

• descriptions of how the variables in the data are mapped to visual properties
(aesthetics) of the geometric objects, and an associated scale (e. g., linear,
logarithmic, rank),

• a facet specification, i.e. the use of multiple similar subplots to look at

subsets of the same data,

• one or more coordinate systems,

• optional parameters that affect the layout and rendering, such text size, font
and alignment, legend positions.

• statistical summarization rules

---(Holmes and Huber, 2021 (https://web.stanford.edu/class/bios221/book/Chap-

Graphics.html))

ggplot(data = <DATA>) +
<GEOM_FUNCTION>(
mapping = aes(<MAPPINGS>),
stat = <STAT>
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) +
<FACET_FUNCTION> +
<COORDINATE SYSTEM> +
<THEME>

While we added stat_ellipse() to our PCAs, scatter plots do not have a built in statistical
transformation. Today, we will talk more about statistical transformations and how these impact
our plot representations. For a list of available statistical transformations in ggplot2 see https://
ggplot2-book.org/layers.html?q=stat#stat (https://ggplot2-book.org/layers.html?q=stat#stat).

Libraries
In this lesson, we will continue to use the ggplot2 package for plotting. To use ggplot2, we first
need to load it into our R work environment using the library command.

library(ggplot2)

The Data
In this lesson we will use data obtained from a study that examined the effect that dietary
supplements at various doses have on guinea pig tooth length. This data set is built into R, so if
you want to take a look for yourself you can type data("ToothGrowth") either in the console
or in a script. But in this exercise, we will import the data to our R work environment because it
is more likely that we will import our own data for analysis.

Here, we are going to import two data sets using read.delim. The file data1.txt contains raw
data from the tooth growth study. The file data2.txt is summary level data with mean tooth length
and standard deviation pre-computed. We will assign data1.txt to object a1 and data2.txt to
object a2. Within read.delim we use sep='\t' to indicate that the columns in the data are
tab separated. After importing, we use the head command to look at the first 6 rows of each
data set.

a1 <- read.delim("./data/data1.txt", sep='\t')

a2 <- read.delim("./data/data2.txt", sep='\t')
head(a1)

## len supp dose

## 1 4.2 VC 0.5
## 2 11.5 VC 0.5
## 3 7.3 VC 0.5
## 4 5.8 VC 0.5

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## 5 6.4 VC 0.5
## 6 10.0 VC 0.5

head(a2)

## supp dose treat mean_len sd

## 1 OJ 0.5 OJ0.5 13.23 4.459709
## 2 OJ 1.0 OJ1 22.70 3.910953
## 3 OJ 2.0 OJ2 26.06 2.655058
## 4 VC 0.5 VC0.5 7.98 2.746634
## 5 VC 1.0 VC1 16.77 2.515309
## 6 VC 2.0 VC2 26.14 4.797731

The tooth growth data set measured tooth length for two supplement types (OJ - orange juice,
VC - vitamin c) at three different doses (0.5, 1, and 2). Each supplement and dose combination
has 10 measurements so we have a total of 60 measurements in this data set. In a1, we have
the raw data. On the other hand, in a2, we pre-computed the mean tooth length and standard
deviation for the 10 measurements taken at each supplement and dose combination.

The column headings (colnames(a1),colnames(a2)) in the raw data (a1) and summary
level data (a2) are as follows:

• len (tooth length - in raw data)

• supp (supplement type)
• dose
• treat (treatment, which is a concatenation of supp and dose - in summary level data only)
• mean_len (mean tooth length - in summary level data only)
• sd (standard deviation - in summary level data only)

Variable Types
Before diving into the construction of bar plot, box & whisker plot, and histogram, we should do
a quick review of the types of variables that we commonly work with in data analysis.

• Categorical variables are qualitative, for instance

• patient gender (male or female)
• disease status (case or control)
• Discrete variables are quantifiable and can take on a finite number of of values, for
instance
• number of male or female patients
• number of controls or disease cases
• medication dose
• Continuous variables can take on an infinite number of values

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65 Stat Transformations: Bar plots, box plots, and histograms

• age
• height
• weight
• Independent variable is a variable whose variation does not depend on another
• Dependent variable is a variable whose variation depends on another
• Factors are independent variables in which an experimental outcome is dependent on.
Levels are variations in the factors.

The stat argument

Until this point we have been plotting raw data with geom_point(), but now we will be
introducing geoms that transform and plot new values from your data.

Many graphs, like scatterplots, plot the raw values of your dataset. Other graphs,
like bar charts, calculate new values to plot:

• bar charts, histograms, and frequency polygons bin your data and then plot
bin counts, the number of points that fall in each bin.

• smoothers fit a model to your data and then plot predictions from the model.

• boxplots compute a robust summary of the distribution and then display a

specially formatted box.---R4DS

Let's explore the tooth growth data using plots. The tooth growth data has two independent
variables, supplement and dose (variables supp and dose, respectively) for which the
dependent variable tooth length (len) is measured. We would like to use the scatter plot to learn
about tooth growth as a function of both dose and supplement (supp). Remember from Lesson
3 that a scatter plot can be generated using geom_point.

Within the aesthetic mapping in geom_point, we assign dose to the x axis, len (tooth length)
to y, and the second independent variable supp (supplement) by assigning it to color. This
will give us a scatter plot where dose is plotted along the x axis and len plotted along the y axis.
The color code indicating which supplement (supp) each of the points or measurements came
from is provided in the legend. In short, the color argument allows us to visualize how the
dependent variable changes as a function of two independent variables.

Within geom_point, position=position_dodge is included to separate the points by

supplement (supp), otherwise the points for the two supplements will overlap each other. The
parameter width in position_dodge allows us to specify how far apart we want the points
for the different supplement groups to be.

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ggplot(data=a1)+
geom_point(mapping=aes(x=dose,y=len,color=supp),
position=position_dodge(width=0.25))

Bar Plot
A barplot is used to display the relationship between a numeric and a categorical
variable. --- R Graph Gallery (https://r-graph-gallery.com/barplot.html)

The tooth growth data can also be visualized via bar plot using geom_bar. However, if we try to
plot tooth length (len) across each dose using the code below, we will get an error. We get this
error because geom_bar uses stat_count by default where it counts the number of cases at
each x position. Thus, by default, geom_point does not require y axis.

ggplot(data=a1)+
geom_bar(mapping=aes(x=dose,y=len))

## Error in `f()`:
## ! stat_count() can only have an x or y aesthetic.

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We received an error referencing stat_count().

stat = count

Let's take a look at a bar plot constructed using the default stat="count" transformation.
Below, we plot the number of tooth length measurements taken at each dose. Setting
color="black" allows us to include a black outline to the bars for better readability. In
accordance with the description of this data, we see from the plot that 20 measurements were
taken at each dose.

ggplot(data=a1)+
geom_bar(mapping=aes(x=dose), color="black")

Given the above plot, how many of the 20 measurements taken at each dose came from the OJ
or VC group. To find out, we can set fill=supp. Like color in the scatter plot, fill, allows
us to include a second independent variable in our graph. The plot below tells us that 10
measurements were taken from the OJ group and 10 were taken from the VC group at each
dose.

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ggplot(data=a1)+
geom_bar(mapping=aes(x=dose, fill=supp), color="black")

By default, geom_bar stacks bars from different groups. If we do not like the arrangement, we
can use position_dodge to arrange the bars from the OJ and VC groups side-by-side.

ggplot(data=a1)+
geom_bar(mapping=aes(x=dose,fill=supp), color="black",
position=position_dodge())

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stat_count() is a great way to compare differences in sample sizes between treatment

categories or other variables and can be a great way to explore and represent metadata
associated with -omics data.

stat = identity
Above, we learned about the number of tooth length measurements taken at each dose and
supplement combination using the default stat="count" transformation of geom_bar. But
what if we want to specify a y axis and plot exactly the values of our dependent variable in the y
axis? This can be done in geom_bar by setting stat="identity".

Below, we are plotting the mean tooth length (mean_len) across each of the treatment groups
(treat) using the summary level data, a2. Using stat="identity", the exact y value or
mean_len is plotted.

ggplot(data=a2)+
geom_bar(mapping=aes(x=treat,y=mean_len),stat="identity")

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What if we wanted to look at the raw values across the treatment groups using a bar plot? We
still use geom_bar but the aesthetic mapping will be similar to the scatter plot except we are
filling the bars to provide color coding for the supplements using fill. Again, because we
want ggplot2 to plot exact value in the y axis, we specify stat="identity" inside geom_bar.
To avoid stacking the values, we can use position_dodge2 in geom_bar to visualize each of
the 10 measurements taken at each supplement and dose combination arranged side-by-side.

ggplot(data=a1)+
geom_bar(mapping=aes(x=dose,y=len,fill=supp),stat="identity",
position=position_dodge2(),color="black")

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Note that because R interprets dose as numeric continuous variable (class(a2$dose)) ggplot2
gives us an extra dose of 1.5. But, the study did not measure tooth length at a dose of 1.5 for
either of the supplements. Thus, we would like to remove this dose.

