Question: After running
cellranger aggr, I got this warning "Low Post-Normalization Read Depth 40%. Ideal > 50%. There may be large differences in sequencing depth across the input libraries. Application performance may be affected." What should I do?
Answer: Normally, this warning in
cellranger aggr occurs when there is a large discrepancy in sequencing depth among the input libraries.
cellranger aggr subsamples each library so that all libraries have the same effective sequencing depth, measured as confidently mapped reads per cell. If one (or more) library has more reads per cell compared to other libraries,
cellranger aggr will subsample that library to match the lowest depth library. In some cases, this may trigger the "[Alert] Low Post-Normalization Read Depth".
If all the libraries were sequenced to sufficient saturation such that downsampling them does not affect the UMI counts, this warning can be safely ignored. It may also be useful to check the saturation curve for each library for effect of lower saturation on metrics such as median genes per cell. The curve can be found in the 'ANALYSIS' tab of the web_summary.html file generated in the original
cellranger count output, or 'Sample' view of the web_summary.html file generated in the original
cellranger multi output.
You may also choose to use
cellranger aggr without normalization (