---
title: "Get Started with DeconvoBuddies"
author: 
  - name: Louise Huuki-Myers
    affiliation:
    - &libd Lieber Institute for Brain Development, Johns Hopkins Medical Campus
    email: lahuuki@gmail.com
output: 
  BiocStyle::html_document:
    self_contained: yes
    toc: true
    toc_float: true
    toc_depth: 2
    code_folding: show
date: "`r doc_date()`"
package: "`r pkg_ver('DeconvoBuddies')`"
vignette: >
  %\VignetteIndexEntry{Get Started with DeconvoBuddies}
  %\VignetteEncoding{UTF-8}  
  %\VignetteEngine{knitr::rmarkdown}
editor_options: 
  markdown: 
    wrap: 72
---

```{r setup, include = FALSE}
knitr::opts_chunk$set(
    collapse = TRUE,
    comment = "#>",
    crop = NULL ## Related to https://stat.ethz.ch/pipermail/bioc-devel/2020-April/016656.html
)
```

```{r vignetteSetup, echo=FALSE, message=FALSE, warning = FALSE}
## Bib setup
library("RefManageR")

## Write bibliography information
bib <- c(
    R = citation(),
    AnnotationHub = citation("AnnotationHub")[1],
    BiocFileCache = citation("BiocFileCache")[1],
    dplyr = citation("dplyr")[1],
    ExperimentHub = citation("ExperimentHub")[1],
    ggplot2 = citation("ggplot2")[1],
    graphics = citation("graphics")[1],
    grDevices = citation("grDevices")[1],
    matrixStats = citation("matrixStats")[1],
    methods = citation("methods")[1],
    purrr = citation("purrr")[1],
    rafalib = citation("rafalib")[1],
    RColorBrewer = citation("RColorBrewer")[1],
    reshape2 = citation("reshape2")[1],
    S4Vectors = citation("S4Vectors")[1],
    scran = citation("scran")[1],
    SingleCellExperiment = citation("SingleCellExperiment")[1],
    spatialLIBD = citation("spatialLIBD")[1],
    stats = citation("stats")[1],
    stringr = citation("stringr")[1],
    SummarizedExperiment = citation("SummarizedExperiment")[1],
    tibble = citation("tibble")[1],
    utils = citation("utils")[1],
    Biobase = citation("Biobase")[1],
    BiocStyle = citation("BiocStyle")[1],
    BisqueRNA = citation("BisqueRNA")[1],
    covr = citation("covr")[1],
    HDF5Array = citation("HDF5Array")[1],
    knitr = citation("knitr")[1],
    RefManageR = citation("RefManageR")[1],
    rmarkdown = citation("rmarkdown")[1],
    sessioninfo = citation("sessioninfo")[1],
    testthat = citation("testthat")[1],
    tidyr = citation("tidyr")[1],
    tidyverse = citation("tidyverse")[1],
    DeconvoBuddies = citation("DeconvoBuddies")[1],
    DeconvoBuddiespaper = citation("DeconvoBuddies")[2]
)
```

# Introduction

`DeconvoBuddies` is an R package developed to assist in running bulk RNA-seq 
deconvolution. This package provides functions to access a data set designed to 
evaluate deconvolution method performance, find marker genes, and create plots
useful in deconvolution. 

This package is associated with "Benchmark of cellular deconvolution methods 
using a multi-assay reference dataset from postmortem human prefrontal cortex" 
from Huuki-Myers et al.
(10.1101/2024.02.09.579665v2)[https://www.biorxiv.org/content/10.1101/2024.02.09.579665v2].


# Basics

## Install `DeconvoBuddies`

`R` is an open-source statistical environment which can be easily
modified to enhance its functionality via packages.
`r Biocpkg("DeconvoBuddies")` is a `R` package available via the
[Bioconductor](http://bioconductor.org) repository for packages. `R` can
be installed on any operating system from
[CRAN](https://cran.r-project.org/) after which you can install
`r Biocpkg("DeconvoBuddies")` by using the following commands in your
`R` session:

```{r "install", eval = FALSE}
if (!requireNamespace("BiocManager", quietly = TRUE)) {
    install.packages("BiocManager")
}

BiocManager::install("DeconvoBuddies")

## Check that you have a valid Bioconductor installation
BiocManager::valid()
```

## Required knowledge

`r Biocpkg("DeconvoBuddies")` is based on many other packages and in
particular in those that have implemented the infrastructure needed for
dealing with snRNA-seq data. That is, packages like
`r Biocpkg("SingleCellExperiment")`.

