Hello!
I’m experiencing issues while running the fragment-insertion sepp plugin on Qiime2 within a Windows Subsystem for Linux (WSL 2) environment. The SEPP process repeatedly generates the error 'non-zero exit status 1' and when I try run with deblur, the zombie processes (Z state), appear to cause the process to stall indefinitely. I’ve tried several troubleshooting steps but haven’t resolved the issue, so any insights would be greatly appreciated!
System Details
- OS: Windows Subsystem for Linux (WSL 2), Ubuntu 20.04
- Qiime2 Version: 2024.10
- Memory & Swap: 16GB memory, 12GB swap
- Processor: 20 cores allocated in WSL
Command
qiime2-amplicon-2024.10) lisou@DESKTOP-OO5NL0A:~/Nested$ qiime fragment-insertion sepp \
--i-representati> --i-representative-sequences Nested-rep-seqs.qza \
> --i-reference-database sepp-refs-silva-128.qza \
> --p-threads 4 \
> --o-tree Nested-merged-sepp-tree.qza \
> --o-placements Nested-merged-sepp-placements.qza \
Plugin error from fragment-insertion:
Command '['run-sepp.sh', '/tmp/qiime2/lisou/data/35e66cf8-05f4-462a-b349-4325ba34a5ef/data/dna-sequences.fasta', 'q2-fragment-insertion', '-x', '8', '-A', '1000', '-P', '5000', '-a', '/tmp/qiime2/lisou/data/e44b5e78-31e5-4a0f-9041-494bc3ca2df2/data/aligned-dna-sequences.fasta', '-t', '/tmp/qiime2/lisou/data/e44b5e78-31e5-4a0f-9041-494bc3ca2df2/data/tree.nwk', '-r', '/tmp/qiime2/lisou/data/e44b5e78-31e5-4a0f-9041-494bc3ca2df2/data/raxml-info.txt']' returned non-zero exit status 1.
Debug info has been saved to /tmp/qiime2-q2cli-err-2swu9_nc.log
Log info:
Removing /tmp/tmp.0gkiL3LLUi/sepp-tmp-RZvEqxZmcq
Traceback (most recent call last):
File "/home/lisou/anaconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2cli/commands.py", line 530, in call
results = self._execute_action(
File "/home/lisou/anaconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2cli/commands.py", line 602, in _execute_action
results = action(**arguments)
File "", line 2, in sepp
File "/home/lisou/anaconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/qiime2/sdk/action.py", line 299, in bound_callable
outputs = self.callable_executor(
File "/home/lisou/anaconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/qiime2/sdk/action.py", line 570, in callable_executor
output_views = self._callable(**view_args)
File "/home/lisou/anaconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_fragment_insertion/_insertion.py", line 75, in sepp
_run(str(representative_sequences.file.view(DNAFASTAFormat)),
File "/home/lisou/anaconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_fragment_insertion/_insertion.py", line 54, in _run
subprocess.run(cmd, check=True, cwd=cwd)
File "/home/lisou/anaconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/subprocess.py", line 526, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run-sepp.sh', '/tmp/qiime2/lisou/data/35e66cf8-05f4-462a-b349-4325ba34a5ef/data/dna-sequences.fasta', 'q2-fragment-insertion', '-x', '4', '-A', '1000', '-P', '5000', '-a', '/tmp/qiime2/lisou/data/e44b5e78-31e5-4a0f-9041-494bc3ca2df2/data/aligned-dna-sequences.fasta', '-t', '/tmp/qiime2/lisou/data/e44b5e78-31e5-4a0f-9041-494bc3ca2df2/data/tree.nwk', '-r', '/tmp/qiime2/lisou/data/e44b5e78-31e5-4a0f-9041-494bc3ca2df2/data/raxml-info.txt']' returned non-zero exit status 1.
I've tried reducing the thread set --p-threads to 2 (from 8 originally), thinking this might reduce memory pressure or process conflicts. This change did not resolve the issue.
I've also checked and increased the memory and CPU: There’s plenty of available memory and swap, so I don’t believe the issue is due to resource exhaustion.
I am also unable to run debug, as QIIME stalls.
Command with debug
qiime fragment-insertion sepp \
--i-representative-sequences Nested-rep-seqs.qza \
--i-reference-database sepp-refs-silva-128.qza \
--p-threads 2 \
--o-tree Nested-merged-sepp-tree.qza \
--o-placements Nested-merged-sepp-placements.qza \
--verbose \
--p-debug
When I run top shows that some of the run_sepp.py processes enter a zombie state after a few minutes. The command then stalls indefinitely, and CPU usage drops to zero, indicating that SEPP is no longer progressing. Here’s what I observed in top:
Tasks: 13 total, 1 running, 10 sleeping, 0 stopped, 2 zombie
%Cpu(s): 0.0 us, 0.0 sy, 0.0 ni,100.0 id, 0.0 wa, 0.0 hi, 0.0 si, 0.0 st
MiB Mem : 26060.9 total, 16683.2 free, 6164.4 used, 3213.3 buff/cache
MiB Swap: 12288.0 total, 12288.0 free, 0.0 used. 19536.0 avail Mem
PID USER PR NI VIRT RES SHR S %CPU %MEM TIME+ COMMAND
1 root 20 0 2296 1324 1108 S 0.0 0.0 0:00.18 init
8 root 20 0 2164 360 0 S 0.0 0.0 0:00.00 init
9 root 20 0 2172 360 0 S 0.0 0.0 0:00.00 init
10 lisou 20 0 10172 5108 3276 S 0.0 0.0 0:00.04 bash
496 root 20 0 2644 564 0 S 0.0 0.0 0:00.00 init
497 root 20 0 2644 564 0 S 0.0 0.0 0:00.05 init
498 lisou 20 0 10172 5176 3404 S 0.0 0.0 0:00.01 bash
517 lisou 20 0 10872 3696 3144 R 0.0 0.0 0:00.32 top
635 lisou 20 0 5456316 2.4g 133432 S 0.0 9.5 0:30.78 qiime
664 lisou 20 0 8624 3224 2948 S 0.0 0.0 0:00.00 run-sepp.sh
668 lisou 20 0 3945268 3.6g 11572 S 0.0 14.0 2:11.24 run_sepp.py
670 lisou 20 0 0 0 0 Z 0.0 0.0 0:01.33 run_sepp.py
671 lisou 20 0 0 0 0 Z 0.0 0.0 0:01.39 run_sepp.py
Any help would be appreciated. I have looked at the other posts on the non-zero exit status 1 for WSL environments and the only solution I've seen is switching to OS. And I'm stumped being unable to run deblur!