Question: I have run my data with Cell Ranger 2 and Cell Ranger 3. Why do I see more cells that are MT (mitochondrial gene) enriched from Cell Ranger 3 results? Which result should I trust?
Answer: Cell Ranger versions 3 or higher have a cell calling algorithm that is more sensitive than the cell calling algorithm in version 2. It is therefore expected to detect cells that were missed by previous version of our cell calling algorithm. These additional cells detected by the new cell calling algorithm may be particularly low RNA content cells in heterogeneous samples. The figure below shows an example barcode rank plot for same data run with Cell Ranger 2.2 and 3.0. It shows additional lower UMI count barcodes called as cells (in blue shaded portion of the curve) in Cell Ranger 3.0.
See this page for more details on the new cell calling algorithm.
This increased sensitivity also results in Cell Ranger 3.0 more easily picking up cells that may be exhibiting signs of stress or higher expression of MT genes, which 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.
If only a single or a few clusters of cells have very few up-regulated genes (indicating low overall gene expression) other than MT genes, this cluster or clusters most likely represents a population of cells that are either about to enter or are already going through apoptosis or necrosis pathways.
For example, in the figure below, barcodes with low UMIs cluster together (dark blue in UMI plot on lower left, blue and lime green in lower right t-SNE plot). These two clusters with the extra cells identified by Cell Ranger 3.0 are enriched for mitochondrial genes, which could be indicative of dying or stressed cells.
In summary, a potential side effect of the new cell calling algorithm is that it may include more of the poor quality cells that are MT enriched due to the higher sensitivity of the algorithm.
You can choose to exclude these cells (as explained here) from downstream analysis.
Products: Single Cell Gene Expression