If you are asking yourself the question "Where do I start using
Bioconductor?" you might be interested in [this blog
post](http://lcolladotor.github.io/2014/10/16/startbioc/#.VkOKbq6rRuU).

## Asking for help

As package developers, we try to explain clearly how to use our packages
and in which order to use the functions. But `R` and `Bioconductor` have
a steep learning curve so it is critical to learn where to ask for help.
The blog post quoted above mentions some but we would like to highlight
the [Bioconductor support site](https://support.bioconductor.org/) as
the main resource for getting help: remember to use the `DeconvoBuddies`
tag and check [the older
posts](https://support.bioconductor.org/tag/DeconvoBuddies/). Other
alternatives are available such as creating GitHub issues and tweeting.
However, please note that if you want to receive help you should adhere
to the [posting
guidelines](http://www.bioconductor.org/help/support/posting-guide/). It
is particularly critical that you provide a small reproducible example
and your session information so package developers can track down the
source of the error.

## Citing `DeconvoBuddies`

We hope that `r Biocpkg("DeconvoBuddies")` will be useful for your
research. Please use the following information to cite the package and
the overall approach. Thank you!

```{r "citation"}
## Citation info
citation("DeconvoBuddies")
```

# Quick start to using `DeconvoBuddies`

Let's load some packages we'll use in this vignette.

```{r "load packages", message=FALSE, warning=FALSE}
suppressMessages({
    library("DeconvoBuddies")
    library("SummarizedExperiment")
    library("dplyr")
    library("tidyr")
    library("tibble")
})
```

## Access Data

Use `fetch_deconvo_data` to download RNA sequencing data from the [Human
DLPFC](https://github.com/LieberInstitute/Human_DLPFC_Deconvolution) `r Citep(bib[["DeconvoBuddiespaper"]])`.

-   `rse_gene`: 110 samples of bulk RNA-seq. [110 bulk RNA-seq samples x
    21k genes] (41 MB).

-   `sce` : snRNA-seq data from the Human DLPFC. [77k nuclei x 36k
    genes] (172 MB)

-   `sce_DLPFC_example`: Sub-set of `sce` useful for testing. [10k
    nuclei x 557 genes] (49 MB)

```{r `access data}
## Access and snRNA-seq example data
if (!exists("sce_DLPFC_example")) sce_DLPFC_example <- fetch_deconvo_data("sce_DLPFC_example")

## Explore snRNA-seq data in sce_DLPFC_example
sce_DLPFC_example

## Access Bulk RNA-seq data
if (!exists("rse_gene")) rse_gene <- fetch_deconvo_data("rse_gene")

## Explore bulk data in rse_gene
rse_gene
```

For more details on this dataset, and an example deconvolution run check
out the [Vignette: Deconvolution Benchmark in Human
DLPFC](https://research.libd.org/DeconvoBuddies/articles/Deconvolution_Benchmark_DLPFC.html).

## Marker Finding

### Using *MeanRatio* to Find Cell Type Markers

Accurate deconvolution requires highly specific marker genes for each
cell type to be defined. To select genes specific for each cell type,
you can evaluate the *MeanRatio* for each gene x each cell type, where
`MeanRatio = mean(Expression  of target cell type) / mean(Expression of highest non-target cell type)`.


These values can be calculated for a single cell RNA-seq dataset using
`get_mean_ratio()`. This can also work for spatially-resolved transcriptomics
datasets. That is, `get_mean_ratio()` can also work with
`SpatialExperiment::SpatialExperiment()` objects.

```{r `get_mean_ratio demo`}
## find marker genes with get_mean_ratio
marker_stats <- get_mean_ratio(
    sce_DLPFC_example,
    cellType_col = "cellType_broad_hc",
    gene_name = "gene_name",
    gene_ensembl = "gene_id"
)

## explore tibble output, gene with high MeanRatio values are good marker genes
marker_stats
```

For more discussion of finding marker genes with `DeconvoBuddies` check
out the [Vignette: Finding Marker Genes with
DeconvoBuddies.](https://research.libd.org/DeconvoBuddies/articles/Marker_Finding.html)

## Plotting Tools

### Creating A Cell Type Color palette

As you work with single-cell data and deconvolution outputs, it is very
useful to establish a consistent color palette to use across different
plots. The function `create_cell_colors()` returns a named vector of hex
values, corresponding to the names of cell types. This list is
compatible with functions like `ggplot2::scale_color_manual()`.

