---
title: "Example Workflow for Single-Cell Annotation with easybio"
author: "[cw](https://cying.org)"
date: "`{r} Sys.Date()`"
output: html
vignette: >
  %\VignetteEngine{litedown::vignette}
  %\VignetteIndexEntry{Example Workflow for Single-Cell Annotation with easybio}
  %\VignetteEncoding{UTF-8}
---

## Introduction

This vignette demonstrates the powerful and intuitive workflow for single-cell RNA-seq annotation provided by the `easybio` package. The process is designed to combine the speed of automated database matching with the reliability of interactive verification and manual curation.

The core workflow follows three logical steps:

1.  **Automated Annotation**: Use `matchCellMarker2()` to quickly get a list of potential cell types for each cluster based on its marker genes.
2.  **Verification & Exploration**: Interactively investigate the automated results using `check_marker()` and `plotSeuratDot()` to build confidence in the annotations. This step helps answer two critical questions:
    *   "**Why** was this annotation made?" (Which of my genes matched the database?)
    *   "Is this annotation **correct**?" (Are the canonical markers for this cell type expressed in my cluster?)
3.  **Final Curation**: Based on the evidence gathered, use `finsert()` to assign the final, high-confidence cell type labels.

You can also view the R script for this workflow by running:
`fs::file_show(system.file(package = 'easybio', 'example-single-cell.R'))`

## Setup

First, let's load the necessary libraries and the example marker data included with `easybio`. This data is derived from the 10x Genomics 3k PBMC dataset.

```{r}
litedown::reactor(warning = FALSE) # vignette setting

library(easybio)
library(Seurat)
library(data.table)

# The pbmc.markers dataset is included in easybio
head(pbmc.markers)
```

## Step 1: Automated Annotation with `matchCellMarker2`

We begin by feeding the cluster markers (from `Seurat::FindAllMarkers`) into `matchCellMarker2()`. This function compares our markers against the CellMarker2.0 database and returns a ranked list of potential cell types for each cluster.

```{r}
marker_matched <- matchCellMarker2(marker = pbmc.markers, n = 50, spc = "Human")

# Let's look at the top 2 potential cell types for each cluster
marker_matched[, head(.SD, 2), by = cluster]
```

The output table gives us `uniqueN` (the number of unique matching markers) and `N` (the total number of matches), which helps rank the potential annotations.

We can create a quick preliminary annotation by taking the top hit for each cluster.

```{r}
cl2cell_auto <- marker_matched[, head(.SD, 1), by = .(cluster)]
cl2cell_auto <- setNames(cl2cell_auto[["cell_name"]], cl2cell_auto[["cluster"]])
print("Initial automated annotation:")
cl2cell_auto
```

We can also get a global view of all possible annotations using `plotPossibleCell`.

```{r}
#| fig.width=10
plotPossibleCell(marker_matched[, head(.SD), by = .(cluster)], min.uniqueN = 2)
```

## Step 2: Verification and Exploration

This is the most critical step. Instead of blindly trusting the automated result, we use `easybio`'s tools to verify it.

### Answering "Why was this annotation made?"

To see the evidence behind an annotation, we use `check_marker()` with `cis = TRUE`. This shows us which of **our own marker genes** from our data matched the database for a given annotation.

```{r}
# Let's investigate clusters 1, 5, and 7
local_evidence <- check_marker(marker_matched, cl = c(1, 5, 7), topcellN = 2, cis = TRUE)
print(local_evidence)
```

### Answering "Is this annotation correct?"

To validate an annotation, we use `check_marker()` with `cis = FALSE` (the default). This fetches the **canonical markers** for the suggested cell type from the database. We can then check if these well-known markers are expressed in our cluster.

```{r}
canonical_markers <- check_marker(marker_matched, cl = c(1, 5, 7), topcellN = 2, cis = FALSE)
print(canonical_markers)
```

### Visual Confirmation with `plotSeuratDot`

The best way to check marker expression is visually. `plotSeuratDot` is designed to work seamlessly with `check_marker`.

