qiime itsxpress trim-single fatal error: file too small

Hello -

I am trying to trim single-read (R1) ITS sequences (generated using the EMP protocol) with Q2-ITSxpress prior to running dada2. I have run this script successfully on other datasets, but am running into an error this time and I’m not sure what the problem is. I am using Qiime2-2019.4 on Amazon AWS.

Here is the command:

qiime itsxpress trim-single --i-per-sample-sequences demux_forward.qza --p-region ITS1 --p-taxa F --o-trimmed ITSxpress_trimmed.qza --verbose

Here is the output:

vsearch v2.7.0_linux_x86_64, 67.2GB RAM, 8 cores

Fatal error: File too small

ERROR:root:Could not perform clustering with Vsearch. Error from Vsearch was:
vsearch v2.7.0_linux_x86_64, 67.2GB RAM, 8 cores

Fatal error: File too small
Traceback (most recent call last):
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/itsxpress/main.py”, line 557, in cluster
p2.check_returncode()
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/subprocess.py”, line 369, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command ‘[‘vsearch’, ‘–cluster_size’, ‘/tmp/itsxpress_j7kb6gum/seq.fq.gz’, ‘–centroids’, ‘/tmp/itsxpress_j7kb6gum/rep.fa’, ‘–uc’, ‘/tmp/itsxpress_j7kb6gum/uc.txt’, ‘–strand’, ‘both’, ‘–id’, ‘0.995’, ‘–threads’, ‘1’]’ returned non-zero exit status 1.
Traceback (most recent call last):
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2cli/commands.py”, line 311, in call
results = action(**arguments)
File “</home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/decorator.py:decorator-gen-273>”, line 2, in trim_single
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 231, in bound_callable
output_types, provenance)
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 365, in callable_executor
output_views = self._callable(**view_args)
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py”, line 119, in trim_single
cluster_id=cluster_id)
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py”, line 198, in main
sobj.cluster(threads=threads, cluster_id=cluster_id)
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/itsxpress/main.py”, line 560, in cluster
raise e
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/itsxpress/main.py”, line 557, in cluster
p2.check_returncode()
File “/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/subprocess.py”, line 369, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command ‘[‘vsearch’, ‘–cluster_size’, ‘/tmp/itsxpress_j7kb6gum/seq.fq.gz’, ‘–centroids’, ‘/tmp/itsxpress_j7kb6gum/rep.fa’, ‘–uc’, ‘/tmp/itsxpress_j7kb6gum/uc.txt’, ‘–strand’, ‘both’, ‘–id’, ‘0.995’, ‘–threads’, ‘1’]’ returned non-zero exit status 1.

Plugin error from itsxpress:

Command ‘[‘vsearch’, ‘–cluster_size’, ‘/tmp/itsxpress_j7kb6gum/seq.fq.gz’, ‘–centroids’, ‘/tmp/itsxpress_j7kb6gum/rep.fa’, ‘–uc’, ‘/tmp/itsxpress_j7kb6gum/uc.txt’, ‘–strand’, ‘both’, ‘–id’, ‘0.995’, ‘–threads’, ‘1’]’ returned non-zero exit status 1.

See above for debug info.

I have checked other discussions on the forum (this and this), and it seems like /tmp/itsxpress_j7kb6gum/seq.fq.gz’ may not have any sequences? Maybe the plug-in is trying to merge reads, despite running the trim-single command? Here is the demuxed qzv demux_forward.qzv (292.4 KB) I validated my demuxed sequences using the qiime tools validate function and all looked good. So, I’m not sure what the problem is. Any insights you could provide would be greatly appreciated!

Chelsea

Hello, I’m just checking back in on this. Any thoughts on what might be the issue and how to proceed? I tried running my demuxed samples through dada2 just to see, and that worked fine, but ITSxpress is still throwing that same error. Thanks!

Hi @ccarey,
Sorry for the delay and thank you for your patience.

I am cc:ing @Adam_Rivers to see if he has any thoughts about this.

Yes that was the issue. I fixed it a few weeks ago, along with a feature addition, but I never pushed out the new version. I’ll try to do that next week, you can get the fixed code from GitHub if you need it right away.

1 Like

Great, thank you! Very helpful.