Single Cell RNA Analysis Overview
This section covers the analysis pipeline for single cell sequencing data, including scRNA-seq and related modalities.
What is Single Cell Analysis?
Single cell sequencing allows gene expression (and other molecular features) to be measured in individual cells rather than in bulk. This reveals cellular heterogeneity, rare cell populations, and developmental trajectories that are masked in bulk assays.
scRNA course link
This is a link to a course on single cell RNA-seq analysis that covers the analysis pipeline in more detail, including hands-on exercises and examples. It was held in Cambridge and covered all the steps from library QC to differential expression analysis. The course materials are available online for self-paced learning.
This covers it far better than I can in this overview, so I recommend checking it out if you want to learn more about single cell RNA-seq analysis.
Common Modalities
- scRNA-seq - Single cell RNA sequencing (10x Genomics Chromium, Smart-seq2)
- snRNA-seq - Single nucleus RNA sequencing, suitable for frozen/fixed tissue
- scATAC-seq - Chromatin accessibility at single cell resolution
- Multiome - Simultaneous RNA + ATAC from the same cell
Workflow Steps
- Library QC - FastQC, MultiQC on raw reads
- Alignment & Quantification - Cell Ranger (10x), STARsolo, Salmon/Alevin
- Quality Filtering - Remove low-quality cells, doublets (Scrublet, DoubletFinder)
- Normalisation - Scran, SCTransform
- Dimensionality Reduction - PCA, UMAP, tSNE
- Clustering - Graph-based clustering (Louvain, Leiden)
- Cell Type Annotation - Marker genes, automated tools (SingleR, Azimuth)
- Differential Expression - Between clusters or conditions
Applications
- Characterising cell type composition in tissues
- Identifying rare or novel cell populations
- Mapping developmental or differentiation trajectories
- Comparing cell states across conditions or time points
Typical Outputs
- Feature-barcode matrices (raw counts)
- Seurat or AnnData objects
- UMAP/tSNE plots
- Marker gene lists
- Differential expression results