Using factors

class(a1$dose)

## [1] "numeric"

As it turns out, dose is really an experimental factor, so if we specify factor(dose) it will be

interpreted as categorical or discrete. Before fixing the x axis in the above plot, let's diverge
briefly and speak about factors in R.

Using the factor function we see that there are three levels for dose (0.5, 1, and 2)

factor(a1$dose)

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## [1] 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1 1 1 1 1 1
## [20] 1 2 2 2 2 2 2 2 2 2 2 0.5 0.5 0.5 0.5 0.5 0.5 0.5
## [39] 0.5 0.5 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2
## [58] 2 2 2
## Levels: 0.5 1 2

To remove the dose of 1.5, we can set the x axis to factor(dose).

ggplot(data=a1)+
geom_bar(mapping=aes(x=factor(dose),y=len,fill=supp),stat="identity"
,position=position_dodge2(),color="black")

We can reorder factors using the function factor. For instance if we want to plot the doses
backwards from highest to lowest on the x axis (i.e., 2, 1, 0.5) we can set the x axis in aesthetic
mapping of geom_bar to factor(dose,levels=c(2,1,0.5)), where in the levels
parameter, we are reassigning the order of the levels.

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ggplot(data=a1)+
geom_bar(mapping=aes(x=factor(dose,
levels=c(2,1,0.5)),y=len,fill=supp),stat="identity",
position=position_dodge2(),color="black")

Adding error bars

Often, we will use bar plots to illustrate mean plus minus standard deviation in our data so we
should learn how to incorporate error bars in our plots. We will learn to do this using the
summary level data (a2) where the mean and standard deviation have been pre-computed and
then with the raw data (a1) to take advantage of more statistical transformations.

First, we use the summary level data (a2) to plot the mean tooth length (mean_len) across the
treatment groups, remembering to set stat="identity" because we are providing a y axis
and position=position_dodge to arrange the bars side-by-side.

ggplot(data=a2, mapping=aes(x=factor(dose),y=mean_len,fill=supp))+
geom_bar(stat="identity", position=position_dodge())

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Next, we add geom_errorbar to incorporate error bars that illustrate plus/minus 1 standard
deviation from the mean. To set the upper and lower bounds of the error bar, we simply set
ymax=mean_len+sd and ymin=mean_len-sd within the aesthetic mapping of
geom_errorbar. Setting position=position_dodge in geom_errorbar again allows us
to separate the error bars from each of the supplement groups. Within the position_dodge
argument we set width=0.9 to help center the error bars with their respective bars. The
width parameter within geom_errorbar allows us to adjust the error bar width (set to 0.1
here).

ggplot(data=a2, mapping=aes(x=factor(dose),y=mean_len,fill=supp))+
geom_bar(stat="identity", position=position_dodge())+
geom_errorbar(aes(ymax=mean_len+sd,ymin=mean_len-sd),
position=position_dodge(width=0.9), width=0.1)

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Histogram
Understanding data distribution can help us decide appropriate downstream steps in analysis
such as which statistical test to use. A histogram is a good way to visualize distribution. It
divides the data into bins or increments and informs the number of occurrences in each of the
bins. Thus, the default statistical transformation for geom_histogram is stat_bin, which bins
the data into a user specified integer (default is 30) and then counts the occurrences in each
bin. In geom_histogram we have the ability to control both the number of bins through the
bins argument or binwidth through the binwidth argument. Important to note is that
stat_bin only works with continuous variables.

Below we constructed a basic histogram using the len column in a1 (the raw data for the Tooth
Growth study). Note that within geom_histogram we do not need to explicitly state
stat="bin" because it is default. The histogram below is not very aesthetically pleasing -
there are gaps and difficult to see the separation of the bins.

ggplot(data=a1, mapping=aes(x=len))+
geom_histogram()

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## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

First, we will use the color argument in geom_histogram to assign a border color to help
distinguish the bins. Then we use the fill argument in geom_histogram to change the bars
associated with the bins to a color other than gray. Below we have a histogram of tooth length
with a default bin of 30.

ggplot(data=a1, mapping=aes(x=len))+
geom_histogram(color="black", fill="cornflowerblue")

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

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Let's alter the number of bins

ggplot(data=a1, mapping=aes(x=len))+
geom_histogram(color="black", fill="cornflowerblue", bins=7)

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From the above, we see that altering the number of bins alters the binwidth (ie. the range in
which occurrence are counted). Thus, altering the bins can influence the distribution that we
see. The histograms above seem to be left skewed and a lot of the tooth length values fall
between 22.5 and 27.5 when 7 bins were used.

Box plot
Box and whisker plots also show data distribution. Unlike a histogram, we can readily see
summary statistics such as median, 25th and 75th percentile, and maximum and minimum.

The default statistical tranformation of a box plot in ggplot2 is stat_boxplot which calculates
components of the boxplot. To construct a box plot in ggplot2, we use the geom_boxplot
argument. Note that within geom_boxplot we do not need to explicitly state stat="boxplot"
because it is default. Below, we have a default boxplot depicting tooth length across the
treatment groups. Potential outliers are presented as points.

ggplot(data=a1, mapping=aes(x=factor(dose), y=len, color=supp))+

geom_boxplot()

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Rather than showing the outliers, we could instead add on geom_point to overlay the data
points. Here, in geom_boxplot we set outlier.shape=NA to remove the outliers for the
purpose of avoiding duplicating a data point. Within geom_point we set the position of the
points to position_jitterdodge to avoid overlapping of points whose values are close
together (set by jitter.width) and overlapping of points from measurements derived from
different supplements (dodge.width).

ggplot(data=a1, mapping=aes(x=factor(dose), y=len, color=supp))+

geom_boxplot(outlier.shape=NA)+
geom_point(position=position_jitterdodge(jitter.width=0.3,
dodge.width=0.8))

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The box plot above shows several things

• Tooth length appears to be longer for the OJ treated group at doses of 0.5 and 1
• Tooth length appears to be equal for both the OJ and VC groups at a dose of 2
• At a dose of 0.5 and 1, the median (line inside the box) for the OJ group is larger than the
VC group
• At a dose of 0.5 and 1, the interquartile range (IQR) which is defined by the lower (25th
percentile) and upper (75th percentile) bounds of the box along the vertical axis is larger
for the OJ group as compared to VC - so there is more variability in the OJ group
measurements.
• At a dose of 2, the median for both the OJ and VC group are roughly equal
• At a dose of 2, the IQR for the VC group is larger than that for the OJ group

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81 Visualizing clusters with heatmaps

Visualizing clusters with heatmaps

Objectives

1. Introduce the heatmap and dendrogram as tools for visualizing clusters in data.

2. Learn to construct cluster heatmap using the package pheatmap.

3. Learn how to save a non-ggplot2 plot.

4. Introduce ggplotify to convert non-ggplots to ggplots.

5. Introduce heatmaply for constructing interactive heatmaps.

What is a heatmap?
A heatmap is a graphical representation of data where the individual values
contained in a matrix are represented as colors. --- R Graph Gallery (https://r-
graph-gallery.com/heatmap.html)

Heatmaps are appropriate when we have lots of data because color is easier to interpret and
distinguish than raw values. --- Dundas BI (https://www.dundas.com/resources/blogs/best-
practices/when-and-why-to-use-heat-maps)

Heatmap can be used to visualize the following

• gene expression across samples (Figure 1)
• correlation (Figure 2)
• disease cases (Figure 3)
• hot/cold zones
• topography

What is a dendrogram?
A dendrogram (or tree diagram) is a network structure and can be used to visualize
hierarchy or clustering in data. --- R Graph Gallery (https://r-graph-gallery.com/
dendrogram.html)

Applications of dendrograms
Dendrograms are used in phylogenetics to help visualize relatedness of or dissimilarities
between species.

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In RNA sequencing, dendrogram can be combined with heatmap to show clustering of samples
by gene expression or clustering of genes that are similarly expressed (Figure 1).

Figure 1: Heatmap and dendrogram showing clustering of samples with similar gene expression
and clustering of genes with similar expression patterns.

Further heatmap and dendrogram can be used as a diagnostic tool in high throughput
sequencing experiments. As an example, we can look at the heatmap and dendrogram in
Figure 2. In Figure 2, the heatmap shows correlation of RNA sequencing samples with the idea
that biological replicates should be more highly correlated compared to samples between
treatment groups. The dendrogram clusters similar samples together. Figure 2 tells us that
heatmaps can also be used to visualize correlation.

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Figure 2: Heatmap and dendrogram showing sample correlation in an RNA sequecing

experiment. This heatmap and dendrogram is generated using DeepTools (https://
deeptools.readthedocs.io/en/develop/content/tools/plotCorrelation.html).

