---
title: "_spatialHeatmap_: Visualizing Spatial Assays in Anatomical Images and Network Graphs"
author: "Authors: Jianhai Zhang, Jordan Hayes, Le Zhang, Bing Yang, Wolf B. Frommer, Julia Bailey-Serres, and Thomas Girke"
date: "Last update: `r format(Sys.time(), '%d %B, %Y')`"
output:
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    self_contained: true
fontsize: 14pt
bibliography: bibtex.bib
package: spatialHeatmap
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---

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# Introduction 

## Motivation

The _spatialHeatmap_ package provides functionalities for visualizing cell-,
tissue- and organ-specific data of biological assays by coloring the
corresponding spatial features defined in anatomical images according to a
numeric color key. The color scheme used to represent the assay values can be
customized by the user. This core functionality of the package is called a
_spatial heatmap_ (SHM) plot. It is enhanced with visualization tools for groups 
of measured items (_e.g._ gene modules) sharing related abundance profiles, including 
matrix heatmaps combined with hierarchical clustering dendrograms and network representations. 
The functionalities of _spatialHeatmap_ can be used either in a command-driven mode
from within R or a graphical user interface (GUI) provided by a Shiny App that
is also part of this package. While the R-based mode provides flexibility to
customize and automate analysis routines, the Shiny App includes a variety of
convenience features that will appeal to experimentalists and other users less
familiar with R. Moreover, the Shiny App can be used on both local computers as
well as centralized server-based deployments (_e.g._ cloud-based or custom
servers) that can be accessed remotely as a public web service for using
_spatialHeatmap's_ functionalities with community and/or private data. The
functionalities of the `spatialHeatmap` package are illustrated in Figure
\@ref(fig:illus).   


```{r illus, echo=FALSE, fig.wide=TRUE, out.width="100%", fig.cap=("Overview of spatialHeatmap. (A) The _saptialHeatmap_ package plots numeric assay data onto spatially annotated images. A wide range of omics technologies is supported including genomic, transcriptomic, proteomic and metabolomic profiling data. The assay data can be provided as numeric vectors, tabular data, or _SummarizedExperiment_ objects. The latter is a widely used data container for organizing both assay data as well as associated annotation and experimental design data. (B) Anatomical and other spatial images need to be provided as annotated SVG (aSVG) files where the spatial features and the corresponding data components of the assay data have matching labels (_e.g._ tissue labels). (C) The assay data are used to color the matching spatial features in one or more aSVG images according to a color key. The result is called a spatial heatmap (SHM) plot. Multiple measurements can be visualized in the same plot, such as several factors (_e.g._ genes, proteins, metabolites), treatment conditions, growth stages and more. (D) Data mining graphics, such as matrix heatmaps and network graphs, are integrated to facilitate the identification of factors with similar assay profiles. The functionalities of _spatialHeatmap_ can be accessed from local computers via the R console or a graphical user interface based on Shiny. In addtion, the latter can be deployed as a web service on custom servers or cloud-based systems.")}

include_graphics('img/spatialHeatmap_Design.jpg')

```

As anatomical images the package supports both tissue maps from public
repositories and custom images provided by the user. In general any type of
image can be used as long as it can be provided in SVG (Scalable Vector
Graphics) format, where the corresponding spatial features have been defined
(see [aSVG](#term) below). The numeric values plotted onto an SHM
are usually quantitative measurements from a wide range of profiling
technologies, such as microarrays, next generation sequencing (_e.g._ RNA-Seq
and scRNA-Seq), proteomics, metabolomics, or many other small- or large-scale
experiments. For convenience, several preprocessing and normalization methods
for the most common use cases are included that support raw and/or preprocessed
data. Currently, the main application domains of the _spatialHeatmap_ package
are numeric data sets and spatially mapped images from biological, agricultural
and biomedical areas. Moreover, the package has been designed to also work with
many other spatial data types, such a population data plotted onto geographic
maps. This high level of flexibility is one of the unique features of
_spatialHeatmap_. Related software tools for biological applications in this
field are largely based on pure web applications [@Lekschas2015-gd; @Papatheodorou2018-jy; @Winter2007-bq; @Waese2017-fx] or local tools [@Maag2018-gi; @Muschelli2014-av] that typically
lack customization functionalities. These restrictions limit users to utilizing
pre-existing expression data and/or fixed sets of anatomical image collections.
To close this gap for biological use cases, we have developed _spatialHeatmap_
as a generic R/Bioconductor package for plotting quantitative values onto any
type of spatially mapped images in a programmable environment and/or in an
intuitive to use GUI application.

## Design {#design}

The core feature of [`spatialHeatmap`](#shm) is to map assay values (_e.g._
gene expression data) of one or many items (_e.g._ genes) measured under
different conditions in form of numerically graded colors onto the
corresponding cell types or tissues represented in a chosen SVG image. In the
gene profiling field, this feature supports comparisons of the expression
values among multiple genes by plotting their SHMs next to each
other. Similarly, one can display the expression values of a single or multiple
genes across multiple conditions in the same plot (Figure \@ref(fig:mul)). This level of flexibility is
very efficient for visualizing complicated expression patterns across genes,
cell types and conditions. In case of more complex anatomical images with
overlapping multiple layer tissues, it is important to visually expose the
tissue layer of interest in the plots. To address this, several default and
customizable layer viewing options are provided. They allow to hide features in
the top layers by making them transparent in order to expose features below
them. This transparency viewing feature is highlighted below in the mouse
example (Figure \@ref(fig:musshm)). Moreover, one can plot multiple distinct 
aSVGs in a single SHM plot as shown in Figure \@ref(fig:arabshm). This is 
particularly useful for displaying abundance trends across multiple development 
stages, where each is represented by its own aSVG image. In addition to
static SHM representations, one can visualize them in form of dynamic animations
embedded in interactive HTML files or generate videos for them.

To maximize reusability and extensibility, the package organizes large-scale
omics assay data along with the associated experimental design information in a
`SummarizedExperiment` object (Figure \@ref(fig:illus)A). The latter is one of the core S4 classes within
the Bioconductor ecosystem that has been widely adapted by many other software
packages dealing with gene-, protein- and metabolite-level profiling data
[@SummarizedExperiment]. In case of gene expression data, the `assays` slot of
the `SummarizedExperiment` container is populated with a gene expression
matrix, where the rows and columns represent the genes and tissue/conditions,
respectively, while the `colData` slot contains sample data including replicate
information. The tissues and/or cell type information in the object maps via
`colData` to the corresponding features in the SVG images using unique
identifiers for the spatial features (_e.g._ tissues or cell types). This
allows to color the features of interest in an SVG image according to the
numeric data stored in a `SummarizedExperiment` object. For simplicity the
numeric data can also be provided as numeric `vectors` or `data.frames`. This
can be useful for testing purposes and/or the usage of simple data sets that
may not require the more advanced features of the `SummarizedExperiment` class,
such as measurements with only one or a few data points. The details about how to
access the SVG images and properly format the associated expression data are
provided in the [Supplementary Section](#data_form) of this vignette.

## Image Format: SVG {#term}
SHMs are images where colors encode numeric values in features of
any shape. For plotting SHMs, Scalable Vector Graphics (SVG) has
been chosen as image format since it is a flexible and widely adapted vector
graphics format that provides many advantages for computationally embedding
numerical and other information in images. SVG is based on XML formatted text
describing all components present in images, including lines, shapes and
colors. In case of biological images suitable for SHMs, the shapes
often represent anatomical or cell structures. To assign colors to specific
features in SHMs, _annotated SVG_ (aSVG) files are used where the
shapes of interest are labeled according to certain conventions so that they
can be addressed and colored programmatically. SVGs and aSVGs of anatomical structures can
be downloaded from many sources including the repositories described [below](#data_repo).
Alternatively, users can generate them themselves with vector graphics software
such as [Inkscape](https://inkscape.org/){target="_blank"}. Typically, in aSVGs one or more 
shapes of a feature of interest, such as the cell shapes of an organ, are
grouped together by a common feature identifier. Via these group identifiers
one or many feature types can be colored simultaneously in an aSVG according to
biological experiments assaying the corresponding feature types with the
required spatial resolution. Correct assignment of image features and assay
results is assured by using for both the same feature identifiers. The color
gradient used to visually represent the numeric assay values is controlled by a
color gradient parameter. To visually interpret the meaning of the colors, the
corresponding color key is included in the SHM plots.  Additional
details for properly formatting and annotating both aSVG images and assay data
are provided in the [Supplementary Section](#sup) section of this vignette. 

