single_cell_workshop/index.Rmd at main · phipsonlab/single_cell_workshop · GitHub

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---
output: github_document
---

<!-- index.md is generated from index.Rmd. Please edit that file -->

```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = FALSE)
```

# Single-Cell RNA-seq Analysis Workshop

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## Welcome

This workshop provides a comprehensive introduction to single-cell RNA-seq analysis using R and Bioconductor. Over two sessions, you will learn the complete workflow from raw count matrices to differential expression analysis.
We analyse **single-nucleus RNA-sequencing (snRNA-seq) data from human heart tissue** across three developmental stages: foetal, young, and adult. The dataset comes from [Sim et al. (2021)](https://doi.org/10.1161/CIRCULATIONAHA.120.051921) published in *Circulation*. Workshop materials are adapted from the [original analysis workflow](https://bphipson.github.io/Human_Development_snRNAseq/).

---

## Workshop Overview

```
Raw Data → QC → Normalisation → Integration → Clustering → Annotation → DE
                                                             ↘
                         Φ-Space → Pseudotime → Co-expression networks
```

| Session | Duration | Topics |
|---------|----------|--------|
| **Session 1** | 3 hours | QC, Normalisation, Integration, Clustering, Annotation, DE |
| **Session 2** | 3 hours | Φ-Space soft annotation, pseudotime, cell-specific co-expression networks |

---

## Session 1: Single-Cell RNA-seq Fundamentals

| Module | Topic | Duration | Description |
|--------|-------|----------|-------------|
| [**Module 0**](articles/00_setup.html) | Setup | Pre-workshop | Installation and verification |
| [**Module 1**](articles/01_quality_control.html) | Quality Control | 45 min | QC metrics, cell filtering |
| [**Module 2**](articles/02_integration_clustering.html) | Integration & Clustering | 50 min | SCTransform, Harmony, UMAP, clustering |
| [**Module 3**](articles/03_cell_type_annotation.html) | Cell Type Annotation | 20 min | Marker genes, manual annotation |
| [**Module 4**](articles/04_differential_expression.html) | Differential Expression | 55 min | Pseudobulk DE, composition analysis |

---

## Session 2: Trajectory and Gene Regulation

| Module | Topic | Duration | Description |
|--------|-------|----------|-------------|
| [**Module 5**](articles/05_phispace_annotation.html) | Continuous Phenotyping with Φ-Space | 45 min | Soft cell-type + stage scores via PLS on a reference atlas |
| [**Module 6**](articles/06_pseudotime.html) | Pseudotime Trajectory Analysis | 45 min | Slingshot and DPT on PCA and Φ-Space, with clustering sensitivity on the CM lineage |
| [**Module 7**](articles/07_nnet.html) | Cell-specific Co-expression Networks | 45 min | NeighbourNet meta-networks from maturation-associated target genes |

---

## Before You Start

**Complete the setup before the workshop:**
1. Check system requirements (8GB RAM minimum, 16GB recommended)
2. Follow the [installation guide](articles/00_setup.html)
3. Download workshop data from [Zenodo](https://zenodo.org/records/18237749) (~300 MB)
4. Verify with `scWorkshop::check_installation()`

---

## Dataset

| Group | Samples | Age Range | Nuclei |
|-------|---------|-----------|--------|
| Foetal | 3 | 19-20 weeks | ~18,000 |
| Young | 3 | 4-14 years | ~18,000 |
| Adult | 3 | 35-42 years | ~18,000 |

**Total:** 9 samples, ~54,000 nuclei

---

## Methods

| Step | Method | Package |
|------|--------|---------|
| QC & Filtering | Per-cell metrics | Seurat |
| Normalisation | SCTransform v2 | Seurat |
| Integration | Harmony | harmony |
| Clustering | Louvain | Seurat |
| Annotation | Marker-based | Seurat |
| DE Analysis | Pseudobulk + limma-voom | edgeR, limma |
| Composition | propeller | speckle |
| Soft annotation | PLS on reference atlas | PhiSpace |
| Co-expression networks | Cell-specific networks + meta-networks | NeighbourNet |

---

## Installation

```r
# Install from GitHub
remotes::install_github("phipsonlab/single_cell_workshop")
```

See [Module 0: Setup](articles/00_setup.html) for detailed instructions.

---

## Citation

If you use these materials, please cite:

> Sim CB, Phipson B, Ziemann M, et al. Sex-Specific Control of Human Heart Maturation by the Progesterone Receptor. *Circulation*. 2021;143(10):1614-1628.

**Original analysis:** [bphipson.github.io/Human_Development_snRNAseq](https://bphipson.github.io/Human_Development_snRNAseq/)