Steps to take to analyze scRNA-seq, scATAC-seq and Multiome data
Read the previous section: Intro to scRNA-seq, scATAC-seq and Multiome
The data analysis of Multiome data can be divided into two phases.
The first phase is the Cell Ranger phase. Cell Ranger is a set of software developed by 10X Genomics that performs demultiplexing (separating data from different cells), first pass quantification (gene expression quantification for scRNA-seq and peak calling for scATAC-seq), and the first round of quality control (QC) of the data.
The second phase of data analysis for Multiome data is the Seurat and Signac phase. Seurat and Signac are software packages developed by Rahul Satija's group at the New York Genome Center specifically for scRNA-seq and scATAC-seq data analysis. This phase includes an additional quality control step that removes likely doublets (mixtures of data from more than one cell), scATAC-seq data with low enrichment scores for transcription start sites (TSS), and cells with high mitochondria content which are likely to be of low quality. It also includes a normalization step to balance the read counts across cells and a clustering step that groups cells based on their similarity in gene expression and chromatin accessibility profiles. Dimension reduction techniques are first applied to the genes (from the scRNA-seq data) and the peaks (from the scATAC-seq data) that show the highest levels of variability across cells, and weighted nearest neighbor metrics are calculated among cells. Clustering is then performed jointly for the gene expression and chromatin accessibility data, followed by Uniform Manifold Approximation and Projection (UMAP) visualization, which reveals subpopulations of cells with distinct joint gene expression and chromatin accessibility profiles.
The Seurat and Signac phase also includes other functional analyses, such as the analysis of enrichment of transcription factor binding motifs (TFBMs). This can reveal hints about what transcription factors may be present at open chromatin regions. The phase also includes differential analysis, which includes differential expression analysis for scRNA-seq data and differential peak analysis for scATAC-seq data.
- Intro to scRNA-seq, scATAC-seq and Multiome
- Steps to take to analyze scRNA-seq, scATAC-seq and Multiome data
- Significance of single-cell analysis
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Read next: Significance of single-cell analysis
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