flowchart LR
A[Raw FASTQ] --> B[Cell Ranger]
B --> C[QC & Filtering]
C --> D[Normalization]
D --> E[Integration]
E --> F[Clustering]
F --> G[Annotation]
style A fill:#e74c3c,color:white
style B fill:#f39c12,color:white
style C fill:#3498db,color:white
style D fill:#9b59b6,color:white
style E fill:#1abc9c,color:white
style F fill:#27ae60,color:white
style G fill:#2c3e50,color:white
Single-cell RNA-seq Pipeline
Comprehensive analysis of single-cell transcriptomics data
Overview
This pipeline covers the analysis of single-cell RNA-sequencing data from raw reads to cell type annotation and downstream analysis.
Pipeline Steps
1. Cell Ranger Processing
Demultiplexing, alignment, and UMI counting with Cell Ranger.
Cell Ranger FASTQ 10x Genomics
2. Ambient RNA Correction
Removing ambient RNA contamination using CellBender.
cellbender Ambient RNA correction QC
3. Data processing and Integration
Normalization, identification of highly variable genes, and data integration.
Integration Seurat HVGs
4. Copy Number Variation Inference
For Tumor samples, infer copy number variations using Numbat.
PCA UMAP Louvain
Required Inputs
| Input | Description | Source |
|---|---|---|
| Raw FASTQ files | Sequencing reads | Sequencer |
| Reference genome | Cell Ranger reference | 10x Genomics |
| Sample metadata | Sample information | User-provided |
Expected Outputs
- Quality control reports
- Filtered count matrices
- UMAP/tSNE visualizations
- Cluster assignments
- Cell type annotations
- Marker gene lists
- Differential expression between clusters