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Figure 3: Maryland cancer cases - source: https://statecancerprofiles.cancer.gov

Methods available to produce a heatmap in R

ggplot2

• We would use geom_tile to construct the heatmap

• A disadvantage to this approach is that we have to generate the dendrogram separately,
then merge and align the dendrogram with the heatmap.

heatmap built into R

• It appears that this by default does not generate a legend showing the correlation
between values and color.
• It also appears that assigning distance calculation and clustering methods are not
intuitive for the users.

heatmap.2 from the gplots package

• It appears assigning distance calculation and clustering methods are not intuitive for the
users.
• Click here to learn about heatmap.2 (https://cran.r-project.org/web/packages/gplots/
index.html)

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ComplexHeatmap

• There is no scaling option so the user will have to scale the data separately using scale.
(see https://support.bioconductor.org/p/68340/ and https://github.com/jokergoo/
ComplexHeatmap/issues/313 for a discussion on scaling in ComplexHeatmap).
• Click here to learn about ComplextHeatmap (https://www.bioconductor.org/packages/
release/bioc/html/ComplexHeatmap.html).

pheatmap

• This is a versatile package that draws clustered heatmaps.

• This package has built-in scaling function, provides ways to incorporate legends, and
many features that allows for customization and construction of a publication quality
figure.
• Click here to learn about pheatmap (https://cran.r-project.org/web/packages/pheatmap/
pheatmap.pdf)

heatmaply

• This package generates interactive heatmaps that allows the user to mouse over a tile to
see information such as sample id, gene, and expression value.
• Click here to learn about heatmaply (https://cran.r-project.org/web/packages/heatmaply/
vignettes/heatmaply.html)

While most of the tools listed above can be used to produce publication quality heatmaps, we
find that pheatmap is perhaps the most comprehensive. Therefore, in this class, we will show
how to construct heatmaps using pheatmap. Because heatmaps can be filled with a lot of data,
we will also demonstrate the use of heatmaply to construct interactive heatmaps that you could
use to explore your data more efficiently.

Load the libraries

library(pheatmap) ## for heatmap generation

library(tidyverse) ## for data wrangling
library(ggplotify) ## to convert pheatmap to ggplot2
library(heatmaply) ## for constructing interactive heatmap

Import data
The data that we will be working with comes from the airway study that profiled the
transcriptome of several airway smooth muscle cell lines under either control or dexamethasone
treatment Himes et al 2014 (http://www.ncbi.nlm.nih.gov/pubmed/24926665) . The dataset is
available from Bioconductor (https://bioconductor.org/packages/release/data/experiment/html/
airway.html).

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Specifically, our dataset represents the normalized (log2 counts per million or log2 CPM) of
count values from the top 20 differential expressed genes. This data is saved as the comma
separated file RNAseq_mat_top20.csv and thus we will be using the read.csv command to
import.

As a refresher, inside the read.csv command we have the following arguments

• "./data/RNAseq_mat_top20.csv" is the file path

• header=TRUE indicates that we have column headings
• we set the first column of our dataset, which contains gene names as the row names in
the imported data using row.names=1, where 1 indicates the column number that we
want to import as row names
• we set check.names=FALSE so that R leaves the column headings alone

The data is assigned to R object mat.

mat<-read.csv("./data/RNAseq_mat_top20.csv",header=TRUE,row.names=1,
check.names=FALSE)

We will now use head to look at the first 6 rows of mat. The column headings represent sample
names and the row names are the genes.

head(mat)

## 508 509 512 513 516 517 52

## WNT2 4.694554 1.332858 5.983720 2.898648 2.1105784 -0.1655252 4.282851
## DNM1 6.180735 4.441965 5.660024 3.981513 5.8002923 3.9603755 6.285300
## ZBTB16 -1.863523 5.257967 -1.777152 4.902223 -2.9319722 4.2830866 -0.515040
## DUSP1 4.936551 8.019074 5.607568 8.302196 5.0283417 7.7229898 5.143245
## HIF3A 1.013991 3.374457 1.575250 4.154740 0.4928878 2.8335879 2.250124
## MT2A 6.248514 8.276988 5.782855 8.070846 5.7583897 8.2015855 5.916362
## 521
## WNT2 1.237000
## DNM1 4.488955
## ZBTB16 5.779173
## DUSP1 8.396709
## HIF3A 4.790581
## MT2A 7.933492

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Heatmap of the top 20 genes from differential

expression analysis
Below we generate a basic heatmap using the pheatmap package. We use the pheatmap
command and include the data that we want to construct a heatmap of as the argument. In the
heatmap below, we have the sample IDs plotted along the bottom horizontal axis, while the
genes names are presented long the right vertical axis. Each tile in the heatmap corresponds to
the expression of a gene for the corresponding sample and as mentioned earlier, the
expression level is indicated by the color scale. Note that later on in this lesson, we can add an
additional legend that color codes the treatment group that each sample belongs to. The
dendrogram that spans the columns indicates how samples are clustered together, while that
which spans the rows tells us how the genes are clustered together.

pheatmap(mat)

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Distance, clustering, and scaling

When generating a heatmap and dendrogram to show clustering three parameters to consider
are

• method for calculating distance between objects (ie. experimental samples)

• method for clustering
• scaling of data prior to inputting into heatmap generating algorithm

Distance calculation
The idea behind cluster analysis is to calculate some sort of distance between objects in order
to identify the ones that are closer together. When two objects have a small distance, we can
conclude they are closer and should cluster together. On the other hand, two objects that are
further apart will have a larger distance. There are various approaches to calculating distance in
cluster analysis so considerations should be taken for choosing the appropriate one. To learn
more about distance calculation methods as well as advantages and disadvantages of each
see Shirkhorshidi et al, PLOS ONE, 2015 (https://journals.plos.org/plosone/article?id=10.1371/
journal.pone.0144059). In pheatmap, we can specify the clustering distance using either the
clustering_distance_rows argument or clustering_distance_cols depending on
whether we would like to cluster by row or column variables.

Cluster generation
After the distance matrix has been calculated, it is time to perform the actual clustering and
again, various approaches can be used to generate clusters. The following resources are good
for learning about the variouse hierarchical clustering methods. In pheatmap, the clustering
method is specified by the clustering_method argument.

• https://hlab.stanford.edu/brian/forming_clusters.htm (https://hlab.stanford.edu/brian/
forming_clusters.htm)
• https://dataaspirant.com/hierarchical-clustering-algorithm/ (https://dataaspirant.com/
hierarchical-clustering-algorithm/)
• https://www.learndatasci.com/glossary/hierarchical-clustering/ (https://
www.learndatasci.com/glossary/hierarchical-clustering/)

Scaling
Prior to sending our data into the heatmap generating algorithm, it is a good idea to sacle.
There are several reasons for doing this

• Variables in the data might not have the same units, thus without scaling we will be, to
borrow a phrase, comparing apples to oranges

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• Scaling allows us to discern patterns in variables with low values when plotting on the
color scale. Without scaling, variables with large values will drown out the signal from
those with low values. We will see an example of this using the mtcars data.
• Scaling also prevents variables with large values from contributing too much weight to
distance https://medium.com/analytics-vidhya/why-is-scaling-required-in-knn-and-k-
means-8129e4d88ed7 (https://medium.com/analytics-vidhya/why-is-scaling-required-in-
knn-and-k-means-8129e4d88ed7). Without scaling, it will hard to discern whether
variables with lower values contribute to separation.

A common method for scaling is to use the z score (see z score formula), which tells us how
many standard deviations away from the mean is a given value in our data. This is the scaling
method for pheatmap.

z score=(individual value - mean) \ (standard deviation)

Below, we will use the mtcars data to look at how scaling influences a heatmap. Because
mtcars is built into R, we can use the data command to load it and we will save this as an
object named cars. Next, we will use the head command to view the first few rows of the cars
data.

data(mtcars)
cars <- mtcars
head(cars)

## mpg cyl disp hp drat wt qsec vs am gear carb

## Mazda RX4 21.0 6 160 110 3.90 2.620 16.46 0 1 4 4
## Mazda RX4 Wag 21.0 6 160 110 3.90 2.875 17.02 0 1 4 4
## Datsun 710 22.8 4 108 93 3.85 2.320 18.61 1 1 4 1
## Hornet 4 Drive 21.4 6 258 110 3.08 3.215 19.44 1 0 3 1
## Hornet Sportabout 18.7 8 360 175 3.15 3.440 17.02 0 0 3 2
## Valiant 18.1 6 225 105 2.76 3.460 20.22 1 0 3 1

Note that variables like disp and hp has larger magnitudes as compared to one like mpg. Also,
the variables in this data does not have the same units. If we constructed a heatmap of the
mtcars data without scaling, we will not be able to discern patterns in variables like mpg among
the samples. This is because the values for mpg are small in comparison to those for disp and
hp, they get squeeze towards the bottom of color scale.

pheatmap(cars)

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However, if we scaled then it becomes easier to observe differences in values for each of the
variables. We are interested in the differences in each variable across the car types, thus, we
scale by column because the sample names (ie. car brands) are listed down the rows. If the car
brands were listed across columns, we would have scaled by row.

pheatmap(cars, scale="column")

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Getting back to RNA seq and something more biologically relevant, we can scale by row in mat.

pheatmap(mat, scale="row")

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Working with color customization

pheatmap(mat,scale="row",
color=colorRampPalette(c("navy", "white", "red"))(50))

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Adding treatment group information to samples (annotation_col,

annotation_colors)
The code below generates a data frame, dfh, that contains information on the treatment group
in which a sample was assigned. To create a data frame in R, we use the data.frame
command. In the data.frame command, we set the sample to the column names of the data
mat using colnames(mat) to extract the mat column headings. We then want to convert the
column headings in mat to character using as.character. Next, also in the data.frame
command, we set the column dex to "Treatment".