## Data Repositories {#data_repo}

If not generated by the user, SHMs can be generated with data downloaded from
various public repositories. This includes gene, protein and metabolic
profiling data from databases, such as [GEO](https://www.ncbi.nlm.nih.gov/gds){target="_blank"},
[BAR](http://bar.utoronto.ca/){target="_blank"} and [Expression
Atlas](https://www.ebi.ac.uk/gxa/home){target="_blank"} from EMBL-EBI [@Papatheodorou2018-jy]. A
particularly useful resource, when working with `spatialHeatmap`, is the EBI
Expression Atlas. This online service contains both assay and anatomical
images. Its assay data include mRNA and protein profiling experiments for
different species, tissues and conditions. The corresponding anatomical image
collections are also provided for a wide range of species including animals and
plants. In `spatialHeatmap` several import functions are provided to work with
the expression and [aSVG repository](#svg_repo) from the Expression Atlas
directly. The aSVG images developed by the `spatialHeatmap` project are
available in its own repository called [spatialHeatmap aSVG
Repository](https://github.com/jianhaizhang/spatialHeatmap_aSVG_Repository){target="_blank"},
where users can contribute their aSVG images that are formatted according to
our guidlines.

## Tutorial Overview {#sample_data}

The following sections of this vignette showcase the most important
functionalities of the `spatialHeatmap` package using as initial example a simple
to understand toy data set, and then more complex mRNA profiling data from the
Expression Atlas and GEO databases. First, SHM plots are generated for both the toy
and mRNA expression data. The latter include gene expression data sets from
RNA-Seq and microarray experiments of [Human Brain](#hum), [Mouse
Organs](#mus), [Chicken Organs](#chk), and [Arabidopsis Shoots](#shoot). The
first three are RNA-Seq data from the [Expression
Atlas](https://www.ebi.ac.uk/gxa/home){target="_blank"}, while the last one is a microarray data
set from [GEO](https://www.ncbi.nlm.nih.gov/geo/){target="_blank"}.  Second, gene context
analysis tools are introduced, which facilitate the visualization of
gene modules sharing similar expression patterns. This includes the
visualization of hierarchical clustering results with traditional matrix
heatmaps ([Matrix Heatmap](#mhm)) as well co-expression network plots
([Network](#net)). Third, an overview of the corresponding [Shiny App](#shiny)
is presented that provides access to the same functionalities as the R
functions, but executes them in an interactive GUI environment [@shiny;
@shinydashboard]. Fourth, more advanced features for plotting customized
SHMs are covered using the Human Brain data set as an example.  


# Getting Started  

## Installation  

The `spatialHeatmap` package should be installed from an R (version $\ge$ 3.6)
session with the `BiocManager::install` command.  

```{ eval=FALSE, echo=TRUE, warnings=FALSE} 
if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("spatialHeatmap")
```

## Packages and Documentation

Next, the packages required for running the sample code in this vignette need to be loaded.  

```{r, eval=TRUE, echo=TRUE, warnings=FALSE, results='hide'}
library(spatialHeatmap); library(SummarizedExperiment); library(ExpressionAtlas); library(GEOquery)
```

The following lists the vignette(s) of this package in an HTML browser. Clicking the corresponding name will open this vignette. 

```{r, eval=FALSE, echo=TRUE, warnings=FALSE}
browseVignettes('spatialHeatmap')
```

# Spatial Heatmaps (SHMs) {#shm}

## Toy Example {#toy}

SHMs are plotted with the `spatial_hm` function. To provide a quick
and intuitive overview how these plots are generated, the following uses a
generalized toy example where a small vector of random numeric values is
generated that are used to color features in an aSVG image. The image chosen
for this example is an aSVG depicting the human brain. The corresponding image
file 'homo_sapiens.brain.svg' is included in this package for testing purposes.
The path to this image on a user\'s system, where `spatialHeatmap` is
installed, can be obtained with the `system.file` function.

### aSVG Image

The following commands obtain the directory of the aSVG collection and the full path 
to the chosen target aSVG image on a user's system, respectively.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
svg.dir <- system.file("extdata/shinyApp/example", package="spatialHeatmap")
svg.hum <- system.file("extdata/shinyApp/example", 'homo_sapiens.brain.svg', package="spatialHeatmap")
```

To identify feature labels of interest in annotated aSVG images, the `return_feature`
function can be used. The following searches the aSVG images stored in `dir`
for the query terms 'lobe' and 'homo sapiens' under the `feature` and `species`
fields, respectively. The identified matches are returned as a `data.frame`.   


```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df <- return_feature(feature=c('lobe'), species=c('homo sapiens'), remote=FALSE, dir=svg.dir)
feature.df
fnames <- feature.df[, 1]
```

### Numeric Data

The following example generates a small numeric toy vector, where the data slot
contains four numbers and its name slot is populated with the three feature
names obtained from the above aSVG image. In addition, a non-matching entry
(here 'notMapped') is included for demonstration purposes. Note, the numbers
are mapped to features via matching names among the numeric vector and the aSVG,
respectively. Accordingly, only numbers and features with matching name
counterparts can be colored in the aSVG image.  Entries without name matches
are indicated by a message printed to the R console, here "notMapped". This
behavior can be turned off with `verbose=FALSE` in the corresponding function
call. In addition, a summary of the numeric assay to feature mappings is stored
in the result `data.frame` returned by the `spatial_hm` function (see below). 


```{r eval=TRUE, echo=TRUE, warnings=FALSE }
my_vec <- sample(1:100, length(unique(fnames))+1)
names(my_vec) <- c(unique(fnames), 'notMapped')
my_vec
```

### Plot SHM

Next, the SHM is plotted with the `spatial_hm` function (Figure
\@ref(fig:toyshm)). Internally, the numbers in `my_vec` are translated into
colors based on the color key assigned to the `col.com` argument, and then
painted onto the corresponding features in the aSVG, where the path to the image
file is defined by `svg.path=svg.hum`. The remaining arguments used here include:
`ID` for defining the title of the plot; `ncol` for setting the column-wise layout 
of the plot excluding the feature legend plot on the right; and `height` for defining 
the height of the SHM relative to its width. In the given example
(Figure \@ref(fig:toyshm)) only three features in `my_vec` ('occipital lobe', 
'parietal lobe', and 'temporal lobe') have matching entries in the corresponding 
aSVG.   

```{r toyshm, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=("SHM of human brain with toy data. The plots from left to right represent: color key, SHM and legend. The colors in the first two plots depict the user provided numeric values, whereas in the legend plot they are used to map the feature labels to the corresponding spatial regions in the image. "), out.width="100%" }
shm.df <- spatial_hm(svg.path=svg.hum, data=my_vec, ID='toy', ncol=1, height=0.9, width=0.8, sub.title.size=20, legend.nrow=2)
```

The named numeric values in `my_vec`, that have name matches with the features in the 
chosen aSVG, are stored in the `mapped_feature` slot.

```{r eval=TRUE, echo=TRUE, warnings=FALSE} 
# The SHM and mapped features are stored in a list
names(shm.df)
# Mapped features
shm.df[['mapped_feature']]
```

## Human Brain {#hum} 

This subsection introduces how to find cell- and tissue-specific assay data in
the Expression Atlas database. After choosing a gene expression experiment, the
data is downloaded directly into a user\'s R session. Subsequently, the 
expression values for selected genes can be plotted onto a chosen aSVG image with 
or without prior preprocessing steps (_e.g._ normalization). For querying and 
downloading expression data from the Expression Atlas database, functions from 
the `ExpressionAtlas` package are used [@ebi]. 

### Gene Expression Data

The following example searches the Expression Atlas for expression data derived from
specific tissues and species of interest, here _'cerebellum'_ and _'Homo sapiens'_,
respectively.