There are three palettes to choose from to generate colors or users can provide 
their own color palette:

-   "classic" (default): classic set of 8 cell type colors from LIBD, checked 
for visability and color blind accessibility.

-   "gg": Equi-distant hues, same process for selecting colors as
    `ggplot` - no maximum number

-   "tableau":
    [tableau20](https://jrnold.github.io/ggthemes/reference/tableau_color_pal.html)
    color set - max 20 colors

```{r `create_cell_colors demo 1`}
test_cell_types <- c("cell_A", "cell_B", "cell_C", "cell_D", "cell_E")

## Preview "classic" colors
test_cell_colors_classic <- create_cell_colors(
    cell_types = test_cell_types,
    palette_name = "classic",
    preview = TRUE
)

## Preview "gg" colors
test_cell_colors_gg <- create_cell_colors(
    cell_types = test_cell_types,
    palette_name = "gg",
    preview = TRUE
)

## Preview "tableau" colors
test_cell_colors_tableau <- create_cell_colors(
    cell_types = test_cell_types,
    palette_name = "tableau",
    preview = TRUE
)

## Check the color hex codes for "tableau"
test_cell_colors_tableau

## Provide a palette from RColorBrewer
test_cell_colors_brew <- create_cell_colors(
    cell_types = test_cell_types,
    palette = RColorBrewer::brewer.pal(n = length(test_cell_types), name = "Dark2"),
    preview = TRUE
)
```

If there are sub-cell types with consistent delimiters, the `split`
argument creates a scale of related colors. This helps expand on the
maximum number of colors and makes your palette flexible when considering
different 'resolutions' of cell types. This works by ignoring any prefixes after
the `split` character. In this example below, `Excit_01` and `Excit_02` will
just be considered as `Excit` since `split = "_"`.

```{r create_cell_colors demo 2`}
my_cell_types <- levels(sce_DLPFC_example$cellType_hc)
## Ignore any suffix after the "_" character by using the "split" argument
my_cell_colors <- create_cell_colors(
    cell_types = my_cell_types,
    palette_name = "classic",
    preview = TRUE,
    split = "_"
)
```

### Plot Expression of Top Markers

The function `plot_marker_express()` helps quickly visualize expression
of top marker genes, by ordering and annotating violin plots of
expression over cell type. Here we'll plot the expression of the top 6
marker genes for Astrocytes.

```{r `plot_marker_expression demo`}
# plot expression of the top 6 Astro marker genes
plot_marker_express(
    sce = sce_DLPFC_example,
    stats = marker_stats,
    cell_type = "Astro",
    n_genes = 6,
    cellType_col = "cellType_broad_hc",
    color_pal = my_cell_colors
)
```

The violin plots of gene expression confirm the cell type specificity of
these marker genes, most of the nuclei with high expression of these six
genes are astrocytes (Astro).

### Plot Composition Bar Plot

The output of deconvolution are cell type estimates that sum to 1. A
good visulization for these predictions is a stacked bar plot. The
function `plot_composition_bar()` creates a stacked bar plot showing the
cell type proportion for each sample, or the average proportion for a
group of samples. In this example data, the `RNum` is a sample (donor)
identifier and `Dx` is a group variable for the diagnosis status of the donors.

```{r `demo plot_composition_bar`}
# load example data
data("rse_bulk_test")
data("est_prop")

# access the colData of a test rse dataset
pd <- colData(rse_bulk_test) |>
    as.data.frame()

## pivot data to long format and join with test estimated proportion data
est_prop_long <- est_prop |>
    rownames_to_column("RNum") |>
    pivot_longer(!RNum, names_to = "cell_type", values_to = "prop") |>
    left_join(pd)

## explore est_prop_long
est_prop_long

## the composition bar plot shows cell type composition for Sample
plot_composition_bar(est_prop_long,
    x_col = "RNum",
    add_text = FALSE
) +
    ggplot2::scale_fill_manual(values = test_cell_colors_classic)

## the composition bar plot shows the average cell type composition for each Dx
plot_composition_bar(est_prop_long, x_col = "Dx") +
    ggplot2::scale_fill_manual(values = test_cell_colors_classic)
```