Using %>%, we pass the dfh data frame to the column_to_rownames command to set the
rownames of the dfh data frame to the sample IDs. Finally, we will change the values in the dex
column to either untrt (untreated) or trt (treated) using ifelse to check if the row names of dfh
(rownames(dfh)) are samples 508, 512, 516, or 520. If yes, then the value for these samples
in the dex column becomes untrt. In other words, 508, 512, 516, and 520 are untreated
samples. Else, we will assign the samples to the trt group, which indicates they are treated.

#create data frame for annotations

dfh<-data.frame(sample=as.character(colnames(mat)),dex="Treatment")%>%
column_to_rownames("sample")
dfh$dex<-ifelse(rownames(dfh) %in% c("508","512","516","520"),

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"untrt","trt")
dfh

## dex
## 508 untrt
## 509 trt
## 512 untrt
## 513 trt
## 516 untrt
## 517 trt
## 520 untrt
## 521 trt

Add the annotations column

We can add the sample treatment annotation by setting the annotation_col argument to dfh
in pheatmap. We use annotation_col rather than annotation_row because the samples
IDs are listed along the horizontal axis so essentially corresponding to the columns of the
heatmap. The result is that the samples are now color coded by the treatment group in which
they belong and this color coding is provided in the legend.

pheatmap(mat,scale="row", annotation_col = dfh,

color=colorRampPalette(c("navy", "white", "red"))(50))

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Modify the annotations colors

Using the annotation_colors argument, we can reassign the colors of the sample to
treatment mapping legend.

pheatmap(mat,scale="row", annotation_col = dfh,

annotation_colors=list(dex=c(trt="orange",untrt="black")),
color=colorRampPalette(c("navy", "white", "red"))(50))

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Splitting heatmap into multiple columns and rows

It maybe helpful to split the heatmap into different portions to illustrate clusters more efficiently.
Here, we can split the heatmap into two columns using the argument cutree_col and setting
this to 2. Doing so will split the heatmap into a column containing the dexamethasone (dex)
treated samples (trt) and untreated samples (untrt).

pheatmap(mat,scale="row", annotation_col = dfh,

annotation_colors=list(dex=c(trt="orange",untrt="black")),
color=colorRampPalette(c("navy", "white", "red"))(50),
cutree_cols=2)

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If we include cutree_rows=2, then the heatmap will be split into two rows. Note that it is split in a
way that the top row represents genes that are down-regulated in the treated group and up-
regulated in the untreated group. The bottom row represents those genes that are up-regulated
by dexamethasone treatment but down-regulated when not treated.

pheatmap(mat,scale="row", annotation_col = dfh,

annotation_colors =list(dex=c(trt="orange",untrt="black")),
color=colorRampPalette(c("navy", "white", "red"))(50),
cutree_cols=2, cutree_rows=2)

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Add a title to heatmap

A title for our heatmap can be included using the main argument

pheatmap(mat,scale="row", annotation_col = dfh,

annotation_colors =list(dex=c(trt="orange",untrt="black")),
color=colorRampPalette(c("navy", "white", "red"))(50),
cutree_cols=2, cutree_rows=2,
main="Expression and clustering of top 20 DE genes")

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The last few customization we will do with the heatmap is to adjust the fontsize using argument
fontsize. We will adjust the cellwidth to move the treatment legend into the plot canvas.
We also use cellheight to adjust the height of the heatmap to fill more of the plotting canvas.

pheatmap(mat,scale="row", annotation_col = dfh,

annotation_colors=list(dex=c(trt="orange",untrt="black")),
color=colorRampPalette(c("navy", "white", "red"))(50),
cutree_cols=2, cutree_rows=2,
main="Expression and clustering of top 20 DE genes",
fontsize=11, cellwidth=35, cellheight=10.25)

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Saving a non-ggplot

Recall that we can use the ggsave command to save a ggplot. However, heatmaps generated
using the pheatmap package are not ggplots, therefore we need to turn to either the image
export feature (Figure 7) in R studio or use one of the several image saving commands.

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The programmatic ways to save an image include the following commands

• jpeg
• bmp
• tiff
• png
• pdf

All of these take the file name in which we would like to save the image, resolution (res), image
width (width), image height (height), and units of image dimension (unit) as arguments.
Below, we use png to save our heatmap as file pheatmap_1.png at 300 dpi as specified in
res. The workflow is to first create the file using one of the image save commands, then
generate the plot, and set dev.off() to turn off the current graphical device. If we do not set
dev.off(), subsequent plots will overwrite the file that we just saved and will not show up in the
plot pane.

dev.new()
png("./data/pheatmap_1.png", res=300, width=7, height=4.5, unit="in")
pheatmap(mat,scale="row", annotation_col = dfh,
annotation_colors=list(dex=c(trt="orange",untrt="black")),
color=colorRampPalette(c("navy", "white", "red"))(50),
cutree_cols=2, cutree_rows=2,
main="Expression and clustering of top DE genes",
fontsize=11, cellwidth=35, cellheight=10.25)

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dev.off()

## quartz_off_screen
## 3

Make this plot compatible with ggplot2 using the

package ggplotify.

hm_gg<-as.ggplot(pheatmap(mat,scale="row", annotation_col=
dfh,annotation_colors =list(dex=c(trt="orange",
untrt="black")),color=colorRampPalette(c("navy",
"white", "red"))(50)))

Save as an R object
Below, we assign the heatmap to the R object hm_ph and we can import this back to R in the
future.

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hm_ph <- pheatmap(mat,scale="row", annotation_col =

dfh,annotation_colors =list(dex=c(trt="orange",
untrt="black")), color=colorRampPalette(c("navy",
"white", "red"))(50),cutree_cols=2, cutree_rows=2,
main="Expression and clustering of top DE genes",
fontsize=11, cellwidth=35, cellheight=10.25)

saveRDS(hm_ph, file="./data/airways_pheatmap.rds")

We will wrap up lesson 5 with an introduction to the package heatmaply, which can be used to
generate interactive heatmaps. Below is a basic interactive heatmap generated using this
package for the airway top 20 differentially expressed genes (mat).

Similar to pheatmap, we will start by providing heatmaply the data that we would like to plot. We
can also scale by row like we did in pheatmap. Plot margins can also be set to ensure the entire
plot fits on the canvas. Like in pheatmap, we assign the plot title using the argument main.
Setting col_side_colors to the data frame dfh, which contains the sample to treatment
mapping, creates a legend that spans the columns of the heatmap, informing us of the
treatment group to which the samples belong.

heatmaply(mat, scale="row", margins=c(0.5,1,50,1),

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main="Interactively explore airway DE genes",

col_side_colors=dfh)

heatmaply example

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Lesson 6: Multi-figure panel

Objectives
1. Combine multiple plots into a single figure

2. Learn how to use aspects of cowplot and patchwork

The primary purpose of this lesson is to learn how to combine multiple figures into a single
multi-panel figure using patchwork and a few features of cowplot. While we will learn how to
customize and arrange plots in a multi-figure panel, this is not a comprehensive lesson on all
aspects of patchwork and cowplot. If you have something specific in mind for your own
data, I implore you to read the documentation for these packages to understand their full
potential for customization.

Why do we need to learn to combine figures?

Combining multiple figures is advantageous when preparing results for conference
presentations (via poster) or publication. Most journals place limits on the number of figures
permitted per publication.

Example journals and their figure limits:

Journal Impact Factor Number of Figures
Nature Cancer 60.72 5-8

Science 47.73 6

Cancer Cell 31.74 8

Journal of Clinical Oncology 44.54 6

JAMA Oncology 31.78 5

Cell Host and Microbe 21.02 7

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Example Multi-figure panel from Zhang et al.(2022). Longitudinal single-cell RNA-seq analysis
reveals stress-promoted chemoresistance in metastatic ovarian cancer. Science advances, 8(8),
eabm1831.

Load the libraries

There are multiple ways to combine figures using R. In this lesson, we will learn how to combine
figures primarily with patchwork. Though, we will also learn some components of cowplot.

To get started, load the libraries. All packages used today can be installed from CRAN using
install.packages().

#Get patchwork
#To install use install.packages('patchwork')
#load
library(patchwork)

#Get cowplot
#To install use install.packages('cowplot')

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#load
library(cowplot)

#We will also use ggplot2

library(ggplot2)

The Data
This is the sixth lesson in our Data Visualization with R Series. At this point, we have created
quite a few plots. For this lesson, we will focus on the RNA-Seq plots that we created in previous
lessons. We will also include other related plots that were created using the same RNA-Seq
data, but were not created throughout this course series. All plots were saved as R objects
(.rds). To load the data into R, we will need to use the readRDS() function.