```{r eval=TRUE, echo=TRUE, message=FALSE, warnings=FALSE }
all.hum <- searchAtlasExperiments(properties="cerebellum", species="Homo sapiens")
```

The search result is stored in a `DFrame` containing `r nrow(all.hum)`
accessions matching the above query. For the following sample code, the
accession
'[E-GEOD-67196](https://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-67196/){target="_blank"}'
from Prudencio _et al._ [-@Prudencio2015-wd] has been chosen, which corresponds
to an RNA-Seq profiling experiment of _'cerebellum'_ and _'frontal cortex'_ brain
tissue from patients with amyotrophic lateral sclerosis (ALS). Details about the
corresponding record can be returned as follows.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
all.hum[2, ]
```

The `getAtlasData` function allows to download the chosen RNA-Seq experiment
from the Expression Atlas and import it into a `RangedSummarizedExperiment`
object of a user\'s R session.   


```{r eval=TRUE, echo=TRUE, warnings=FALSE }
rse.hum <- getAtlasData('E-GEOD-67196')[[1]][[1]]
```

The design of the downloaded RNA-Seq experiment is described in the `colData` slot of 
`rse.hum`. The following returns only its first five rows and columns.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
colData(rse.hum)[1:5, 1:5]
```

### aSVG Image  

The following example shows how to download from the above described [SVG
repositories](#data_repo) an aSVG image that matches the tissues and species
assayed in the gene expression data set downloaded in the previous subsection.
The `return_feature` function queries the repository for feature- and
species-related keywords, here `c('frontal cortex', 'cerebellum')` and `c('homo
sapiens', 'brain')`, respectively. To return matching aSVGs, the argument
`keywords.any` is set to `TRUE` by default. When `return.all=FALSE`, only aSVGs
matching the query keywords are returned and saved under `dir`. Otherwise, all
aSVGs are returned regardless of the keywords. To avoid overwriting of existing
SVG files, it is recommended to start with an empty target directory, here
`tmp.dir`. To search a local directory for matching aSVG images, the argument
setting `remote=FALSE` needs to be used, while specifying the path of the
corresponding directory under `dir`. All or only matching features are returned
if `match.only` is set to `FALSE` or `TRUE`, respectively.


<!--
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```{r eval=FALSE, echo=TRUE, warnings=FALSE }
tmp.dir <- paste0(normalizePath(tempdir(check=TRUE), winslash="/", mustWork=FALSE), '/shm')  # Create empty directory
feature.df <- return_feature(feature=c('frontal cortex', 'cerebellum'), species=c('homo sapiens', 'brain'), keywords.any=TRUE, return.all=FALSE, dir=tmp.dir, remote=TRUE, match.only=TRUE, desc=FALSE) # Query aSVGs
feature.df[1:8, ] # Return first 8 rows for checking
unique(feature.df$SVG) # Return all matching aSVGs
```

To build this vignettes according to the R/Bioconductor package requirements, the
following code section uses the aSVG file instance included in the
`spatialHeatmap` package rather than the downloaded instance from the previous
example. 


```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df <- return_feature(feature=c('frontal cortex', 'cerebellum'), species=c('homo sapiens', 'brain'), keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE)
```

Note, the target tissues `frontal cortex` and `cerebellum` are included in both
the experimental design slot of the downloaded expression data as well as the 
annotations of the aSVG. This way these features can be colored in the downstream
SHM plots. If necessary users can also change from within R the feature identifiers 
and names in an aSVG. Details on this utility are provided in the [Supplementary Section](#update).  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df
```

Since the Expression Atlas supports the [cross-species anatomy
ontology](http://uberon.github.io/){target="_blank"}, the corresponding UBERON identifiers are
included in the `id` column of the `data.frame` returned by the above function
call of `return_feature` [@Mungall2012-ma]. This ontology is also supported
by the `rols` Bioconductor package [@rols].


### Experimental Design

For organizing experimental designs and downstream plotting purposes, it can be
desirable to customize the text in certain columns of `colData`. This way one can
use the source data for displaying 'pretty' sample names in columns and legends
of all downstream tables and plots, respectively, in a consistent and automated
manner. To achieve this, the following example imports a 'targets' file that
can be generated and edited by the user in a text or spreadsheet program. In
the following example the target file content is used to replace the text in the
`colData` slot of the `RangedSummarizedExperiment` object with a version containing
shorter sample names that are more suitable for plotting purposes. 


The following imports a custom target file containing simplified sample labels 
and experimental design information.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
hum.tar <- system.file('extdata/shinyApp/example/target_human.txt', package='spatialHeatmap')
target.hum <- read.table(hum.tar, header=TRUE, row.names=1, sep='\t')
```

Load custom target data into `colData` slot.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
colData(rse.hum) <- DataFrame(target.hum)
```

A slice of the simplified `colData` object is shown below, where the `disease`
column contains now shorter labels than in the original data set. Additional
details for generating and using target files in `spatialHeatmap` are provided
in the [Supplementary Section](#data_form) of this vignette.  


```{r eval=TRUE, echo=TRUE, warnings=FALSE}
colData(rse.hum)[c(1:3, 41:42), 4:5]
```

### Preprocess Assay Data

The actual gene expression data of the downloaded RNA-Seq experiment is stored
in the `assay` slot of `rse.hum`. Since it contains raw count data, it can be
desirable to apply basic preprocessing routines prior to plotting spatial
heatmaps. The following shows how to normalize the count data, aggregate
replicates and then remove genes with unreliable expression responses.  These
preprocessing steps are optional and can be skipped if needed. For this,
the expression data can be provided to the `spatial_hm` function directly, where
it is important to assign to the `sam.factor` and `con.factor` arguments 
the corresponding sample and condition column names (Table \@ref(tab:arg)).  

For normalizing raw count data from RNA-Seq experiments, the `norm_data`
function can be used. It supports the following pre-existing functions from
widely used packages for analyzing count data in the next generation sequencing
(NGS) field: `calcNormFactors` (CNF) from `edgeR` [@edgeR1]; as well as
`estimateSizeFactors` (ESF),  `varianceStabilizingTransformation` (VST), and
`rlog` from DESeq2 [@DESeq2].  The argument `norm.fun` specifies one of the
four internal normalizing methods: `CNF`, `ESF`, `VST`, and `rlog`. If
`norm.fun='none'`, no normalization is applied.  The arguments for each
normalizing function are provided via a `parameter.list`, which is a `list`
with named slots. For example, `norm.fun='ESF'` and
`parameter.list=list(type='ratio')` is equivalent to
`estimateSizeFactors(object, type='ratio')`.  If `paramter.list=NULL`, the
default arguments are used by the normalizing function assigned to `norm.fun`.
For additional details, users want to consult the help file of the `norm_data`
function by typing `?norm_data` in the R console. 

The following example uses the `ESF` normalization option. This method has been 
chosen mainly due to its good time performance.   

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se.nor.hum <- norm_data(data=rse.hum, norm.fun='ESF', data.trans='log2')
```

Replicates are aggregated with the `aggr_rep` function, where the summary
statistics can be chosen under the `aggr` argument (_e.g._ `aggr='mean'`). The
columns specifying replicates can be assigned to the `sam.factor` and
`con.factor` arguments corresponding to samples and conditions, respectively.
For tracking, the corresponding sample/condition labels are used as column
titles in the aggregated `assay` instance, where they are concatenated with a
double underscore as separator. In addition, the corresponding rows in the
`colData` slot are collapsed accordingly.


```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se.aggr.hum <- aggr_rep(data=se.nor.hum, sam.factor='organism_part', con.factor='disease', aggr='mean')
assay(se.aggr.hum)[1:3, ]
```

To remove unreliable expression measures, filtering can be applied.
The following example retains genes with expression values
larger than 5 (log2 space) in at least 1% of all samples (`pOA=c(0.01, 5)`), and
a coefficient of variance (CV) between 0.30 and 100 (`CV=c(0.30, 100)`).  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se.fil.hum <- filter_data(data=se.aggr.hum, sam.factor='organism_part', con.factor='disease', pOA=c(0.01, 5), CV=c(0.3, 100), dir=NULL)
```

To inspect the results, the following returns three selected rows of the fully
preprocessed data matrix (Table \@ref(tab:humtab)). 

```{r eval=FALSE, echo=TRUE, warnings=FALSE }
assay(se.fil.hum)[c(5, 733:734), ]
```

```{r humtab, eval=TRUE, echo=FALSE, warnings=FALSE}
kable(assay(se.fil.hum)[c(5, 733:734), ], caption='Slice of fully preprocessed expression matrix.')
```

### SHM: Single Gene

The preprocessed expression values for any gene in the `assay` slot of
`se.fil.hum` can be plotted as an SHM. The following uses gene
`ENSG00000268433` as an example. The chosen aSVG is a depiction of the human
brain where the assayed featured are colored by the corresponding expression
values in `se.fil.hum`.  

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-->
<a name='hum_single'></a>
```{r humshm, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=("SHM of human brain. Only cerebellum and frontal cortex are colored, because they are present in both the aSVG and the expression data. The legend plot on the right maps the feature labels to the corresponding spatial regions in the image."), out.width="100%", fig.show='hide' }
shm.df <- spatial_hm(svg.path=svg.hum, data=se.fil.hum, ID=c('ENSG00000268433'), height=0.7, legend.r=1.5, legend.key.size=0.02, legend.text.size=12, legend.nrow=2)
```

The plotting instructions of the SHM along with the corresponding 
mapped features are stored as a `list`, here named `shm.df`. Its components
can be accessed as follows.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
names(shm.df)
# Mapped features
shm.df[['mapped_feature']]
```

In the above example, the normalized expression values of gene `ENSG00000268433` 
are colored in the frontal cortex and cerebellum, where the different conditions, 
here normal and ALS, are given in separate SHMs plotted next to 
each other. The color and feature mappings are defined
by the corresponding color key and legend plot on the left and right, respectively.

