Results interpretation - overestimation?

Hi,

I would like to have some help: I’m running CellBender on a “problematic” sample (trial of new nuclei isolation protocol , resulted in lower quality). We know that many cells need to be discarded from this sample (achieved with stringent filtering for nUMI and %mt). I decided to try CellBender to see if I could get a better estimation on the keepable cell for downstream analysis, but CellBender seems to keep all the cells. I’m not sure what actually is cell and what is ambient/low quality cell from the Cell ranger output, I used the following parameters:

cellbender remove-background \
>                  --input ./raw_S2R2/raw_feature_bc_matrix.h5 \
>                  --output ./raw_S2R2/cellbender_matrix.h5 \
>                  --expected-cells 800 \
>                  --total-droplets-included 6000 \
>                  --fpr 0.01 \
>                  --epochs 150

and got the attached pdf output. s2r2.pdf

Now my questions are:

• What does this output mean? Is this sample not suitable for CellBender? or I just used the wrong parameters?

• Since the outputs of cellranger and CellBender are comparable in the # of cells kept, can the output of CellBender be considered cleaner because it should have also removed some background from the new count matrix, or the results of CellBender are not trustable?

I hope it is clear and thanks very much in advance!

[sjfleming]

Huh, I did not expect that… well, thanks for letting me know! I should keep that in mind in future…

[giorgiatosoni]

Oh I see… thanks for the explanation!