Let's load and view our plots. To view our plots, we can simply call the objects by name.

pca<-readRDS("./data/airwaypca.rds")

volcano<-readRDS("./data/volcanoplot.rds")

hmap<-readRDS("./data/airwayhm.rds")

sc<-readRDS("./data/stripchart.rds")

#view objects
pca

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volcano

## Warning: Using alpha for a discrete variable is not advised.

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hmap

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sc

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What is cowplot?
The cowplot package provides various features that help with creating publication-
quality figures, such as a set of themes, functions to align plots and arrange them
into complex compound figures, and functions that make it easy to annotate plots
and or mix plots with images. The package was originally written for internal use in
the Wilke lab, hence the name (Claus O. Wilke’s plot package). --- cowplot 1.1.1
(https://wilkelab.org/cowplot/index.html)

The cowplot documentation (https://wilkelab.org/cowplot/index.html) is very user friendly, so

be sure to check it out. Until recently cowplot has been my go to for arranging and combining
plots in a multi-figure plot panel. Today we will primarily focus on patchwork, described below,
for this purpose because it is, in my opinion, far easier to use for simple figure alignment in a
grid format. However, cowplot does have some features that are notable, especially related to
label customization, unique plot arrangements, and image drawing. The drawing functions
(https://wilkelab.org/cowplot/articles/drawing_with_on_plots.html), which allow you to easily
integrate outside images onto your plots, are especially useful, so take a look at the linked
documentation. Some of the more difficult features of cowplot, such as getting a combined
legend, have been simplified by wrapper functions from other packages (See ggarrange()
from ggpubr).

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Using cowplot to arrange figures

The main function to combine figures using cowplot is plot_grid(). Let's check out the help
documentation using ?plot_grid(). The first and most important parameter is the list of plots
we want to combine, plotlist.

Let's check out the basic use of this function by calling the plots we want to combine and by
providing labels using the labels argument.

plot_grid(pca,volcano,hmap,sc, labels="AUTO")

## Warning: Using alpha for a discrete variable is not advised.

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This figure isn't bad. Though, we would likely want to change the relative sizes of the plots and
work on the plot alignments. This can be done with rel_heights, rel_widths, align, and
axis. We are not going to work with these today because cowplot does not do well with plot
alignments when a given plot's aspect ratio has been fixed (e.g., coord_fixed(),
coord_equal()).

However, I do want to point out some of the options for label customization before moving on to
ggdraw().

Plotting labels with cowplot

There are quite a few parameters to adjust figure labels. To re-position labels, see label_x,
label_y, hjust, and vjust. These each take either a single value to move all labels or a
vector of values, one for each subplot. We can also change the size of the labels
(label_size), the font (label_fontface), the label color (label_colour), and the font
type (label_fontfamily). In general, there seem to be more options for plot customization
(without adding ggplot2 layers) with cowplot.

plot_grid(pca,volcano,hmap,sc, labels="AUTO",label_size = 14,

label_fontface = "bold.italic", label_colour ="blue",
label_fontfamily ="Times New Roman",
label_y=0.25)

## Warning: Using alpha for a discrete variable is not advised.

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Here we changed the size of the labels to 14 pt and the color to blue. Also, the labels are now
bold and italicized, and the font is Times New Roman. We also re-positioned the labels.

Other notable features of cowplot

You can nest figures by combining figures using plot_grid(), saving that to an object, and
then plotting those pre-combined figures with another figure using plot_grid() again. Shared
legends can be obtained using cowplot's get_legend(). cowplot also has its own function
to save plots (save_plot()), which is a bit more dynamic for multi-figure panels, but you may
also use ggsave().

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Taking advantage of ggdraw

An extremely useful feature of cowplot is ggdraw() combined with draw_plot(),

draw_grob(), draw_image() and draw_label(). These can be used to add or combine
text, images, and plots and create more complicated plot arrangements. ggdraw() can be
used directly with any package that creates grid grobs. It can also be used with lattice and
base R graphics. The output of these functions can be treated like ggplot2 objects.

Note: Grobs are graphical objects that you can make and change with grid
graphics functions. The ggplot2 package is built on top of grid graphics, so the grid
graphics system “plays well” with ggplot2 objects. --- Pang, Kross, and Andersen,
2020 (https://bookdown.org/rdpeng/RProgDA/the-grid-package.html)

Let's look at the basics of drawing an image.

Note: Drawing an image requires library(magick).

plos <- "./images/himesetal2014_plos.png" #save our image file path

#Create an image to add to a multi-figure plot.

a<-ggdraw() + #create blank canvas
draw_image(plos,scale=1) # draw the image and save to object

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ggdraw() creates a new ggplot2 canvas without visible axes or background grid.
The draw_* functions are simply wrappers around regular geoms. --- cowplot
documentation (https://wilkelab.org/cowplot/articles/drawing_with_on_plots.html)

Let's add this to the background of our PCA.

pca + draw_image(plos,x=-15,y=-2,width=30,height=20)

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Using cowplot we can get an image such as this:

p1<-plot_grid(NULL,a,NULL,(pca+theme(legend.position="right")),labels=c("","","

bc<-plot_grid(volcano,sc,labels = c("B","C"),align="h",axis="b")
## Warning: Using alpha for a discrete variable is not advised.

p2<-plot_grid(p1,NULL,bc,ncol=1,align="v",axis="l",rel_heights = c(1,0.05,1.75)

p2b<-plot_grid(hmap,NULL,nrow=1,align="h",axis="b",rel_widths=c(0.65,0.25))

p3<-plot_grid(NULL,p2,NULL,p2b,ncol=1,labels=c("","","D"),align="v",axis="l",re

labp<-ggdraw(p3)+
draw_label("Airway Data", color = "Black", size = 14, x=0.1,y=0.97,hjust=0.6

labp

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What is patchwork?
The goal of patchwork is to make it ridiculously simple to combine separate ggplots
into the same graphic. As such it tries to solve the same problem as
gridExtra::grid.arrange() and cowplot::plot_grid but using an API that incites
exploration and iteration, and scales to arbitrily complex layouts. ---Thomas Lin
Pederson, Patchwork documentation (https://patchwork.data-imaginist.com/
index.html).

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Patchwork allows users to combine plots using simple mathematic operations such as + and /.

Combining two plots

Let's combine our PCA and volcano plots.

pca + volcano

## Warning: Using alpha for a discrete variable is not advised.

The last plot included in patchwork statements is considered the active plot, to which we can
add additional ggplot2 layers. Notice the seamless alignment of these plots. Without any
additional parameters, coord_fixed() is maintained in the pca plot. patchwork does a lot
better with a fixed aspect plot.

Let's customize the legend position of the active plot.

pca + volcano +
theme(legend.position="right")

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## Warning: Using alpha for a discrete variable is not advised.

We can easily add ggplot2 layers within our patchwork code.

We can continue to add plots using the + symbol, and patchwork will try to form a grid,
proceeding from left to right row-wise. Let's see this in action.

pca + volcano + hmap + sc

## Warning: Using alpha for a discrete variable is not advised.

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The plot layout can be controlled further with additional operators. The | symbol is used to
place plots side by side, while the / symbol is used to stack plots vertically. Plotting layouts
kind of follow the rules designated by the order of operations (Remember back to PEMDAS),
the / occurs before | and +.

#vertical stacking
pca / volcano

## Warning: Using alpha for a discrete variable is not advised.

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#horizontal
pca | volcano

## Warning: Using alpha for a discrete variable is not advised.

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pca | volcano / sc

## Warning: Using alpha for a discrete variable is not advised.

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For specific layouts, it is a good idea to use parentheses for correct evaluation.

For example, what if we want our pca and heatmap side by side stacked on top of our box plot?

Compare the following:

pca | hmap / sc

((pca | hmap) / sc)

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Using plot_layout()

The function plot_layout() can be used to control the layout, combine legends, and
overwrite plot titles.

pca1 <-pca + ggtitle("(A)")

pca1

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sc1 <- sc + ggtitle("(B)")

sc1

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#combine legends
pca1 + sc1 + plot_layout(ncol =1, guides = 'collect')

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#Can control the relative heights and widths

pca1 + guides(shape = guide_legend(nrow=4)) + sc1 +
plot_layout(nrow=1,guides = 'collect',widths=c(1.5,2))

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Changing the relative sizes of the figures alters the alignment. Units may also be specified to
control the height or width.

It is possible to design unique (non-grid) layouts using patchwork, but it seems a bit more
difficult than cowplot in that regard.

Adding a spacer

We can add a spacer using plot_spacer() to add blank sections to our plot. These blank
sections are the size of our figure panels and may be different depending on how the plot is
arranged.

