doublet detection errors with troublet: non-zero exit status and index out of bounds

[shirshuj]

I completed the tutorial and two samples of my own without error but now I'm consistently getting this problem with subsequent samples:

running souporcell doublet detection
Traceback (most recent call last):
  File "/opt/souporcell/souporcell_pipeline.py", line 596, in <module>
    doublets(args, ref_mtx, alt_mtx, cluster_file)
  File "/opt/souporcell/souporcell_pipeline.py", line 541, in doublets
    subprocess.check_call([directory+"/troublet/target/release/troublet", "--alts", alt_mtx, "--refs", ref_mtx, "--clusters", cluster_file], stdout = dub, stderr = err)
  File "/usr/local/envs/py36/lib/python3.6/subprocess.py", line 311, in check_call
    raise CalledProcessError(retcode, cmd)
subprocess.CalledProcessError: Command '['/opt/souporcell/troublet/target/release/troublet', '--alts', 'S8C_souporcell_2/alt.mtx', '--refs', 'S8C_souporcell_2/ref.mtx', '--clusters', 'S8C_souporcell_2/clusters_tmp.tsv']' returned non-zero exit status 101.

I checked the doublets.err file which says:

thread 'main' panicked at 'index out of bounds: the len is 0 but the index is 95256', src/main.rs:328:22
note: run with `RUST_BACKTRACE=1` environment variable to display a backtrace

But I'm not sure how to correct this issue. Any advice or things to try is appreciated. Thank you!

[wheaton5]

Probably upstream of doublet detection if len is 0. Can you give me the first few lines of alt.mtx and the cluster_tmp.tsv?

[shirshuj]

First lines of alt.mtx

%%MatrixMarket matrix coordinate real general
% written by sprs
14492 6794880 457068
1 95257 1
1 337008 1
1 1023389 1
1 1158281 1
1 2192702 1
1 3038255 1
1 3261750 1
1 3548729 1
1 3729811 1
1 3736516 1
1 3893785 1
1 4267166 1
1 4336965 1

But cluster_tmp.tsv is empty

[wheaton5]

Right so u are using the unfiltered barcodes file. Only use barcodes that are cells according to cellranger. This file should be in outs/filtered/ or something like that

[shirshuj]

The filepath is going to the filtered ones for each sample but maybe something got corrupted in them. I'll try some different samples to see if they will run and I will try taking a closer look at the filtered barcodes files that aren't working.

[wheaton5]

Well that alt.mtx says you have 6.79 million cells

[shirshuj]

That doesn't sound right at all but I'm looking into it. Thanks for your help in figuring out where I need to look for troubleshooting these runs!

[megannguyen1009]

I'm having this same exact error. Did you ever figure out how to fix it?

[wheaton5]

Use the filtered barcodes not the raw barcodes

[wheaton5]

In cellranger output there is a filtered folder and raw folder. Get it from the filtered folder

[megannguyen1009]

Hello thank you for your quick reply. My barcodes.tsv file is from the filtered folder, and I still got those errors. Do you know any other possibilities that could be causing this error?

[wheaton5]

Give me the first 3 lines of alt.mtx

[megannguyen1009]

My first few lines of alt.mtx

%%MatrixMarket matrix coordinate real general % written by sprs 2112270 2035673 50014710 1 64536 1 1 140738 0 1 239400 0 1 251279 1 1 254260 0 1 279530 0 1 518165 0

[wheaton5]

Right. This is due to an unflitered barcodes file. This says u have 2 million cells