Cellranger Reanalyze NO barcode rank plot in web_summary.html

Question

[el24]

Hi everyone,

I have a question regarding running cellranger count on the data, I get the following warning after running it:

Low Fraction Reads in Cells

68.5%   Ideal > 70%. 

Application performance may be affected. Many of the reads were not assigned to cell-associated barcodes. This could be caused by high levels of ambient RNA or by a significant population of cells with a low RNA content, which the algorithm did not call as cells. The latter case can be addressed by inspecting the data to determine the appropriate cell count and using --force-cells.

Then, as suggested I run cellranger reanalyze using the following command to get 900 cells:

cellranger reanalyze --id=reanalysis_900 
--force-cells=900 
--matrix=/PATH/outs/raw_feature_bc_matrix.h5    #cellranger count output

When I check web_summry.html it doesn't include the summary of the data or any plots in the "summary tab", it's just a blank page with trivial information. I have tried another number of for the force cells (600), but it didn't help. (I originally got about 1200 cells when running cellarnger count in the web_symmary.html output)

Could anyone give me a clue if I'm doing anything wrong? Do you have any recommendations to get the barcode rank plot and summary information in my web_summary.html?

I appreciate it! Thanks a lot!

[swbarnes2]

I just checked a reanalyze of mine, and it's the same. Nothing on the front tab. New t-SNE on the second tab.

[el24]

Thanks for checking, do you have any idea how can I safely remove the cells with fewer read values (which are not reliable) so that I can get the barcode rank plot and summary information in my web_summary.html? Also, do you usually recommend using any other parameters in "cellranger reanalyze" or you think my script is fine?

I appreciate it if you have any recommendations! Thanks!

Answer 1

[swbarnes2]

The web summary information would be almost exactly the same as what you already have. Unless you are absolutely sure what you are doing, I'd let 10X decide the barcode cut-off. Being a percent and a half under the QC cutoff isn't much of a reason to second guess them.

[el24]

Sound good! Thanks for the information!