### SHM: Multiple Genes

SHMs for multiple genes can be plotted by providing the
corresponding gene IDs under the `ID` argument as a character vector. The
`spatial_hm` function will then sequentially arrange the SHMs for
each gene in a single composite plot. To facilitate comparisons among expression
values across genes and/or conditions, the `lay.shm` parameter can be assigned
`'gene'` or `'con'`, respectively. For instance, in Figure \@ref(fig:mul) the
SHMs of the genes `ENSG00000268433` and `ENSG00000006047` are organized
by condition in a horizontal view. This functionality is particularly useful when
comparing gene families. Users can also customize the order of the SHM subplots, by
assigning `lay.shm='none'`. With this setting the SHM subplots are organized according 
to the gene and condition ordering under `ID` and `data`, respectively.  </font> 

```{r mul, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=("SHMs of two genes. The subplots are organized by \"condition\" with the `lay.shm='con'` setting."), out.width="100%" }
spatial_hm(svg.path=svg.hum, data=se.fil.hum, ID=c('ENSG00000268433', 'ENSG00000006047'), lay.shm='con', width=0.8, height=1, legend.r=1.5, legend.nrow=2)
```

### SHM: HTML and Video

SHMs can be saved to interactive HTML files as well as video files. To trigger
this export behavior, the argument `out.dir` needs to be assinged a directory
path where the HTML and video files will be stored. Each HTML file
contains an interactive SHM with zoom in and out functionality. Hovering over
graphics features will display data, gene, condition and other information. The
video will play the SHM subplots in the order specified under the `lay.shm`
argument. 

The following example saves the interactive HTML and video files under  
a directory named `tmp.dir`.  

<!--
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-->
```{r eval=FALSE, echo=TRUE, warnings=FALSE }
tmp.dir <- paste0(normalizePath(tempdir(check=TRUE), winslash="/"), '/shm')
spatial_hm(svg.path=svg.hum, data=se.fil.hum, ID=c('ENSG00000268433', 'ENSG00000006047'), lay.shm='con', width=0.8, height=1, legend.r=1.5, legend.nrow=2, out.dir=tmp.dir)
```

### SHM: Customization

To provide a high level of flexibility, the `spatial_hm` contains many arguments.
An overview of important arguments and their utility is provided in Table \@ref(tab:arg).

```{r arg, eval=TRUE, echo=FALSE, warnings=FALSE}
arg.df <- read.table('file/spatial_hm_arg.txt', header=TRUE, row.names=1, sep='\t')
kable((arg.df), escape=TRUE, caption="List of important argumnets of \'spatial_hm\'.")
```

## Mouse Organs {#mus}

This section generates an SHM plot for mouse data from the Expression Atlas.
The code components are very similar to the previous [Human Brain](#hum)
example. For brevity, the corresponding text explaining the code has
been reduced to a minimum. 

### Gene Expression Data

The chosen mouse RNA-Seq data compares tissue level gene expression across 
mammalian species [@Merkin2012-ak]. The following searches the Expression 
Atlas for expression data from _'heart'_ and _'Mus musculus'_.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
all.mus <- searchAtlasExperiments(properties="heart", species="Mus musculus")
```

Among the many matching entries, accession 'E-MTAB-2801' will be downloaded.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
all.mus[7, ]
rse.mus <- getAtlasData('E-MTAB-2801')[[1]][[1]]
```

The design of the downloaded RNA-Seq experiment is described in the `colData` slot of 
`rse.mus`. The following returns only its first three rows.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
colData(rse.mus)[1:3, ]
```

### aSVG Image  

The following example shows how to download from the above described [SVG
repositories](#data_repo) an aSVG image that matches the tissues and species
assayed in the gene expression data set downloaded in the previous subsection.
As before the image is saved to a directory named `tmp.dir`.

<!--
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```{r eval=FALSE, echo=TRUE, warnings=FALSE }
tmp.dir <- paste0(normalizePath(tempdir(check=TRUE), winslash="/", mustWork=FALSE), '/shm')
feature.df <- return_feature(feature=c('heart', 'kidney'), species=c('Mus musculus'), keywords.any=TRUE, return.all=FALSE, dir=tmp.dir, remote=TRUE, match.only=FALSE)
```

To build this vignettes according to the R/Bioconductor package requirements, the
following code section uses the aSVG file instance included in the
`spatialHeatmap` package rather than the downloaded instance from the example in
the previous step. 

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df <- return_feature(feature=c('heart', 'kidney'), species=NULL, keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE, match.only=FALSE) 
```

Return the names of the matching aSVG files.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
unique(feature.df$SVG)
```

The following first selects `mus_musculus.male.svg` as target aSVG, then
returns the first three rows of the resulting `feature.df`, and finally prints
the unique set of all aSVG features.   

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df <- subset(feature.df, SVG=='mus_musculus.male.svg')
feature.df[1:3, ]
unique(feature.df[, 1])
```

Obtain path of target aSVG on user system.   

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
svg.mus <- system.file("extdata/shinyApp/example", "mus_musculus.male.svg", package="spatialHeatmap")
```

### Experimental Design

The following imports a sample target file that is included in this package.
To inspect its content, the first three rows of the target file are printed to the 
screen.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
mus.tar <- system.file('extdata/shinyApp/example/target_mouse.txt', package='spatialHeatmap')
target.mus <- read.table(mus.tar, header=TRUE, row.names=1, sep='\t')
target.mus[1:3, ]
unique(target.mus[, 3])
```

Load custom target data into `colData` slot.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
colData(rse.mus) <- DataFrame(target.mus)
```

### Preprocess Assay Data

The raw RNA-Seq count are preprocessed with the following steps: (1)
normalization, (2) aggregation of replicates, and (3) filtering of reliable
expression data. The details of these steps are explained in the sub-section 
above using data from human.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se.nor.mus <- norm_data(data=rse.mus, norm.fun='ESF', data.trans='log2') # Normalization
se.aggr.mus <- aggr_rep(data=se.nor.mus, sam.factor='organism_part', con.factor='strain', aggr='mean') # Aggregation of replicates
se.fil.mus <- filter_data(data=se.aggr.mus, sam.factor='organism_part', con.factor='strain', pOA=c(0.01, 5), CV=c(0.6, 100), dir=NULL) # Filtering of genes with low counts and variance 
```

### SHM: Transparency

The pre-processed expression data for gene 'ENSMUSG00000000263' is plotted in form
of an SHM. In this case the plot includes expression data for 8 tissues across 3 
mouse strains. 

```{r musshm, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=("SHM of mouse organs. This is a multiple-layer image where the shapes of the 'skeletal muscle' is set transparent to expose 'lung' and 'heart'."), out.width="100%" }
spatial_hm(svg.path=svg.mus, data=se.fil.mus, ID=c('ENSMUSG00000000263'), height=0.7, legend.width=0.7, legend.text.size=10, sub.title.size=9, ncol=3, tis.trans=c('skeletal muscle'), legend.nrow=4, line.size=0.2, line.color='grey70')
```

The SHM plots in Figures \@ref(fig:musshm) and below demonstrate
the usage of the transparency feature via the `tis.trans` parameter. The
corresponding mouse organ aSVG image includes overlapping tissue layers. In
this case the skelectal muscle layer partially overlaps with lung and heart
tissues. To view lung and heart in Figure \@ref(fig:musshm), the skelectal
muscle tissue is set transparent with `tis.trans=c('skeletal muscle')`. To view
in the same aSVG the skeletal muscle tissue instead, `tis.trans` is assigned
`NULL` as shown below.  

To fine control the visual effects in feature rich aSVGs, the `line.size` and
`line.color` parameters are useful. This way one can adjust the thickness and 
color of complex structures.


<!--
This image is hidden to meet the 5MB requirement on vignette.
-->
```{r musshm1, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=("SHM of mouse organs. This is a multiple-layer image where the view onto 'lung' and 'heart' is obstructed by displaying the 'skeletal muscle' tissue."), out.width="100%", fig.show='hide' }
spatial_hm(svg.path=svg.mus, data=se.fil.mus, ID=c('ENSMUSG00000000263'), height=0.6, legend.text.size=10, sub.title.size=9, ncol=3, tis.trans=NULL, legend.ncol=2, line.size=0.2, line.color='grey70')
```

## Chicken Organs {#chk}

This section generates an SHM plot for chicken data from the Expression Atlas.
The code components are very similar to the [Human Brain](#hum) example. For
brevity, the corresponding text explaining the code has been reduced to a
minimum. 

### Gene Expression Data

The chosen chicken RNA-Seq experiment compares the developmental changes across 
nine time points of seven organs [@Cardoso-Moreira2019-yq].

The following searches the Expression Atlas for expression data from _'heart'_
and _'gallus'_.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
all.chk <- searchAtlasExperiments(properties="heart", species="gallus")
```

Among the matching entries, accession 'E-MTAB-6769' will be downloaded.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
all.chk[3, ]
rse.chk <- getAtlasData('E-MTAB-6769')[[1]][[1]]
```

The design of the downloaded RNA-Seq experiment is described in the `colData`
slot of `rse.chk`. The following returns only its first three rows.

