Question: My data contains lots of cells that are enriched for mitochondrial (MT) genes. How can I exclude these cells from further analysis?
Answer: High level of mitochondrial gene expression could be an indicator of poor sample quality or biology of a particular sample. Why do I see a high level of mitochondrial gene expression?
Cell Ranger 3.x is more sensitive than earlier cell calling algorithm and sometimes picks up cells that are stressed or dying (mitochondria-enriched).
There is no direct way to exclude mitochondrial rich cells in
cellranger count pipeline. However, you can accomplish this indirectly using either of the 2 methods below.
1) Using Cell Ranger and Loupe Cell Browser
Step 1. Identify the barcodes for the MT enriched cell from
cellranger count output files or using the Loupe Cell Browser:
cellranger count output) Default clustering results (Graph-based) in the output folder correspond to the t-SNE plot in the 'ANALYSIS' tab of the web_summary.html. For example, in the figure below, cells in cluster 1 and 9 are MT enriched. The barcodes for these clusters can be found in this output file:
- (Using Loupe Cell Browser) Use "Export" function in Loupe Cell Browser as below and remove those barcodes that belong to MT enriched cluster.
Step 2. Re-run secondary analysis and generate a new cloupe file without the MT enriched clusters using
cellranger reanalyze. To specify barcodes, you will need to use
2) Using Seurat
The third-party tool such as Seurat could be used to filter cells with a high percentage of mitochondrial gene expression. Please find more information in the vignette here. (section "QC and selecting cells for further analysis")
Note: Seurat is a third-party software product. We are not staffed to answer questions about it or provide support for modifications.