Question: We seem to be observing differences in the number of mitochondrial (MT) and ribosomal protein (RP) genes detected with Single Cell 3’ v3 compared to Single Cell 3’ v2. Could you elaborate?
Answer: When comparing sequencing data from libraries generated with different chemistry versions of the Single Cell Gene Expression Solution (e.g. Single Cell 3’ v2 and Single Cell 3’ v3), you will observe systematic differences in gene expression profiles between these libraries.
Alongside an improvement in the number of Median Genes per Cell and Median UMI Counts per Cell obtained with Single Cell 3’ v3, you will also observe an alteration in the number of Mitochondrial (MT) and Ribosomal Protein (RP) reads relative to Single Cell 3’ v2.
A summary of quality and performance metrics for eight datasets from our publicly available Single Cell Gene Expression Datasets page is provided in table below.
Note: For the demonstration datasets above, we generated libraries with v2 and v3 chemistry from same sample types and analyzed with same Cell Ranger version.
Whilst a proportion of the MT genes you are observing in your dataset will be as a result of the different chemistry versions of the Single Cell Gene Expression Solution, additional factors should also be considered.
Cell Ranger 3.0 (which is used to analyze 3' Single Cell v3 chemistry data) has a new cell calling algorithm that is more sensitive and 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. 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. Higher MT content can be an indicator of:
- Poor sample/cell quality
- The overall biology of the sample, for example tumor biopsies, which may have increased mitochondrial gene expression due to metabolic activity and/or necrosis.