```{r eval=TRUE, echo=TRUE, warnings=FALSE } 
colData(rse.chk)[1:3, ]
```

### aSVG Image  

The following example shows how to download from the above introduced [SVG
repositories](#data_repo) an aSVG image that matches the tissues and species
assayed in the gene expression data set downloaded in the previous subsection.
As before the image is saved to a directory named `tmp.dir`.

<!--
Code chunk not evaluated to save time building this vignette.
-->
```{r eval=FALSE, echo=TRUE, warnings=FALSE }
tmp.dir <- paste0(normalizePath(tempdir(check=TRUE), winslash="/", mustWork=FALSE), '/shm')
# Query aSVGs.
feature.df <- return_feature(feature=c('heart', 'kidney'), species=c('gallus'), keywords.any=TRUE, return.all=FALSE, dir=tmp.dir, remote=TRUE, match.only=FALSE)
```

To build this vignettes according to the R/Bioconductor package requirements, the
following code section uses the aSVG file instance included in the
`spatialHeatmap` package rather than the downloaded instance from the previous
step. 

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df <- return_feature(feature=c('heart', 'kidney'), species=c('gallus'), keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE, match.only=FALSE)
feature.df
```

Obtain path of target aSVG on user system.   

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
svg.chk <- system.file("extdata/shinyApp/example", "gallus_gallus.svg", package="spatialHeatmap")
```

### Experimental Design

The following imports a sample target file that is included in this package.
To inspect its content, the first three rows of the target file are printed to the 
screen.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
chk.tar <- system.file('extdata/shinyApp/example/target_chicken.txt', package='spatialHeatmap')
target.chk <- read.table(chk.tar, header=TRUE, row.names=1, sep='\t')
target.chk[1:3, ]
```

Load custom target data into `colData` slot.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
colData(rse.chk) <- DataFrame(target.chk)
```

Return samples used for plotting SHMs.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
unique(colData(rse.chk)[, 'organism_part'])
```

Return conditions considered for plotting downstream SHM.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
unique(colData(rse.chk)[, 'age'])
```

### Preprocess Assay Data

The raw RNA-Seq count are preprocessed with the following steps: (1)
normalization, (2) aggregation of replicates, and (3) filtering of reliable
expression data. The details of these steps are explained in the above
sub-section on human data.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se.nor.chk <- norm_data(data=rse.chk, norm.fun='ESF', data.trans='log2') # Normalization
se.aggr.chk <- aggr_rep(data=se.nor.chk, sam.factor='organism_part', con.factor='age', aggr='mean') # Replicate agggregation using mean 
se.fil.chk <- filter_data(data=se.aggr.chk, sam.factor='organism_part', con.factor='age', pOA=c(0.01, 5), CV=c(0.6, 100), dir=NULL) # Filtering of genes with low counts and varince
```

### SHM: Time Series

The expression profile for gene `ENSGALG00000006346` is plotted across nine time
points in four organs in form of a composite SHM with 9 panels. Their layout in
three columns is controlled with the argument setting `ncol=3`. 

```{r chkshm, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=("Time course of chicken organs. The SHM shows the expression profile of a single gene across nine time points and four organs."), out.width="100%" }
spatial_hm(svg.path=svg.chk, data=se.fil.chk, ID='ENSGALG00000006346', width=0.9, legend.width=0.9, legend.r=1.5, sub.title.size=9, ncol=3, legend.nrow=2, label=TRUE)
```

## Arabidopsis Shoot {#shoot}

This section generates an SHM for _Arabidopsis thaliana_ tissues with gene expression
data from the Affymetrix microarray technology. The chosen experiment used
ribosome-associated mRNAs from several cell populations of shoots and roots that were
exposed to hypoxia stress [@Mustroph2009-nu]. In this case the expression data
will be downloaded from [GEO](https://www.ncbi.nlm.nih.gov/geo/){target="_blank"} with utilites
from the `GEOquery` package [@geo]. The data preprocessing routines are
specific to the Affymetrix technology. The remaining code components for
generating SHMs are very similar to the previous examples. For brevity, the
text in this section explains mainly the steps that are specific to this data
set. 


### Gene Expression Data

The GSE14502 data set will be downloaded with the `getGEO` function from the
`GEOquery` package. Intermediately, the expression data is stored in an
`ExpressionSet` container [@biobase], and then converted to a
`SummarizedExperiment` object.

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
gset <- getGEO("GSE14502", GSEMatrix=TRUE, getGPL=TRUE)[[1]]
se.sh <- as(gset, "SummarizedExperiment")
```

The gene symbol identifiers are extracted from the `rowData` component to be used 
as row names. Similarly, one can work with AGI identifiers by providing below `AGI` 
under `Gene.Symbol`.

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
rownames(se.sh) <- make.names(rowData(se.sh)[, 'Gene.Symbol'])
```

The following returns a slice of the experimental design stored in the
`colData` slot. Both the samples and conditions are contained in the `title` column.
The samples include promoters (pGL2, pCO2, pSCR, pWOL, p35S), tissues 
and organs (root atrichoblast epidermis, root cortex meristematic zone, root 
endodermis, root vasculature, root_total and shoot_total); and the conditions 
are control and hypoxia.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
colData(se.sh)[60:63, 1:4]
```

### aSVG Image  

In this example, the aSVG image has been generated in Inkscape from 
the corresponding figure in @Mustroph2009-nu. The resulting custom figure
has been included as a sample aSVG file in the `spatialHeatmap` package. Detailed 
instructions for generating custom aSVG images in Inkscape are provided in the 
[SVG tutorial](https://jianhaizhang.github.io/SVG_tutorial_file/){target="_blank"}.   

The annotations in the corresponding aSVG file located under `svg.dir` can be
queried with the `return_features` function.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df <- return_feature(feature=c('pGL2', 'pRBCS'), species=c('shoot'), keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE, match.only=FALSE)
```

The unique set of the matching aSVG files can be returned as follows.   

```{r eval=TRUE, echo=TRUE, warnings=FALSE } 
unique(feature.df$SVG)
```

The aSVG file `arabidopsis_thaliana.shoot_shm.svg` is chosen to generate the SHM in this section.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
feature.df <- subset(feature.df, SVG=='arabidopsis_thaliana.shoot_shm.svg')
feature.df[1:3, ]
```

Obtain full path of target aSVG on user system.   

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
svg.sh <- system.file("extdata/shinyApp/example", "arabidopsis_thaliana.shoot_shm.svg", package="spatialHeatmap")
```

### Experimental Design

The following imports a sample target file that is included in this package.
To inspect its content, four selected rows of this target file are printed to the 
screen.

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
sh.tar <- system.file('extdata/shinyApp/example/target_arab.txt', package='spatialHeatmap')
target.sh <- read.table(sh.tar, header=TRUE, row.names=1, sep='\t')
target.sh[60:63, ]
```

Return all samples present in target file.     

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
unique(target.sh[, 'sample'])
```

Return all conditions present in target file.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
unique(target.sh[, 'condition'])
```

Load custom target data into `colData` slot.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
colData(se.sh) <- DataFrame(target.sh)
```

### Preprocess Assay Data

The downloaded GSE14502 data set has already been normalized with the RMA
algorithm [@affy]. Thus, the pre-processing steps can be restricted to the
aggregation of replicates and filtering of reliably expressed genes. For the
latter, the following code will retain genes with expression values larger than
6 (log2 space) in at least 3% of all samples (pOA=c(0.03, 6)), and with a
coefficient of variance (CV) between 0.30 and 100 (CV=c(0.30, 100)).   

