---
title: "Introduction to scAnnotatR"
author: "Vy Nguyen"
date: "`r Sys.Date()`"
output: rmarkdown::html_vignette
vignette: >
  %\VignetteIndexEntry{1. Introduction to scAnnotatR}
  %\VignetteEngine{knitr::rmarkdown}
  \usepackage[utf8]{inputenc}
---

```{r setup, include = FALSE}
knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>"
)
options(rmarkdown.html_vignette.check_title = FALSE)
```

## Introduction

scAnnotatR is an R package for cell type prediction on single cell 
RNA-sequencing data. Currently, this package supports data in the forms 
of a Seurat object or a SingleCellExperiment object.

More information about Seurat object can be found here: 
https://satijalab.org/seurat/
More information about SingleCellExperiment object can be found here: 
https://osca.bioconductor.org/

scAnnotatR provides 2 main features:

- A set of pretrained and robust classifiers for basic 
immune cells. See the section below.
- A user-friendly and fully customizable framework to train
new classification models. These models can then be easily
saved and reused in the future. Details usage of this framework is explained
in vignettes 2 and 3.

## Installation

The `scAnnotatR` package can be directly installed from Bioconductor:

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

if (!require(scAnnotatR))
  BiocManager::install("scAnnotatR")
```

For more information, see https://bioconductor.org/install/.

## Included models

The `scAnnotatR` package comes with several pre-trained models to classify
cell types.

```{r}
# load scAnnotatR into working space
library(scAnnotatR)
```

The models are stored in the `default_models` object:

```{r}
default_models <- load_models("default")
names(default_models)
```

The `default_models` object is named a list of classifiers. Each classifier 
is an instance of the `scAnnotatR S4 class`. For example:

```{r}
default_models[['B cells']]
```

## Basic pipeline to identify cell types in a scRNA-seq dataset using scAnnotatR   

### Preparing the data

To identify cell types available in a dataset, we need to load the dataset as 
[Seurat](https://satijalab.org/seurat/) or [SingleCellExperiment](https://osca.bioconductor.org/) object.

For this vignette, we use a small sample datasets that is available as a
`Seurat` object as part of the package.

```{r}
# load the example dataset
data("tirosh_mel80_example")
tirosh_mel80_example
```

The example dataset already contains the clustering results as part of the metadata. This is **not** necessary for the
classification process.

```{r}
head(tirosh_mel80_example[[]])
```

### Cell classification

To launch cell type identification, we simply call the `classify_cells` 
function. A detailed description of all parameters can be found through
the function's help page `?classify_cells`.

Here we use only 3 classifiers for B cells, T cells and NK cells to reduce 
computational cost of this vignette. If users want to use all pretrained 
classifiers on their dataset, `cell_types = 'all'` can be used.
```{r}
seurat.obj <- classify_cells(classify_obj = tirosh_mel80_example, 
                             assay = 'RNA', layer = 'counts',
                             cell_types = c('B cells', 'NK', 'T cells'), 
                             path_to_models = 'default')
```

#### Parameters

  * The option **cell_types = 'all'** tells the function to use all available
    cell classification models.
    Alternatively, we can limit the identifiable cell types: 
    * by specifying: `cell_types = c('B cells', 'T cells')`
    * or by indicating the applicable classifier using the **classifiers** option: 
      `classifiers = c(default_models[['B cells']], default_models[['T cells']])`

  * The option **path_to_models = 'default'** is to automatically use the 
    package-integrated pretrained models (without loading the models into the
    current working space). This option can be used to load a local database instead.
    For more details see the vignettes on training your own classifiers.

## Result interpretation

The `classify_cells` function returns the input object
but with additional columns in the metadata table.

```{r}
# display the additional metadata fields
seurat.obj[[]][c(50:60), c(8:ncol(seurat.obj[[]]))]
```
New columns are:

  * **predicted_cell_type**: The predicted cell type, also containing any 
    ambiguous assignments. In these cases, the possible cell types are separated
    by a "/"

  * **most_probable_cell_type**: contains the most probably cell type ignoring any 
    ambiguous assignments.

  * columns with syntax `[celltype]_p`: probability of a cell to belong 
    to a cell type. Unknown cell types are marked as NAs.

### Result visualization

The predicted cell types can now simply be visualized using the matching
plotting functions. In this example, we use Seurat's `DimPlot` function:

```{r fig.width = 4, fig.height = 2.5}
# Visualize the cell types
Seurat::DimPlot(seurat.obj, group.by = "most_probable_cell_type")
```

With the current number of cell classifiers, we identify cells belonging to 2 cell types (B cells and T cells) and to 2 subtypes of T cells (CD4+ T cells and CD8+ T cells). The other cells (red points) are not among the cell types that can be classified by the predefined classifiers. Hence, they have an empty label.

For a certain cell type, users can also view the prediction probability. Here we show an example of B cell prediction probability:

```{r fig.width = 4, fig.height = 2.5}
# Visualize the cell types
Seurat::FeaturePlot(seurat.obj, features = "B_cells_p")
```

Cells predicted to be B cells with higher probability have darker color, while the lighter color shows lower or even zero probability of a cell to be B cells. For B cell classifier, the threshold for prediction probability is currently at 0.5, which means cells having prediction probability at 0.5 or above will be predicted as B cells. 

The automatic cell identification by scAnnotatR matches the traditional cell assignment, ie. the approach based on cell canonical marker expression. Taking a simple example, we use CD19 and CD20 (MS4A1) to identify B cells:

```{r fig.width = 7.25, fig.height = 2.5}
# Visualize the cell types
Seurat::FeaturePlot(seurat.obj, features = c("CD19", "MS4A1"), ncol = 2)
```

We see that the marker expression of B cells exactly overlaps the B cell prediction made by scAnnotatR.

## Session Info
```{r}
sessionInfo()
```
