Question: What is the recommended sequencing depth for Single Cell 3' and 5' Gene Expression libraries?
Answer: For new sample types, we recommend sequencing 50,000 read pairs/cell for Single Cell 3' v2 and Single Cell 5' libraries. As a result of increased performance with Single Cell 3' v3 reagents, we recommend a minimum of 20,000 read pairs/cell.
The sequencing depth required for a particular experiment, however, will depend on:
- Sample type (different samples will have more or less RNA per cell)
- Experimental question being addressed.
After sequencing, the 'Sequencing Saturation' metric reported by Cell Ranger can be used to optimize sequencing depth for specific sample types.
- For instance, with 50,000 read pairs/cell for RNA-rich cells such as cell lines, only 30-50% sequencing saturation may be reached. However, this may be sufficient to cluster your sub-populations of interest during the analysis.
- If you are interested in identifying as many genes as possible in your sample, you may want to sequence deeper to reach around 90% sequencing saturation level.
For more information, see the Technical Note Resolving Cell Types as a Function of Read Depth and Cell Number.
Products: Single Cell 3', VDJ