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se.aggr.sh <- aggr_rep(data=se.sh, sam.factor='sample', con.factor='condition', aggr='mean') # Replicate agggregation using mean
se.fil.arab <- filter_data(data=se.aggr.sh, sam.factor='sample', con.factor='condition', pOA=c(0.03, 6), CV=c(0.30, 100), dir=NULL) # Filtering of genes with low intensities and variance
```

### SHM: Microarray 

The expression profile for the HRE2 gene is plotted for the control and the hypoxia treatment
across six cell types (Figure \@ref(fig:shshm)).

```{r shshm, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=('SHM of Arabidopsis shoots. The expression profile of the HRE2 gene is plotted for control and hypoxia treatment across six cell types.'), out.width="100%"}
spatial_hm(svg.path=svg.sh, data=se.fil.arab, ID=c("HRE2"), height=0.7, legend.nrow=3, legend.text.size=11)
```

### SHM: Development {#mul_svg}

The `spatial_hm` function can arrange multiple aSVGs in a single SHM plot, such
as aSVGs from different development stages. To organize the subplots, the names
of the aSVG files are expected to include the following suffixes: `*_shm1.svg`,
`*_shm2.svg`, *etc*.  The paths to the aSVG files are provided under the
`svg.path` argument.  By default, every aSVG image will have a legend plot on
the right.  The `legend` argument provides fine control over which legend plots
to display. 

As a simple toy example, the following stores random numbers in a 
`data.frame`.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
df.random <- data.frame(matrix(sample(x=1:100, size=50, replace=TRUE), nrow=10))
colnames(df.random) <- c('shoot_totalA__condition1', 'shoot_totalA__condition2', 'shoot_totalB__condition1', 'shoot_totalB__condition2', 'notMapped') # Assign column names
rownames(df.random) <- paste0('gene', 1:10) # Assign row names 
df.random[1:3, ]
```

<font color='blue'>
Next the paths to the aSVG files are obtained, here for younger and older plants using `*_shm1` 
and `*_shm1`, respectively, which are made from @Mustroph2009-nu.   
</font>

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
svg.sh1 <- system.file("extdata/shinyApp/example", "arabidopsis_thaliana.organ_shm1.svg", package="spatialHeatmap")
svg.sh2 <- system.file("extdata/shinyApp/example", "arabidopsis_thaliana.organ_shm2.svg", package="spatialHeatmap")
```

The following generates the corresponding SHMs plot for `gene1`. The orginal 
image dimensions can be preserved by assigning `TRUE` to the `preserve.scale` argument.    

```{r arabshm, eval=TRUE, echo=TRUE, warnings=FALSE, fig.wide=TRUE, fig.cap=('SHMs of Arabidopsis at two growth stages. The expression profile of gene1 under condition1 and condition2 is plotted for two growth stages (top and bottom row).'), out.width="100%"}
spatial_hm(svg.path=c(svg.sh1, svg.sh2), data=df.random, ID=c('gene1'), width=0.7, legend.r=0.9, legend.width=1, preserve.scale=TRUE) 
```

# Matrix Heatmaps {#mhm}

SHMs are suitable for comparing assay profiles among small number of items
(_e.g._ few genes or proteins) across cell types and conditions. To also
support analysis routines of larger number of items, `spatialHeatmap` integrates
functionalities for identifying groups of items with similar and/or dissimilar 
assay profiles, and subsequently analyzing the results with data mining methods 
methods that scale to larger numbers of items than SHMs, such as hierarchical
clustering and network analysis methods. The following introduces both
approaches using as sample data the gene expression data from Arabidopsis
shoots from the previous section.

To identify similar items, the `submatrix` function can be used. The latter
identifies items sharing similar profiles with one or more query items of
interest. The given example uses correlation coefficients as similarity metric.
Three filtering parameters are provided to control the similarity and number of
items in the result. First, the `p` argument allows to restrict the number of
similar items to return based on a percentage cutoff relative to the number of
items in the assay data set (_e.g._ 1% of the top most similar items). Second,
a fixed number `n` of the most similar items can be returned.  Third, all items
above a user definable correlation coefficient cutoff value can be returned,
such as a Pearson correlation coefficient (PCC) of at least 0.6. If several
query items are provided then the function returns the similar genes for each
query, while assuring uniqueness among items in the result. 

The following example uses as query the two genes: 'RCA' and 'HRE2'. The `ann`
argument corresponds to the column name in the `rowData` slot containing gene
annotation information. The latter is only relevant if users want to see these
annotations when mousing over a node in the [interactive network](#inter_net)
plot generated in the next section. 

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
sub.mat <- submatrix(data=se.fil.arab, ann='Target.Description', ID=c('RCA', 'HRE2'), p=0.1)
```

The result from the previous step is the assay matrix subsetted to the genes sharing similar assay profiles
with the query genes 'RCA' and 'HRE2'.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
sub.mat[c('RCA', 'HRE2'), c(1:3, 37)] # Subsetted assay matrix
```

<a name="mhm1"></a>
Subsequently, hierarchical clustering is applied to the subsetted assay matrix
containing only the genes that share profile similarities with the query genes
'RCA' and 'HRE2'.  The clustering result is displayed as a matrix heatmap where
the rows and columns are sorted by the corresponding hierarchical clustering
dendrograms (Figure \@ref(fig:static)). The position of the query genes ('RCA'
and 'HRE2') is indicated in the heatmap by black lines. Setting `static=FALSE`
will launch the interactive mode, where users can zoom into the heatmap by
selecting subsections in the image or zoom out by double clicking.  

```{r static, eval=TRUE, echo=TRUE, warnings=FALSE, fig.cap=("Matrix Heatmap. Rows are genes and columns are samples. The input genes are tagged by black lines."), out.width='100%'}
matrix_hm(ID=c('RCA', 'HRE2'), data=sub.mat, angleCol=80, angleRow=35, cexRow=0.8, cexCol=0.8, margin=c(10, 6), static=TRUE, arg.lis1=list(offsetRow=0.01, offsetCol=0.01))
```   

# Network Graphs {#net}
## Module Identification

<a name="ds"></a>
Network analysis is performed with the WGCNA algorithm [@Langfelder2008-sg;
@Ravasz2002-db] using as input the subsetted assay matrix generated in the 
previous section. The objective is to identify network modules that can be 
visualized in the following step in form of network graphs. Applied to the gene
expression sample data used here, these network modules represent groups of
genes sharing highly similar expression profiles. Internally, the network
module identification includes five major steps. First, a correlation matrix is
computed using distance or correlation-based similarity metrics. Second, the
obtained matrix is transformed into an adjacency matrix defining the
connections among items. Third, the adjacency matrix is used to calculate a
topological overlap matrix (TOM) where shared neighborhood information among
items is used to preserve robust connections, while removing spurious
connections. Fourth, the distance transformed TOM is used for hierarchical
clustering. To maximize time performance, the hierarchical clustering is
performed with the `flashClust` package [@Langfelder2012-nv]. Fifth, network modules
are identified with the `dynamicTreeCut` package [@dynamicTreeCut]. Its `ds`
(`deepSplit`) argument can be assigned integer values from `0` to `3`, where
higher values increase the stringency of the module identification process.  To
simplify the network module idenification process with WGCNA, the individual
steps can be executed with a single function called `adj_mod`. The result is a
list containing the adjacency matrix and the final module assignments stored in
a `data.frame`. Since the [interactive network](#inter_net) feature used in the
visualization step below performs best on smaller modules, only modules are
returned that were obtained with stringent `ds` settings (here `ds=2` and `ds=3`). 

```{r eval=TRUE, echo=TRUE, warnings=FALSE, results=FALSE}
adj.mod <- adj_mod(data=sub.mat)
```

A slice of the adjacency matrix is shown below.    

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
adj.mod[['adj']][1:3, 1:3]
```

The module assignments are stored in a `data frame`. Its columns contain the results
for the `ds=2` and `ds=3` settings. Integer values $>0$ are the module labels, while $0$ 
indicates unassigned items. The following returns the first three rows of the module 
assignment result.  

```{r eval=TRUE, echo=TRUE, warnings=FALSE}
adj.mod[['mod']][1:3, ] 
```


## Module Visualization

Network modules can be visualized with the `network` function. To plot a module
containing an item (gene) of interest, its ID (_e.g._ 'HRE2') needs to be
provided under the corresponding argument.  Typically, this could be one of the
items chosen for the above SHM plots. There are two modes to visualize the
selected module: static or interactive. Figure \@ref(fig:inter) was generated
with `static=TRUE`. Setting `static=FALSE` will generate the interactive
version. In the network plot shown below the nodes and edges represent genes
and their interactions, respectively. The thickness of the edges denotes the
adjacency levels, while the size of the nodes indicates the degree of
connectivity of each item in the network. The adjacency and connectivity levels
are also indicated by colors. For example, in Figure \@ref(fig:inter)
connectivity increases from "turquoise" to "violet", while the adjacency
increases from "yellow" to "blue".  If a gene of interest has been assigned
under `ID`, then its ID is labeled in the plot with the suffix tag: '*_target'.

```{r inter, eval=TRUE, echo=TRUE, warnings=FALSE, fig.cap=("Static network. Node size denotes gene connectivity while edge thickness stands for co-expression similarity.") }
network(ID="HRE2", data=sub.mat, adj.mod=adj.mod, adj.min=0.90, vertex.label.cex=1.2, vertex.cex=2, static=TRUE)
```   

<a name="inter_net"></a>
Setting `static=FALSE` launches the interactive network. In this mode there
is an interactive color bar that denotes the gene connectivity. To modify it,
the color lables need to be provided in a comma separated format, _e.g._: 
`yellow, orange, red`. The latter would indicate that the gene connectivity 
increases from yellow to red. 