We can see that the mean proportions of cell types A through E are very similar 
across the `Dx` groups (`Case` and `Control`). In this case, this is expected
given that we are using simulated data. Although if you look across each donor
with `RNum` we can see more variability across the simulated data.

Since you are now familiar with the basic overview of `DeconvoBuddies`, you are
now ready to dive deeper into:

* the [selection of marker genes](https://research.libd.org/DeconvoBuddies/articles/Marker_Finding.html),
* followed by the application of `DeconvoBuddies` to the [Human Brain (DLPFC) Deconvolution dataset](https://research.libd.org/DeconvoBuddies/articles/Deconvolution_Benchmark_DLPFC.html).

# Reproducibility

The `r Biocpkg("DeconvoBuddies")` package `r Citep(bib[["DeconvoBuddies"]])` was
made possible thanks to:

* R `r Citep(bib[["R"]])`
* `r Biocpkg("AnnotationHub")` `r Citep(bib[["AnnotationHub"]])`
* `r Biocpkg("BiocFileCache")` `r Citep(bib[["BiocFileCache"]])`
* `r CRANpkg("dplyr")` `r Citep(bib[["dplyr"]])`
* `r Biocpkg("ExperimentHub")` `r Citep(bib[["ExperimentHub"]])`
* `r CRANpkg("ggplot2")` `r Citep(bib[["ggplot2"]])`
* `r CRANpkg("graphics")` `r Citep(bib[["graphics"]])`
* `r CRANpkg("grDevices")` `r Citep(bib[["grDevices"]])`
* `r CRANpkg("matrixStats")` `r Citep(bib[["matrixStats"]])`
* `r CRANpkg("methods")` `r Citep(bib[["methods"]])`
* `r CRANpkg("purrr")` `r Citep(bib[["purrr"]])`
* `r CRANpkg("rafalib")` `r Citep(bib[["rafalib"]])`
* `r CRANpkg("reshape2")` `r Citep(bib[["reshape2"]])`
* `r Biocpkg("S4Vectors")` `r Citep(bib[["S4Vectors"]])`
* `r Biocpkg("scran")` `r Citep(bib[["scran"]])`
* `r Biocpkg("SingleCellExperiment")` `r Citep(bib[["SingleCellExperiment"]])`
* `r Biocpkg("spatialLIBD")` `r Citep(bib[["spatialLIBD"]])`
* `r CRANpkg("stats")` `r Citep(bib[["stats"]])`
* `r CRANpkg("stringr")` `r Citep(bib[["stringr"]])`
* `r Biocpkg("SummarizedExperiment")` `r Citep(bib[["SummarizedExperiment"]])`
* `r CRANpkg("tibble")` `r Citep(bib[["tibble"]])`
* `r CRANpkg("utils")` `r Citep(bib[["utils"]])`

This vignette was generated using `r Biocpkg("BiocStyle")`
`r Citep(bib[["BiocStyle"]])` with `r CRANpkg("knitr")`
`r Citep(bib[["knitr"]])` and `r CRANpkg("rmarkdown")`
`r Citep(bib[["rmarkdown"]])` running behind the scenes.

Citations made with `r CRANpkg("RefManageR")`
`r Citep(bib[["RefManageR"]])`.

This package was developed using `r BiocStyle::Biocpkg("biocthis")`.

`R` session information.

```{r reproduce3, echo=FALSE}
## Session info
library("sessioninfo")
options(width = 120)
session_info()
```

# Bibliography

```{r vignetteBiblio, results = "asis", echo = FALSE, warning = FALSE, message = FALSE}
## Print bibliography
PrintBibliography(bib, .opts = list(hyperlink = "to.doc", style = "html"))
```