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# Spatial Transcriptomics Nanocourse

| Audience | Computational skills required| Duration |
:----------|:----------|:----------|
| Biologists | [Introduction to R](https://hbctraining.github.io/Intro-to-R-flipped/) | 1 session in-person (~1h 45 min of trainer-led time)|


### Description
This repository contains materials for a module which is part of a Nanocourse organized by the [Single Cell Core at HMS](https://singlecellcore.hms.harvard.edu/). The nanocourse titled "Spatial Transcriptomics: Key Technologies, Experimental Considerations, and Data Analysis" introduces the fundamentals of data analysis for spatial transcriptomics, including common techniques and tools. **In this module we walk through the analysis workflow for a Visium HD experiment.**

### Learning Objectives

* Describe the steps in a Visium HD analysis workflow
* Use Seurat and associated tools to perform analysis of spatial transcriptomics data, including QC metric evaluation, normalization, clustering, and marker identification

### Dataset
The dataset is located in "Dataset for workshop" -\> "Day 1- NGS-based- VisiumHD" in the course DropBox.

### Lessons

<!-- - [Loupe Browser Demonstration](lessons/loupe_demo.qmd) -->
[Visium HD Analysis with Seurat](lessons/visium_hd.qmd)

### Installation Requirements

#### Applications
Download the most recent versions of R and RStudio for your laptop:

 - [R](http://lib.stat.cmu.edu/R/CRAN/) **(version 4.4.0 or above)**
 - [RStudio](https://www.rstudio.com/products/rstudio/download/#download)

#### Packages for R

::: callout-note
# Notes for installing packages

**Note 1: Install the packages in the order listed below.**

**Note 2: All the package names listed below are case sensitive!**

**Note 3**: **If you have a Mac**, download and install [this gfortan tool](https://mac.r-project.org/tools/gfortran-12.2-universal.pkg) before installing your packages if using **R versions 4.3.0 up to 4.4.3** or [this gfortran tool](https://mac.r-project.org/tools/gfortran-14.2-universal.pkg) if using **R versions after 4.4.3**.

**Note 4**: At any point (especially if you’ve used R/Bioconductor in the past), in the console **R may ask you if you want to update any old packages by asking Update all/some/none? [a/s/n]:**. 
If you see this, **type "a" at the prompt and hit Enter** to update any old packages. _Updating packages can sometimes take quite a bit of time to run, so please account for that before you start with these installations._  

**Note 5:** If you see a message in your console along the lines of “binary version available but the source version is later”, followed by a question, **“Do you want to install from sources the package which needs compilation? y/n”, type n for no, and hit enter**.

:::

**(1)** Install the following packages with **CRAN** using the `install.packages()` function:

```{r}
install.packages("ggplot2")
install.packages("tidyverse")
install.packages("Seurat")
install.packages("patchwork")
install.packages("qs2")
install.packages("quadprog")
install.packages("remotes")
install.packages("devtools")
install.packages("BiocManager")
```


**(2)** Install the packages listed below from **Bioconductor** using the `BiocManager::install()` function.


```{r}
library(BiocManager)

BiocManager::install("scuttle")
BiocManager::install("scater")
BiocManager::install("bluster")
BiocManager::install("Voyager")
BiocManager::install("glmGamPoi")
BiocManager::install("SpatialFeatureExperiment")
BiocManager::install("scattermore")
BiocManager::install("sfarrow")
```

**(3)** Install the packages listed below from GitHub using the given `remotes:install_github` or `devtools::install_github` command.

```{r}
#| eval: false
library(remotes)
library(devtools)
options(timeout = 600000000)

remotes::install_github("satijalab/seurat-wrappers")
remotes::install_github("prabhakarlab/Banksy@devel")
devtools::install_github("dmcable/spacexr", build_vignettes = FALSE)
```

**(4)** Finally, please check that all the packages were installed successfully by **loading them one at a time** using the `library()` function.  

```{r}
library(SpatialFeatureExperiment)
library(scuttle)
library(scater)
library(bluster)
library(Voyager)
library(ggplot2)
library(tidyverse)
library(Seurat)
library(patchwork)
library(qs2)
library(quadprog)
library(remotes)
library(devtools)
library(BiocManager)
library(glmGamPoi)
library(SeuratWrappers)
library(Banksy)
library(spacexr)
library(sfarrow)
library(scattermore)
```

**(5)** Once all packages have been loaded, run sessionInfo().  

```{r}
sessionInfo()
```

Copy the output of `sessioninfo()` and paste it in the input text box below or on [the website](https://hcbc.connect.hms.harvard.edu/Spatial_scRNAseq_nanocourse_sessionInfo_check/?showcase=0). Then click the “Check my sessionInfo()” button. It will let you know if you are missing any packages.
Take a screenshot of the message confirming that all packages have been successfully installed ( you will get a message letting you know that “no packages are missing”), and email it to us before the first session.


<p align="center">

<iframe src="https://hcbc.connect.hms.harvard.edu/Spatial_scRNAseq_nanocourse_sessionInfo_check/?showcase=0" width="800px" height="800px" data-external="1">

</iframe>

</p>