If the subsetted expression matrix contains gene/protein annotation information
in the last column, then it will be shown when moving the cursor over a network
node.    

```{r eval=FALSE, echo=TRUE, warnings=FALSE}
network(ID="HRE2", data=sub.mat,  adj.mod=adj.mod, static=FALSE)
```

# Shiny App {#shiny}

In additon to running `spatialHeatmap` from R, the package includes a [Shiny
App](https://shiny.rstudio.com/) that provides access to the same
functionalities from an intuitive-to-use web browser interface. Apart from
being very user-friendly, this App conveniently organizes the results of the
entire visualization workflow in a single browser window with options to adjust
the parameters of the individual components interactively. For instance, genes
can be selected and replotted in the SHM simply by clicking the corresponding
rows in the expression table included in the same window. 
This representation is very efficient in guiding the interpretation of the results 
in a visual and user-friendly manner. For testing purposes, the `spatialHeatmap` 
Shiny App also includes ready-to-use sample expression data and aSVG images 
along with embedded user instructions.

## Local System 

The Shiny App of `spatialHeatmap` can be launched from an R session with the following
function call. 

```{r eval=FALSE, echo=TRUE, warnings=FALSE}
shiny_all()
```   

The dashboard panels of the Shiny App are organized as follows:

1. Left Side Panel: menu for instructions, and selecting input files and parameters
2. Data Matrix: scrollable assay matrix uploaded by users
3. Spatial Heatmap: spatially colored images based on aSVG images selected and/or uploaded by user
4. Matrix Heatmap: hierarchical clustering results of user selected items along with nearest neighbors showing most similar abundance profiles 
5. Network Graph: interactive network graph for selected module 
6. Parameter Control Menus: postioned on top of each visualization panel 


A screenshot is shown below depicting an SHM plot generated with the Shiny App of `spatialHeatmap` 
(Figure \@ref(fig:shiny)).

```{r shiny, echo=FALSE, fig.wide=TRUE, fig.cap=("Screenshot of spatialHeatmap's Shiny App."), out.width="100%"}
include_graphics('img/shiny.png')
```  

After launching, the Shiny App displays by default one of the included data sets. 

The assay data and aSVG images are uploaded to the Shiny App as tabular files
(_e.g._ in CSV or TSV format) and SVG files, respectively. To also allow users
to upload gene expression data stored in `SummarizedExperiment` objects, one
can export it from R to a tabular file with the `filter_data` function, where
the user specifies the directory path under the `dir` argument. This will create
in the target directory a tablular file named `customData.txt` in TSV format. 
The column names in this file preserve the experimental design information from the
`colData` slot by concatenating the corresponding sample and condition
information separated by double underscores. An example of this format is shown
in Table \@ref(tab:humtab). 

```{r eval=FALSE, echo=TRUE, warnings=FALSE}
se.fil.arab <- filter_data(data=se.aggr.sh, ann="Target.Description", sam.factor='sample', con.factor='condition', pOA=c(0.03, 6), CV=c(0.30, 100), dir='./')
```

To interactively access gene-, transcript- or protein-level annotations in the plots and
tables of the Shiny App, such as viewing functional descriptions by moving the
cursor over network nodes, the corresponding annotation column needs to be
present in the `rowData` slot and its column name assigned to the `ann`
argument. In the exported tabular file, the extra annotation column is appended
to the expression matrix.   


## Server Deployment

As most Shiny Apps, `spatialHeatmap` can be deployed as a centralized web
service. A major advantage of a web server deployment is that the
functionalities can be accessed remotely by anyone on the internet without the
need to use R on the user system. For deployment one can use custom web
servers or cloud services, such as AWS, GCP or
[shinysapps.io](https://www.shinyapps.io/){target="_blank"}. An example web instance for testing
`spatialHeatmap` online is available
[here](https://www.plantsecretome.org/software/spatialheatmap){target="_blank"}.

## Custom Shiny App

The `spatialHeatmap` package also allows users to create customized Shiny App
instances using the `custom_shiny` function. This function provides options to include
custom example data and aSVGs, and define default values within most visualization
panels (*e.g.* color schemes, image dimensions). For details users want
to consult the help file of the `custom_shiny` function. To maximize
flexibilty, the default parameters are stored in a yaml file on the Shiny App.
This makes it easy to refine and optimize default parameters simply by changing
this yaml file.

# Supplementary Section {#sup} 

To generate SHMs with custom data, proper formatting of the numeric (assay)
data and aSVG images is required. A tabular target file describing the
numeric data can also be provided but is optional. This section provides
additional details on these three input types.

## Numeric Data {#data_form}

The numceric data used to color the features in aSVG images can be provided as
three different object types including `vector`, `data.frame` and
`SummerizedExperiment`. When working with complex omics-based assay data then
the latter provides the most flexibility, and thus should be the preferred
container class for managing numeric data in `spatialHeatmap`. Both
`data.frame` and `SummarizedExperiment` can hold data from many measured items,
such as many genes or proteins. In contrast to this, the
`vector` class is only suitable for data from single items. Due to its
simplicity this less complex container is often useful for testing or when
dealing with simple data sets.

### Object Types

#### `vector`

When using numeric vectors as input to `spatial_hm`, then their name slot needs
to be populated with strings matching the feature names in the corresponding aSVG. 
To also specify conditions, their labels need to be appended to the feature names
with double underscores as separator, _i.e._ 'feature__condition'. 

The following example replots the [toy example](#toy) for two spatial features 
('occipital lobe' and 'parietal lobe') and two conditions ('1' and '2'). 

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
vec <- sample(x=1:100, size=5) # Random numeric values
names(vec) <- c('occipital lobe__condition1', 'occipital lobe__condition2', 'parietal lobe__condition1', 'parietal lobe__condition2', 'notMapped') # Assign unique names to random values
vec
```

With this configuration the resulting plot contains two spatial heatmap plots
for the human brain, one for 'condition 1' and another one for 'contition 2'.
To keep the build time and storage size of this package to a minimum, the 
`spatial_hm` function call in the code block below is not evaluated. Thus,
the corresponding SHM is not shown in this vignette. 

```{r vecshm, eval=FALSE, echo=TRUE, warnings=FALSE, fig.wide=FALSE, fig.cap=c('SHMs on a vector. \'occipital lobe\' and \'parietal lobe\' are 2 aSVG features and \'condition1\' and \'condition2\' are conditions.')}
spatial_hm(svg.path=svg.hum, data=vec, ID='toy', ncol=1, legend.r=1.2, sub.title.size=14)
```

#### `data.frame`  

Compared to the above vector input, `data.frames` are structured here like row-wise 
appended vectors, where the name slot information in the vectors is stored in the
column names. Each row also contains a name that corresponds to the corresponding
item name, such as a gene ID. The naming of spatial features and conditions in the 
column names follows the same conventions as the naming used for the name slots in 
the above vector example. 

The following illustrates this with an example where a numeric `data.frame` with
random numbers is generated containing 20 rows and 5 columns. To avoid name clashes, 
the values in the rows and columns should be unique. 

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
df.test <- data.frame(matrix(sample(x=1:1000, size=100), nrow=20)) # Create numeric data.frame
colnames(df.test) <- names(vec) # Assign column names
rownames(df.test) <- paste0('gene', 1:20) # Assign row names
df.test[1:3, ]
```

