Question: My data contains lots of cells that are enriched for mitochondrial (MT) genes. How can I exclude these cells from further analysis?
Answer: In some samples, you might see a fraction of cells that may be exhibiting signs of stress or higher expression of MT genes. This could be an indicator of:
- Poor sample quality, leading to a high fraction of apoptotic or lysed cells.
- The overall biology of the sample, for example tumor biopsies, which may have increased mitochondrial gene expression due to metabolic activity and/or necrosis.
This phenotype is discussed in more detail here.
You may choose to exclude these cells from downstream analysis. There is no direct way to exclude mitochondria-rich cells in the Cell Ranger pipeline. However, you can accomplish this indirectly using either of the 3 options below.
Option 1: Using Loupe Browser Filtering and Reclustering Workflow
Loupe Browser 5.0 and later provides an interactive filtering and reclustering workflow. In one of the steps, you can filter cells by mitochondrial fraction -- the percentage of UMIs per barcode associated with mitochondrial genes. See the detailed tutorial here: Filtering and Reclustering Workflow.
Option 2: Using Loupe Browser and Cell Ranger
Step 1. The same as the above option, you need to first filter MT-enriched cells and run re-clustering in Loupe Browser.
Step 2. Export the cell barcodes from the reclustered category using the 'Export' cluster function. Make sure to 'Exclude Unlabeled' cells (which were the MT-enriched cells we filtered out).
Step 3. Re-run secondary analysis and generate a new .cloupe file (and new matrix) without the MT-enriched cells using cellranger reanalyze pipeline. To specify barcodes, you will need to use --barcodes parameter.
Option 3: Using third-party tools
Third-party tools 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 community-developed tool. 10x Genomics does not provide support for community-developed tools and makes no guarantees regarding their function or performance. Please contact tool developers with any questions.