GCP VM Fragment Insertion SEPP error

[horsemant]

Hi,

I've seen the following error on the forum but I haven't found a great fix. I tried my command on QIIME2 2023.5 and installed qiime2-amplicon-2024.5. I am running QIIME2 on a GCP VM with boot disk size of 500GB and in a conda environment. I have tried the command with the GG and SILVA reference databases, redownloading each from what I used previously. Seems like a working/temp directory issue so I created a new tmp directory and directed QIIME2 to it by mkdir -p /tmp/tempdir and
export TMPDIR=/tmp/tempdir. Received the same error. My command and error are still as follows:

qiime fragment-insertion sepp \

--i-representative-sequences mergedexp2repseqs.qza \
--i-reference-database ~/sepp-refs-gg-13-8.qza \
--o-tree exp2tree.qza \
--o-placements exp2insertionplacements.qza \
--p-debug \
--verbose

mktemp: failed to create directory via template ‘/path/to/new/tempdir/tmp.XXXXXXXXXX’: No such file or directory
/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/bin/run-sepp.sh: Can't create temp directory, exiting...
Traceback (most recent call last):
File "/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 520, in call
results = self._execute_action(
File "/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 581, in _execute_action
results = action(**arguments)
File "", line 2, in sepp
File "/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self.callable_executor(
File "/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 576, in callable_executor
output_views = self._callable(**view_args)
File "/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_fragment_insertion/_insertion.py", line 75, in sepp
_run(str(representative_sequences.file.view(DNAFASTAFormat)),
File "/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_fragment_insertion/_insertion.py", line 54, in _run
subprocess.run(cmd, check=True, cwd=cwd)
File "/home/timothy_horseman/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/subprocess.py", line 528, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run-sepp.sh', '/tmp/qiime2/timothy_horseman/data/30027b0b-2e93-4850-8d56-8b1a05fcf63b/data/dna-sequences.fasta', 'q2-fragment-insertion', '-x', '1', '-A', '1000', '-P', '5000', '-a', '/tmp/qiime2/timothy_horseman/data/a14c6180-506b-4ecb-bacb-9cb30bc3044b/data/aligned-dna-sequences.fasta', '-t', '/tmp/qiime2/timothy_horseman/data/a14c6180-506b-4ecb-bacb-9cb30bc3044b/data/tree.nwk', '-r', '/tmp/qiime2/timothy_horseman/data/a14c6180-506b-4ecb-bacb-9cb30bc3044b/data/raxml-info.txt', '-b', '1']' returned non-zero exit status 1.

Plugin error from fragment-insertion:

Command '['run-sepp.sh', '/tmp/qiime2/timothy_horseman/data/30027b0b-2e93-4850-8d56-8b1a05fcf63b/data/dna-sequences.fasta', 'q2-fragment-insertion', '-x', '1', '-A', '1000', '-P', '5000', '-a', '/tmp/qiime2/timothy_horseman/data/a14c6180-506b-4ecb-bacb-9cb30bc3044b/data/aligned-dna-sequences.fasta', '-t', '/tmp/qiime2/timothy_horseman/data/a14c6180-506b-4ecb-bacb-9cb30bc3044b/data/tree.nwk', '-r', '/tmp/qiime2/timothy_horseman/data/a14c6180-506b-4ecb-bacb-9cb30bc3044b/data/raxml-info.txt', '-b', '1']' returned non-zero exit status 1.

See above for debug info.

[colinvwood (Colin Wood)]

Hello @horsemant,

When the command that generated the error log you posted ran, the tempir was set to /path/to/new/tempdir/ as you can see here:

mktemp: failed to create directory via template ‘/path/to/new/tempdir/tmp.XXXXXXXXXX’: No such file or directory

This directory didn't exist and so the command failed. The mktemp command creates files/directories in your default temporary directory (usually stored in $TEMPDIR). Perhaps you are overwriting the value of this environment variable in config file somewhere, or simply got confused when you were changing the $TMPDIR variable?

[horsemant]

Thanks for the response @colinvwood. Came to the conclusion the error stemmed from a VM permissions issue. I ran the fragment-insertion sepp command on an iMAC we have in the office. Although the run time was pretty long, it worked.