Seurat -PCA


As input, give the Seurat R-object (Robj) from the Seurat setup -tool.

After setting up the R-object (.Robj), some quality control plots are drawn, filtering and regression is performed and plots to aid in estimating the statistically significant principal components are drawn.



As input, give the Seurat R-object (Robj) from the Seurat setup -tool.

Principal component analysis (PCA) is performed for the highly variable genes selected in the Filtering, regression and detection of variable genes tool. To overcome the extensive technical noise in any single gene for scRNA-seq data, Seurat clusters cells based on their PCA scores, with each PC essentially representing a 'metagene' that combines information across a correlated gene set. Determining how many PCs to include downstream is therefore an important step, but it can be challenging and uncertain. Few plots are drawn to PCAplots.pdf to aid in this exploration of the primary sources of heterogeneity in the dataset. Based on these plots the user is supposed to decide which principal components to include in the downstream analysis.

In the first plots, the top genes associated with the first two principal components are shown. Second plot shows the principal components 1 and 2 for the cells in the dataset, and the third plot is a heatmap focusing on principal component 1. We also list the top 5 genes associated with high/low loadings for the first 5 PCs in the PCAgenes.txt file.

Next plots can be used to make the decision on which principal components to use: by default, 12 heatmaps are plotted, focusing on the 12 first principal components, in which both cells and genes are sorted by their principal component scores. User can change the number of heatmaps to plot. Explore especially the PCs which you choose for downstream analysis: heatmaps can display the 'extremes' across both genes and cells, and can be useful to help exclude PCs that may be driven primarily by ribosomal/mitochondrial or cell cycle genes.

The last plot shows the standard deviations of the principal components. Here, the cutoff is where there is an elbow in the graph.

For more details, please check the Seurat tutorials.