The entire pipeline from annotation to visualization can be done in a single, elegant pipe:

```{r, fig.width=9, fig.height=5}
# For this example to be runnable, we need a Seurat object.
# We'll create a minimal one. In your real workflow, you would use your own srt object.
marker_genes <- unique(pbmc.markers$gene)
counts <- matrix(
  abs(rnorm(length(marker_genes) * 50, mean = 1, sd = 2)),
  nrow = length(marker_genes),
  ncol = 50
)
rownames(counts) <- marker_genes
colnames(counts) <- paste0("cell_", 1:50)
srt <- CreateSeuratObject(counts = counts)
# Assign clusters that match the pbmc.markers data
srt$seurat_clusters <- sample(0:8, 50, replace = TRUE)
Idents(srt) <- "seurat_clusters"


# Now, let's plot the evidence for clusters 1, 5, and 7
matchCellMarker2(marker = pbmc.markers, n = 50, spc = "Human") |>
  check_marker(cl = c(1, 5, 7), topcellN = 2, cis = TRUE) |>
  plotSeuratDot(srt = srt)
```

This dot plot clearly shows the expression of the genes that led to the annotations for clusters 1, 5, and 7, allowing us to confidently assess the results.

## Step 3: Final Manual Curation

After reviewing the evidence from the dot plots, we can make our final, informed decision. The `finsert` function provides a convenient way to create the final annotation vector.

```{r}
# Based on our exploration, we finalize the annotations
cl2cell_final <- finsert(
  list(
    c(3) ~ "B cell",
    c(8) ~ "Megakaryocyte",
    c(7) ~ "DC",
    c(1, 5) ~ "Monocyte",
    c(0, 2, 4) ~ "Naive CD8+ T cell",
    c(6) ~ "Natural killer cell"
  ),
  len = 9 # Ensure vector length covers all clusters (0-8)
)
print("Final curated annotation:")
cl2cell_final
```

This `cl2cell_final` vector can now be added to your Seurat object's metadata for downstream analysis and plotting.

## Using a Custom Marker Database

For specialized analyses, such as focusing on a specific tissue, working with a non-model organism, or using a proprietary list of markers, you can provide your own custom reference to `matchCellMarker2`.

The reference must be a `data.frame` (or `data.table`) with at least two columns: `cell_name` and `marker`. The easiest way to create this is from a named list.

**Step 1: Create a named list of your custom markers.**
```{r}
custom_ref_list <- list(
  "T-cell" = c("CD3D", "CD3E", "CD3G"),
  "B-cell" = c("CD79A", "MS4A1"),
  "Myeloid" = c("LYZ", "CST3", "AIF1")
)
print(custom_ref_list)
```

**Step 2: Convert the list to the required data.frame format.**
`easybio` provides the `list2dt` helper function for this.
```{r}
custom_ref_df <- list2dt(custom_ref_list, col_names = c("cell_name", "marker"))
head(custom_ref_df)
```

**Step 3: Run `matchCellMarker2` with the `ref` parameter.**
When `ref` is provided, the function ignores the `spc`, `tissueClass`, and `tissueType` parameters for matching.
```{r}
marker_custom <- matchCellMarker2(
  marker = pbmc.markers,
  n = 50,
  ref = custom_ref_df
)
# Note that the cell_name column now contains our custom cell types
marker_custom[, head(.SD, 2), by = cluster]
```

## Additional Utilities

`easybio` also provides functions for direct queries.

### `get_marker()`
Directly retrieve markers for any cell type of interest.
```{r}
get_marker(spc = "Human", cell = c("Monocyte", "Neutrophil"), number = 5, min.count = 1)
```

### `plotMarkerDistribution()`
Check the distribution of a specific marker across all cell types and tissues in the database.
```{r, fig.width=7.5, fig.height=7}
plotMarkerDistribution(mkr = "CD68")
```



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