CellBender: Fails with "not enough empty droplets" error even though --low-count-threshold 2

CellBender fails on my data with “not enough empty droplets” error even though I set --low-count-threshold to 2. The error & log itself are a bit weird (look at the scale of nUMI filtered). I get this exact error (with different UMI numbers) on 2 very different datasets.

cellbender:remove-background: 2019-11-06 00:23:12
cellbender:remove-background: Running remove-background
cellbender:remove-background: Loading data from directory ./rawdata/filtered_feature_bc_matrix/folder
cellbender:remove-background: CellRanger v3 format
cellbender:remove-background: Trimming dataset for inference.
cellbender:remove-background: Prior on counts in empty droplets is 6001
cellbender:remove-background: Prior on counts for cells is 49252
cellbender:remove-background: Excluding barcodes with counts below 4800
Traceback (most recent call last):
  File "/path/to/software/miniconda3/envs/mypyro/bin/cellbender", line 11, in <module>
    load_entry_point('cellbender', 'console_scripts', 'cellbender')()
  File "~/CellBender/cellbender/base_cli.py", line 101, in main
    cli_dict[args.tool].run(args)
  File "~/CellBender/cellbender/remove_background/cli.py", line 92, in run
    main(args)
  File "~/CellBender/cellbender/remove_background/cli.py", line 185, in main
    run_remove_background(args)
  File "~/CellBender/cellbender/remove_background/cli.py", line 143, in run_remove_background
    args.low_count_threshold)
  File "~/CellBender/cellbender/remove_background/data/dataset.py", line 90, in __init__
    gene_blacklist=gene_blacklist)
  File "~/CellBender/cellbender/remove_background/data/dataset.py", line 252, in _trim_dataset_for_analysis
    f"There are no empty droplets with UMI counts over the lower " \
AssertionError: There are no empty droplets with UMI counts over the lower cutoff of 4800.  Some empty droplets are necessary for the analysis.Reduce the --low-count-threshold parameter.

About this issue

Original URL
State: closed
Created 5 years ago
Comments: 15 (7 by maintainers)

Most upvoted comments

@bobermayer Good to know! Yes, I have seen this kind of error happen before in the case where the “filtered” file is used by mistake. I’ve even done it myself… I should probably try to come up with some heuristic check so that a warning is issued if the file looks like it might be “filtered”.

In fact, I will do this, see #99

sjfleming on May 3, 2021

I just ran into the same error until I realized that I had accidentally passed filtered_feature_bc_matrix.h5 instead of raw_feature_bc_matrix.h5 as input file. it looks like the same thing happened here.

bobermayer on Jan 5, 2021

Well, we suggest that the droplets you’ve highlighted in your plot there are the “empty droplets” whose RNA comes from the various sources we describe, including ambient RNA and barcode swapping / chimera formation. Droplets with UMI counts below 100 we believe to be junk from other sources, especially un-corrected barcode sequencing errors. Everything above the line at about 500 would probably be real cells.

sjfleming on Nov 22, 2019