(pca1 + guides(shape = guide_legend(nrow=4),

color = guide_legend(nrow=2))) / plot_spacer() | sc1

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Add our cowplot image

pca2<- pca + guides(shape = guide_legend(nrow=4))

((a |pca2) / sc / hmap) + plot_layout(guides='collect')

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Including a title

To include a title, subtitle, or caption use the function plot_annotation().

patchf<-((a | pca2) / sc / hmap) +

plot_layout(guides='collect',heights=c(1,1,3)) +
plot_annotation(title = 'Airway Data', tag_levels = "A") &
theme(plot.tag = element_text(face = 'bold'),
title= element_text(face = 'bold'))
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patchf

Notice that the assignment of "A" and "B" were automatic with the tag_levels parameter. The
parentheses here are required. Also, & was used to apply theme() to all subplots in the
patchwork.

Save the plot

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ggsave("patchf.png",height=5,width=4.25,dpi=300,units="in",scale=2)

Which package to use?

This is ultimately up to you. In general, patchwork is easier to combine nicely aligned grid
style plots. cowplot seems to include greater opportunities for customization, but you could
easily customize things like titles using ggplot2 layers with either package prior to combining
figures. patchwork also allows integration of ggplot2 layers. Both packages also allow you to
inset figures and add non-ggplot2 figures. As you gain more experience with R, you may find
yourself using both packages to achieve specific goals, or you may look to other packages.
egg (https://cran.r-project.org/web/packages/egg/vignettes/Overview.html) is another popular
package for combining figures. ggarrange() (https://rpkgs.datanovia.com/ggpubr/reference/
ggarrange.html) from the package ggpubris also popular.

Acknowledgements
Content in this tutorial was adapted from information in the cowplot documentation (https://
wilkelab.org/cowplot/articles/plot_grid.html) and patchwork documentation (https://
patchwork.data-imaginist.com/).

Bioinformatics Training and Education Program

Getting the Data
135 Course Data

Course Data
Course data is available in the attached zipped archive: data.zip.

Bioinformatics Training and Education Program

Practice Questions
137 Practice plotting using ggplot2: Lesson 2

Practice plotting using ggplot2: Lesson 2

Load the data

For these exercises, you will explore the titanic data from kaggle.com (https://www.kaggle.com/
c/titanic/data), which was downloaded from here (https://web.stanford.edu/class/archive/cs/
cs109/cs109.1166/problem12.html). You will need to download the data and load into R. As this
is a comma separated file, you will need to explore the read.csv() function.

Description of the data:

Column Description
Survived 0 = No, 1 = Yes

Pclass Ticket Class / Socioeconomic status (1 = 1st, 2 = 2nd, 3 = 3rd)

Name Passenger name

Sex Male / Female

Age Numeric age in years

Siblings/Spouses Aboard # of siblings / spouses aboard the Titanic

Parents/Children Aboard # of parents / children aboard the Titanic

Fare Passenger fare

Get the data here.

Load ggplot2.

library(ggplot2)

Exercise Questions
Question 1

Load titanic.csv and save to an object named titanic.

{{Sdet}}

Possible Solution{{Esum}}

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138 Practice plotting using ggplot2: Lesson 2


titanic <- read.csv("https://web.stanford.edu/class/archive/cs/cs109/cs109.1166

{{Edet}}

Question 2

Explore the data. What is the structure of the data? Try str(). What are the column names? Try
colnames(). How can you get help if you do not know how to use these functions?

{{Sdet}}

Possible Solution{{Esum}}

str(titanic) # get the structure

## 'data.frame': 887 obs. of 8 variables:

## $ Survived : int 0 1 1 1 0 0 0 0 1 1 ...
## $ Pclass : int 3 1 3 1 3 3 1 3 3 2 ...
## $ Name : chr "Mr. Owen Harris Braund" "Mrs. John Bradley
## $ Sex : chr "male" "female" "female" "female" ...
## $ Age : num 22 38 26 35 35 27 54 2 27 14 ...
## $ Siblings.Spouses.Aboard: int 1 1 0 1 0 0 0 3 0 1 ...
## $ Parents.Children.Aboard: int 0 0 0 0 0 0 0 1 2 0 ...
## $ Fare : num 7.25 71.28 7.92 53.1 8.05 ...

colnames(titanic) # get the column names.

## [1] "Survived" "Pclass"

## [3] "Name" "Sex"
## [5] "Age" "Siblings.Spouses.Aboard"
## [7] "Parents.Children.Aboard" "Fare"

?str # get help

?colnames

{{Edet}}

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139 Practice plotting using ggplot2: Lesson 2

Question 3 

Make a simple scatter plot. Is there a relationship between the age of the passenger and the
passenger fare?

{{Sdet}}

Possible Solution{{Esum}}

ggplot(titanic) +
geom_point(aes(x=Age, y=Fare))

{{Edet}}

Question 4
Color the points from question 3 by Pclass. Remember that Pclass is a proxy for socioeconomic
status. While the values are treated as numeric upon loading, they are really categorical and
should be treated as such. You will need to coerce Pclass into a categorical (factor) variable.
See factor() and as.factor().

{{Sdet}}

Bioinformatics Training and Education Program

140 Practice plotting using ggplot2: Lesson 2

Possible Solution{{Esum}} 

ggplot(titanic) +
geom_point(aes(x=Age, y=Fare, color=as.factor(Pclass)))

{{Edet}}

Question 5
Manually scale the colors in question 4. 1st class = yellow, 2nd class = purple, 3rd class =
seagreen. Also change the legend labels (1 = 1st Class, 2 = 2nd Class, 3 = 3rd Class).

{{Sdet}}

Possible Solution{{Esum}}

ggplot(titanic) +
geom_point(aes(x=Age, y=Fare, color=as.factor(Pclass)))+
scale_color_manual(values=c("yellow","purple","seagreen"),
labels=c("1st Class","2nd Class","3rd Class"))

Bioinformatics Training and Education Program

141 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Question 6
Facet the plot made in 5 by the column 'Sex'.

{{Sdet}}

Possible Solution{{Esum}}

ggplot(titanic) +
geom_point(aes(x=Age, y=Fare, color=as.factor(Pclass))) +
scale_color_manual(values=c("yellow","purple","seagreen"),
labels=c("1st Class","2nd Class","3rd Class")) +
facet_wrap(~Sex)

Bioinformatics Training and Education Program

142 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Challenge question 1
Let's use some other geoms. Plot the number of passengers (a simple count) that survived by
ticket class and facet by sex.

{{Sdet}}

Possible Solution{{Esum}}

ggplot(titanic) +
geom_bar(aes(x=Pclass, fill=factor(Survived)),
position=position_dodge()) +
facet_wrap(~Sex)+
labs( y="Number of Passengers", x="Passenger Class",
title="Titanic Survival Rate by Passenger Class")

Bioinformatics Training and Education Program

143 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Challenge question 2
Add a variable to the data frame called age_cat (child = <12, adolescent = 12-17,adult= 18+).
Plot the number of passengers (a simple count) that survived by age_cat, fill by Sex, and facet
by class and survival.

{{Sdet}}

Possible Solution{{Esum}}

library(dplyr)

##
## Attaching package: 'dplyr'

## The following objects are masked from 'package:stats':

##
## filter, lag

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144 Practice plotting using ggplot2: Lesson 2

## The following objects are masked from 'package:base':

##
## intersect, setdiff, setequal, union

titanic %>%
mutate(age_cat= case_when(Age < 12 ~ "child",
Age >= 12 & Age < 18 ~ "adolescent",
Age >= 18 ~ "adult"
)) %>%
ggplot() +
geom_bar(aes(x=age_cat, fill=factor(Sex)),
position=position_dodge()) +
facet_grid(Pclass~Survived)+
labs( y="Number of Passengers", x="Age Category",
title="Titanic Survival")

{{Edet}}

Bioinformatics Training and Education Program

145 Practice plotting using ggplot2: Lesson 2

Want more practice? 

Let's use the dataset mtcars. According to the help documentation (?mtcars), "the data was
extracted from the 1974 Motor Trend US magazine, and comprises fuel consumption and 10
aspects of automobile design and performance for 32 automobiles (1973–74 models)." Each
question below will depend on code from the previous question.

Question 1
Let's check out the structure of the data.

{{Sdet}}

Possible Solution{{Esum}}

str(mtcars)

## 'data.frame': 32 obs. of 11 variables:

## $ mpg : num 21 21 22.8 21.4 18.7 18.1 14.3 24.4 22.8 19.2 ...
## $ cyl : num 6 6 4 6 8 6 8 4 4 6 ...
## $ disp: num 160 160 108 258 360 ...
## $ hp : num 110 110 93 110 175 105 245 62 95 123 ...
## $ drat: num 3.9 3.9 3.85 3.08 3.15 2.76 3.21 3.69 3.92 3.92 ...
## $ wt : num 2.62 2.88 2.32 3.21 3.44 ...
## $ qsec: num 16.5 17 18.6 19.4 17 ...
## $ vs : num 0 0 1 1 0 1 0 1 1 1 ...
## $ am : num 1 1 1 0 0 0 0 0 0 0 ...
## $ gear: num 4 4 4 3 3 3 3 4 4 4 ...
## $ carb: num 4 4 1 1 2 1 4 2 2 4 ...