With the resulting `data.frame` one can generate the same SHM as in the previous
example, that used a named vector as input to the `spatial_hm` function. Additionally,
one can now select each row by its name (here gene ID) under the `ID` argument.

```{r dfshm, eval=FALSE, echo=TRUE, warnings=FALSE, fig.wide=FALSE, fig.cap=c('SHMs on a data frame. \'occipital lobe\' and \'parietal lobe\' are 2 aSVG features and \'condition1\' and \'condition2\' are conditions.')}
spatial_hm(svg.path=svg.hum, data=df.test, ID=c('gene1'), ncol=1, legend.r=1.2, sub.title.size=14)
```

Additional information can be appended to the `data.frame` column-wise, such as
annotation data including gene descriptions. This information can then be displayed 
interactively in the network plots of the Shiny App by placing the curser over 
network nodes.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
df.test$ann <- paste0('ann', 1:20)
df.test[1:3, ]
```


#### `SummarizedExperiment` 

The `SummarizedExperiment` class is a much more extensible and flexible container 
for providing metadata for both rows and columns of numeric data stored in tabular 
format. 

To import experimental design information from tabular files, users can provide
a target file that will be stored in the `colData` slot of the
`SummarizedExperiment` (SE, @SummarizedExperiment) object. In other words, the
target file provides the metadata for the columns of the numeric assay data. Usually,
the target file contains at least two columns: one for the features/samples and
one for the conditions. Replicates are indicated by identical entries in these
columns. The actual numeric matrix representing the assay data is stored in
the `assay` slot, where the rows correspond to items, such as gene IDs.
Optionally, additional annotation information for the rows (_e.g._ gene
descriptions) can be stored in the `rowData` slot. 

For constructing a valid `SummarizedExperiment` object, that can be used by 
the `spatial_hm` function, the target file should meet the following requirements.  

1. It should be imported with `read.table` or `read.delim` into a `data.frame`
   or the `data.frame` can be constructed in R on the fly (as shown below).

2. It should contain at least two columns. One column represents the features/samples 
   and the other one the conditions. The rows in the target file 
   correspond to the columns of the numeric data stored in the `assay` slot. If
   the condition column is empty, then the same condition is assumed under the
   corresponding features/samples entry.
 
3. The feature/sample names must have matching entries in to corresponding aSVG
   to be considered in the final SHM. Note, the double underscore is a special
   string that is reserved for specific purposes in *spatialHeatmap*, and thus
   should be avoided for naming feature/samples and conditions. 

The following example illustrates the design of a valid `SummarizedExperiment`
object for generating SHMs. In this example, the 'occipital lobe' tissue has 2
conditions and each condition has 2 replicates. Thus, there are 4 assays for
`occipital lobe`, and the same design applies to the `parietal lobe` tissue. 

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
sample <- c(rep('occipital lobe', 4), rep('parietal lobe', 4))
condition <- rep(c('condition1', 'condition1', 'condition2', 'condition2'), 2)
target.test <- data.frame(sample=sample, condition=condition, row.names=paste0('assay', 1:8))
target.test
```

The `assay` slot is populated with a `8 x 20` `data.frame` containing random
numbers. Each column corresponds to an assay in the target file (here imported 
into `colData`), while each row corresponds to a gene. 

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
df.se <- data.frame(matrix(sample(x=1:1000, size=160), nrow=20))
rownames(df.se) <- paste0('gene', 1:20)
colnames(df.se) <- row.names(target.test)
df.se[1:3, ]
```

Next, the final `SummarizedExperiment` object is constructed by providing the
numeric and target data under the `assays` and `colData` arguments,
respectively.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se <- SummarizedExperiment(assays=df.se, colData=target.test)
se
```

If needed row-wise annotation information (_e.g._ for genes) can be included in
the `SummarizedExperiment` object as well. This can be done during the
construction of the initial object, or as below by injecting the information
into an existing `SummarizedExperiment` object.

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
rowData(se) <- df.test['ann']
```

In this simple example, possible normalization and filtering steps are skipped.
Yet, the aggregation of replicates is performed as shown below. 

```{r eval=TRUE, echo=TRUE, warnings=FALSE }
se.aggr <- aggr_rep(data=se, sam.factor='sample', con.factor='condition', aggr='mean')
assay(se.aggr)[1:3, ]
```

With the fully configured `SummarizedExperiment` object, a similar SHM is plotted as 
in the previous examples. 

```{r seshm, eval=FALSE, echo=TRUE, warnings=FALSE, fig.wide=FALSE, fig.cap=c('SHMs on a SummarizedExperiment. \'occipital lobe\' and \'parietal lobe\' are 2 aSVG features and \'condition1\' and \'condition2\' are conditions.')}
spatial_hm(svg.path=svg.hum, data=se.aggr, ID=c('gene1'), ncol=1, legend.r=1.2, sub.title.size=14)
```

## aSVG File

### aSVG repository {#svg_repo}

A public aSVG repository, that can be used by `spatialHeatmap` directly, is the one
maintained by the [EBI Gene Expression
Group](https://github.com/ebi-gene-expression-group/anatomogram/tree/master/src/svg).
It contains annatomical aSVG images from different species. The same aSVG
images are also used by the web service of the Expression Atlas project. In addition, the
`spatialHeatmap` has its own repository called [spatialHeatmap aSVG
Repository](https://github.com/jianhaizhang/spatialHeatmap_aSVG_Repository),
that stores custom aSVG files developed for this project (*e.g.* Figure
\@ref(fig:shshm)).  

If users cannot find a suitable aSVG image in these two repositories, we have developed 
a step-by-step [SVG tutorial](https://jianhaizhang.github.io/SVG_tutorial_file/) for creating
custom aSVG images. For example, the [BAR eFP browser](http://bar.utoronto.ca/) at University 
of Toronto contains many anatomical images. These images can be used as templates in the SVG 
tutorial to construct custom aSVGs.  

Moreover, in the future we will add more aSVGs to our repository, and users are welcome to
deposit their own aSVGs to this repository to share them with the community.   


### Update aSVG features {#update}

To create and edit existing feature identifiers in aSVGs, the `update_feature` function
can be used. The demonstration below, creates an empty folder `tmp.dir1` and copies
into it the `homo_sapiens.brain.svg` image provided by the `spatialHeatmap`
package. Subsequently, selected feature annotations are updated in this file. 

<!--
Code chunk not evaluated to save time building this vignette.
-->
```{r eval=FALSE, echo=TRUE, warnings=FALSE }
tmp.dir1 <- paste0(normalizePath(tempdir(check=TRUE), winslash="/", mustWork=FALSE), '/shm1')
if (!dir.exists(tmp.dir1)) dir.create(tmp.dir1)
svg.hum <- system.file("extdata/shinyApp/example", 'homo_sapiens.brain.svg', package="spatialHeatmap") 
file.copy(from=svg.hum, to=tmp.dir1, overwrite=TRUE) # Copy "homo_sapiens.brain.svg" file into 'tmp.dir1'
```

Query the above aSVG with feature and species keywords, and return the resulting matches
in a `data.frame`.

```{r eval=FALSE, echo=TRUE, warnings=FALSE }
feature.df <- return_feature(feature=c('frontal cortex', 'prefrontal cortex'), species=c('homo sapiens', 'brain'), dir=tmp.dir1, remote=FALSE, keywords.any=FALSE)
feature.df
``` 

Subsequently, create a character vector of new feature identifiers corresponding to 
each of the returned features. In the following examples spaces in strings will be 
filled with dots. This character vector must be added to the first column of the 
feature `data.frame`. The latter is used by the `update_feature` function to look 
up new features. 


Sample code that creates new feature names and stores them in a character vector.

```{r eval=FALSE, echo=TRUE, warnings=FALSE } 
f.new <- c('prefrontal.cortex', 'frontal.cortex')
```

Next, new features are added to the first column of the feature `data.frame`.

```{r eval=FALSE, echo=TRUE, warnings=FALSE } 
feature.df.new <- cbind(featureNew=f.new, feature.df)
feature.df.new
```

Finally, the features are updated in the aSVG file(s) located in the `tmp.dir1` directory.

```{r eval=FALSE, echo=TRUE, warnings=FALSE } 
update_feature(feature=feature.df.new, dir=tmp.dir1)
```

<br/>

# Version Informaion 

```{r eval=TRUE, echo=TRUE}
sessionInfo()
```

# Funding

This project has been funded by NSF awards: [PGRP-1546879](https://www.nsf.gov/awardsearch/showAward?AWD_ID=1546879&HistoricalAwards=false){target="_blank"}, [PGRP-1810468](https://www.nsf.gov/awardsearch/showAward?AWD_ID=1810468){target="_blank"}, [PGRP-1936492](https://www.nsf.gov/awardsearch/showAward?AWD_ID=1936492&HistoricalAwards=false){target="_blank"}.

# References
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