{{Edet}}

Question 2
How might we plot automobile weight (wt) versus miles per gallon (mpg).

{{Sdet}}

Possible Solution{{Esum}}

ggplot(mtcars, aes(wt, mpg)) +

geom_point()

Bioinformatics Training and Education Program

146 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Question 3
What if we want to represent the number of cylinders (cyl) by color and shape?

{{Sdet}}

Possible Solution{{Esum}}

ggplot(mtcars, aes(wt, mpg)) +

geom_point(aes(color = factor(cyl),shape = factor(cyl)))

Bioinformatics Training and Education Program

147 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Question 4
Make the size of the points change by the quarter mile time (qsec).

{{Sdet}}

Possible Solution{{Esum}}

ggplot(mtcars, aes(wt, mpg)) +

geom_point(aes(color = factor(cyl),shape = factor(cyl),size=qsec))

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148 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Question 5
Create subplots by transmission (am).

{{Sdet}}

Possible Solution{{Esum}}

ggplot(mtcars, aes(wt, mpg)) +

geom_point(aes(color = factor(cyl),shape = factor(cyl),size=qsec))+
facet_wrap(~am)

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149 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Question 6

Model the trend using geom_smooth(). What is the default method used by geom_smooth()?

{{Sdet}}

Possible Solution{{Esum}}

ggplot(mtcars, aes(wt, mpg)) +

geom_point(aes(color = factor(cyl),shape = factor(cyl),size=qsec))+
facet_wrap(~am) +
geom_smooth()

## `geom_smooth()` using method = 'loess' and formula = 'y ~ x'

Bioinformatics Training and Education Program

150 Practice plotting using ggplot2: Lesson 2

{{Edet}}

Bioinformatics Training and Education Program

151 Practice plotting using ggplot2: Lesson 3

Practice plotting using ggplot2: Lesson 3

The following questions synthesize several of the skills you have learned thus far. It may not be
immediately apparent how you would go about answering these questions. Remember, the R
community is expansive, and there are a number of ways to get help including but not limited to
google search. These questions have multiple solutions, but you should try to stick to the tools
you have learned to use thus far.

Your mission is to make a publishable figure using the iris data set.

Question 1

Start by plotting Petal.Length on the x-axis and Petal.Width on the y-axis.

{{Sdet}}

Possible Solution{{Esum}}

library(ggplot2)
ggplot(iris)+
geom_point(aes(Petal.Length,Petal.Width,color=Species))

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152 Practice plotting using ggplot2: Lesson 3

{{Edet}}

Question 2
Fix the axes so that the dimensions on the x-axis and the y-axis are equal. Both axes should
start at 0. Label the axis breaks every 0.5 units on the y-axis and every 1.0 units on the x-axis.

{{Sdet}}

Possible Solution{{Esum}}

ggplot(iris)+
geom_point(aes(Petal.Length,Petal.Width,color=Species))+
coord_fixed(ratio=1,ylim=c(0,2.75),xlim=c(0,7),expand=FALSE) +
scale_y_continuous(breaks=c(0,0.5,1,1.5,2,2.5)) +
scale_x_continuous(breaks=c(0,1,2,3,4,5,6,7))

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153 Practice plotting using ggplot2: Lesson 3

{{Edet}}

Question 3
Change to color of the points by species to be color blind friendly, and change the legend title
to "Iris Species". Label the x and y axis to eliminate the variable names and add unit information.

{{Sdet}}

Possible Solution{{Esum}}

#multiple ways to find color blind friendly palettes.

#using color brewer scales
RColorBrewer::display.brewer.all(colorblindFriendly=TRUE)

Bioinformatics Training and Education Program

154 Practice plotting using ggplot2: Lesson 3

ggplot(iris)+
geom_point(aes(Petal.Length,Petal.Width,color=Species))+
coord_fixed(ratio=1,ylim=c(0,2.75),xlim=c(0,7),expand=FALSE) +
scale_y_continuous(breaks=c(0,0.5,1,1.5,2,2.5)) +
scale_x_continuous(breaks=c(0,1,2,3,4,5,6,7)) +
scale_color_brewer(palette = "Dark2",name="Iris Species") +
labs(x="Petal Length (cm)", y= "Petal Width (cm)")

Bioinformatics Training and Education Program

155 Practice plotting using ggplot2: Lesson 3

{{Edet}}

Question 4
Play with the theme to make this a bit nicer. Change font style to "Times". Change all font sizes
to 12 pt font. Bold the legend title and the axes titles. Increase the size of the points on the plot
to 2. Bonus: fill the points with color and have a black outline around each point.

{{Sdet}}

Possible Solution{{Esum}}

ggplot(iris)+
geom_point(aes(Petal.Length,Petal.Width,fill=Species),size=2,shape=
coord_fixed(ratio=1,ylim=c(0,2.75),xlim=c(0,7),expand=FALSE) +
scale_y_continuous(breaks=c(0,0.5,1,1.5,2,2.5)) +
scale_x_continuous(breaks=c(0,1,2,3,4,5,6,7)) +
scale_fill_brewer(palette = "Dark2",name="Iris Species") +
labs(x="Petal Length (cm)", y= "Petal Width (cm)") +
theme_bw()+
theme(axis.text=element_text(family="Times",size=12),
axis.title=element_text(family="Times",face="bold",size=12),
legend.text=element_text(family="Times",size=12),

Bioinformatics Training and Education Program

156 Practice plotting using ggplot2: Lesson 3

legend.title = (element_text(family="Times",face="bold",size=

)

{{Edet}}

Question 5

Now, save your plot using ggsave.

{{Sdet}}

Possible Solution{{Esum}}

ggsave("iris.tiff", width=5.5, height=3.5,units="in")

{{Edet}}

Bioinformatics Training and Education Program

157 Lesson 4: Stat Transformations: Bar plots, box plots, and histograms

Lesson 4: Stat Transformations: Bar plots,

box plots, and histograms
The following questions will have you explore the mtcars dataset through creating plots that
were presented in Lesson 4. At the end of these exercises, you should be more comfortable
creating plots that convey statistical summary information about data.

Activate packages

library(ggplot2)

Load the mtcars dataset using the code below. This is a dataset that
comes with R.

data(mtcars)

Question 1
How many cars in this dataset have 4, 6, or 8 cylinders (cyl)?

{{Sdet}}

Solution{{Esum}}

ggplot(mtcars,aes(x=factor(cyl)))+geom_bar(fill="ivory4")

Bioinformatics Training and Education Program

158 Lesson 4: Stat Transformations: Bar plots, box plots, and histograms

{{Edet}}

Question 2
Does the number of cylinders (cyl) that a car has influence it's quarter mile time (qsec)?

{{Sdet}}

Solution{{Esum}}

ggplot(mtcars,aes(x=factor(cyl),y=qsec))+stat_summary(fun=mean,position

## Warning: Ignoring unknown parameters: tion

Bioinformatics Training and Education Program

159 Lesson 4: Stat Transformations: Bar plots, box plots, and histograms

{{Edet}}

Question 3
What is the distribution of fuel efficiency (mpg)? Use 7 bins for this exercise.

{{Sdet}}

Solution{{Esum}}

ggplot(mtcars,aes(x=mpg))+geom_histogram(fill="orange",bins=7)

Bioinformatics Training and Education Program

160 Lesson 4: Stat Transformations: Bar plots, box plots, and histograms

{{Edet}}

Question 4
Can you create a box plot of horsepower (hp) as a function of the number of cylinders (cyl) a
car has?

{{Sdet}}

Solution{{Esum}}

ggplot(mtcars,aes(x=factor(cyl),y=hp))+geom_boxplot(colour="orangered"

Bioinformatics Training and Education Program

161 Lesson 4: Stat Transformations: Bar plots, box plots, and histograms

{{Edet}}

Bioinformatics Training and Education Program

162 Lesson5: Visualizing clusters with heatmap and dendrogram

Lesson5: Visualizing clusters with heatmap

and dendrogram
The following questions will help you gain more confidence in exploring data through heatmap.
We will work with a subset of the Human Brain Reference (HBR) and Universal Human
Reference (UHR) RNA sequencing dataset (https://rnabio.org/module-01-inputs/0001/05/01/
RNAseq_Data/) and use the heatmap to

• Visualize gene expression

• Determine whether subsets of genes can help us differentiate between the HBR and UHR
samples

Load necessary packages

library(pheatmap)
library(tidyverse)

## ── Attaching packages ─────────────────────────────────────── tidyverse 1.3.

## ✔ ggplot2 3.3.6 ✔ purrr 0.3.4
## ✔ tibble 3.1.8 ✔ dplyr 1.0.9
## ✔ tidyr 1.2.0 ✔ stringr 1.4.0
## ✔ readr 2.1.2 ✔ forcats 0.5.1
## ── Conflicts ────────────────────────────────────────── tidyverse_conflicts(
## ✖ dplyr::filter() masks stats::filter()
## ✖ dplyr::lag() masks stats::lag()

Question 1
Could you import the hbr_uhr_normalized_counts.csv file into your workspace?

{{Sdet}}

Solution{{Esum}}

hbr_uhr_normalized_counts <- read.csv("./data/hbr_uhr_normalized_counts.csv"

{{Edet}}

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163 Lesson5: Visualizing clusters with heatmap and dendrogram

Question 2 

Explore this gene expression dataset a bit. How many samples (columns) and genes (row
names) does this dataset have?

{{Sdet}}

Solution{{Esum}}

This dataset contains 6 samples (

• HBR_1.bam
• HBR_2.bam
• HBR_3.bam
• UHR_1.bam
• UHR_2.bam
• UHR_3.bam

The samples with names starting with HBR are from the Human Brain Reference (HBR) and
those with names starting with UHR are from the Universal Human Reference (UHR). Remember
this for a later questions.

hbr_uhr_normalized_counts

## HBR_1.bam HBR_2.bam HBR_3.bam UHR_1.bam UHR_2.bam UHR_3.bam

## SULT4A1 375.0 343.6 339.4 3.5 6.9 2.6
## MPPED1 157.8 158.4 162.6 0.7 3.0 2.6
## PRAME 0.0 0.0 0.0 568.9 467.3 519.2
## IGLC2 0.0 0.0 0.0 488.6 498.0 457.5
## IGLC3 0.0 0.0 0.0 809.7 313.8 688.0
## CDC45 2.6 1.0 0.0 155.0 152.5 149.9
## CLDN5 77.6 88.5 67.2 1.4 2.0 0.0
## PCAT14 0.0 0.0 1.2 139.8 154.4 155.1
## RP5-1119A7.17 53.0 57.6 51.9 0.0 0.0 0.0
## MYO18B 0.0 0.0 0.0 59.5 84.2 56.5
## RP3-323A16.1 0.0 0.0 1.2 51.9 76.2 53.1
## CACNG2 42.7 35.0 56.6 0.0 1.0 0.0

{{Edet}}

Question 3
Create a heatmap for to visualize gene expression for this dataset.

{{Sdet}}

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164 Lesson5: Visualizing clusters with heatmap and dendrogram

Solution{{Esum}} 

pheatmap(hbr_uhr_normalized_counts,scale="row")

{{Edet}}

Question 4

Create a data frame called annotation_df that contains the sample and treatment group
information that we will add to the legend for this heatmap.

{{Sdet}}

Solution{{Esum}}

annotation_df <- data.frame(sample=c("HBR_1.bam","HBR_2.bam","HBR_3.bam"

annotation_df

## treatment
## HBR_1.bam HBR
## HBR_2.bam HBR
## HBR_3.bam HBR

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165 Lesson5: Visualizing clusters with heatmap and dendrogram

## UHR_1.bam UHR 
## UHR_2.bam UHR
## UHR_3.bam UHR

{{Edet}}

Question 5

Add the annotations for the legend and color the HBR samples orangered and the UHR
samples blue. Also, add a title to the heatmap.

{{Sdet}}

Solution{{Esum}}

pheatmap(hbr_uhr_normalized_counts,scale="row", annotation_col =annotation_df

annotation_colors =list(treatment=c(HBR="orangered",UHR="blue"
main="Expression and clustering of top 12 DE genes")

{{Edet}}

Bioinformatics Training and Education Program

Additional Resources
167 For Further Reading

For Further Reading

Getting started with R

1. Hands on Programming with R (https://rstudio-education.github.io/hopr/index.html)
2. R for Data Science (R4DS) (https://r4ds.had.co.nz/index.html)
3. BTEP R Introductory Series (https://bioinformatics.ccr.cancer.gov/docs/rintro/index.html)
4. Base R cheat sheet
5. RStudio cheat sheet

General help with ggplot2

1. ggplot2 cheatsheet
2. R Graph Gallery (https://www.r-graph-gallery.com/)
3. The R Graphics Cookbook (https://r-graphics.org/recipe-quick-bar)
4. R4DS (https://r4ds.had.co.nz/data-visualisation.html)

Troubleshooting error messages

1. Epidemiologist R Handbook (47 Common errors) (https://epirhandbook.com/en/common-
errors.html)
2. R for Graduate Students (Troubleshooting Error Messages) (https://bookdown.org/
yih_huynh/Guide-to-R-Book/trouble.html)

Other Resources
1. Helpful search engine for R: rseek (https://rseek.org/)

Bioinformatics Training and Education Program

DNAnexus (Class
Registrants Only)
169 Navigating DNAnexus

Navigating DNAnexus
DNAnexus is a Cloud-based platform for NextGen Sequence analysis for which
CCR has a "site-license". For this class we are using the platform to provide a
uniform, stable, preinstalled interface for R training. This interface makes use of the
Web version of R-studio. In addition to the R-studio interface this process also
integrates the course-notes for the class in one window.

The following instructions should be followed when using this resource during
formal class time. For using this resource outside class times see the document
entitled "DNAnexus Basics".

Instruction for using DNAnexus for the Intro to R

class
1. Getting a DNAnexus account - every student should go to the main DNAnexus web page
(https://dnanexus.com/) and apply for a "free account". The BTEP staff will associate each
account with the NCI/CCR paid account prior to the first class.
2. Logging into DNAnexus account - Prior to the class each student should log into their
account, and navigate to the R Class project (DataViz_Apr_2023).
3. Starting R - Starting 30 mins before each class there will be a file labelled
"Start_Here.html" at the top level of the project. Select this file by clicking on it, and then
find your name on the list (arranged alphabetically by first name) and click on it. Note: if
there is more than one Start_Here.html file, you will need to select the file that has a
range of letters in which your first name would be included. For example, if there were two
files, Start_Here_A_J.html and Start_Here_K_Z.html, and my name was "Alex", I
would select Start_Here_A_J.html. The names of these files will vary based on the
total number of students in the class.

Bioinformatics Training and Education Program

170 Navigating DNAnexus

Once you select your name from the correct file, a window with the RStudio login page
will open.

Bioinformatics Training and Education Program

171 Navigating DNAnexus

Log in using the username "rstudio" and the password "rstudio". At this point you will be
presented with the RStudio main interface (shown below).
4. Splitting the window - If you wish to integrate the class notes into the same window as the
R-Studio interface, click on the file "Hsplit.html" or "Vsplit.html" (found in the lower right
hand segment) and select the "View in Web Browser" option from the pop-up menu. This
will add the class notes to the top portion of the browser window. There is a horizontal or
vertical bar separating the class notes window from the RStudio interface, and this bar
can be dragged up and down or right to left, depending on which file you selected
(Hsplit.html vs Vsplit.html), to change the size of the window dedicated to each function.

Bioinformatics Training and Education Program

172 Navigating DNAnexus

Bioinformatics Training and Education Program

173 DNAnexus Outside of Class

DNAnexus Outside of Class

Setting up the R-Studio environment outside of Class

hours
These instructions should be followed if you are setting up the R-Studio Web
environment outside the normal class hours. For instructions about using the
resource during class hours follow the instructions found in the document
"Navigating DNAnexus".

Log into DNAnexus

Each student should log into their account, and navigate to the R Class project
(DataViz_Apr_2023). For these instructions to make sense, you should use the "New Version"
of DNAnexus. If you are instead using the "Classic Version", your screen will look like this:

You need to select "Start Using New Version".

Note

Your class project ID WILL NOT be the same as the project ID in the picture.

Your screen should now look like the following:

Bioinformatics Training and Education Program

174 DNAnexus Outside of Class

To Start the Application

To start the application, select "Practicing R - Outside of Class App".

Then follow these steps:

• Step 1: Under Analysis Settings, enter your name in the Execution Name field.

• Step 2: Change Priority from "Normal" to "High".

Bioinformatics Training and Education Program

175 DNAnexus Outside of Class

• Step 3: Select the green button in the upper right labeled "Start Analysis".

• Step 4: Navigate to the "Monitor" tab to check the status of the job.

Bioinformatics Training and Education Program

176 DNAnexus Outside of Class

Open the R-Studio Web interface

Step 5: Wait approximately 5 minutes and then select the link under "Worker URL". The link will
look similar to this: "https://job-g7v6z280k4yp6qg4pbkz1gvb.dnanexus.cloud").

The reason for waiting ~5 mins is to give the system time to get everything in place. If you click
too soon you will see an error message.

Bioinformatics Training and Education Program

177 DNAnexus Outside of Class

Bioinformatics Training and Education Program

178 DNAnexus Outside of Class

Don't PANIC, just wait a little longer and refresh the screen, until you are finally presented with
the RStudio login screen.

Proceed with running content from the R Class

From the login screen, login with the username/passwd "rstudio/rstudio", and proceed from
there.

If you encounter any problems send email to BTEP at [email protected] (mailto:[email protected])

Bioinformatics Training and Education Program