Plugin error from itsxpress

Community Plugin Support
1

Dear All,
I’m trying to extract my ITS1 sequences (Single reads) using itsxpress version 1.8. I used the following command:
qiime itsxpress trim-single --i-per-sample-sequences single_end_demultiplexed_2nd_trial.qza --p-region ITS1 --p-cluster-id 0.99 --o-trimmed ITSX_trial
But unfortunately i received this error:
Plugin error from itsxpress:

  • Command ‘[‘vsearch’, ‘–cluster_size’, ‘/tmp/itsxpress_lk_utmeh/seq.fq.gz’, ‘–centroids’, ‘/tmp/itsxpress_lk_utmeh/rep.fa’, ‘–uc’, ‘/tmp/itsxpress_lk_utmeh/uc.txt’, ‘–strand’, ‘both’, ‘–id’, ‘0.99’, ‘–threads’, ‘1’]’ returned non-zero exit status 1.*

Debug info has been saved to /tmp/qiime2-q2cli-err-0c22bvh3.log

Would you please help
Regards,

2

Hello Marwa,

Welcome to the forums! :qiime2:

Thanks for posting your full command and error message. Could you also open up that .log file and post what's inside? We are still looking for clues about the cause of this error. :female_detective:

Colin

3

Hello Collin,
Thanks very much for welcoming. I'm enjoyiing usinig qiime2 and the forum too.
Hereinafter is the content of the log file.

search v2.7.0_linux_x86_64, 15.5GB RAM, 4 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, 15.5GB RAM, 4 cores

Fatal error: File too small
Traceback (most recent call last):
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/itsxpress/main.py", line 540, in cluster
p2.check_returncode()
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/subprocess.py", line 369, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command '['vsearch', '--cluster_size', '/tmp/itsxpress_87c0glj6/seq.fq.gz', '--centroids', '/tmp/itsxpress_87c$
Traceback (most recent call last):
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/q2cli/commands.py", line 328, in call
results = action(**arguments)
File "</home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/decorator.py:decorator-gen-273>", line 2, in trim_single
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/qiime2/sdk/action.py", line 240, in bound_callable
output_types, provenance)
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/qiime2/sdk/action.py", line 383, in callable_executor
output_views = self._callable(**view_args)
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py", line 123, in trim_single
cluster_id=cluster_id)
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py", line 208, in main
sobj.cluster(threads=threads, cluster_id=cluster_id)
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/itsxpress/main.py", line 543, in cluster
raise e
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/site-packages/itsxpress/main.py", line 540, in cluster
p2.check_returncode()
File "/home/marwa/miniconda3/envs/qiime2-2019.10/lib/python3.6/subprocess.py", line 369, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command '['vsearch', '--cluster_size', '/tmp/itsxpress_87c0glj6/seq.fq.gz', '--centroids', '/tmp/itsxpress_87c$

Regards,
Marwa

4

Hello Marwa,

Looks like part of the process resulted in an input file that had few (or no!) reads in it!

How large is your input file single_end_demultiplexed_2nd_trial.qza?
What region of ITS did you sequence?

Colin

5

All the files have reeds in it and i successfully used the same file in the other step lke denoising and filtering.
The file is 378.1 MB in size and consists of 93 fastq files (93 samples).
The region is ITS1.

Thanks,

6

Hello Marwa,

Ah OK. 378 MB is not very big for 93 samples. I bet some of those samples have very few reads in them, and that might cause errors in this plugin.

How many reads do you have in each of your samples? Maybe you could remove some of your samples with low read counts?

Colin

7

Hello Collin,
The highest number of reads per sample is 102297, and the lowest number of reads is 13799.
I do not really know what to do, it seems that the error is related to the Artifact contents, and a special artifact content works with the plugin, but what this special content is, i don’t know.
It is really frustrating.

1 Like
8

Hello again Marwa,

Those read depths look great! That shouldn’t be the problem.

Let’s see if @Adam_Rivers has any advice for us!

Colin

9

I’m the plugin developer. It looks like an error in the command passed to vsearch by itsxpress for single read data. I’ll look into it more now.

2 Likes
10

I wrote a few more tests for single-end, clustered data but could not reproduce the error. @Marwa100 Would you be willing to send me a link to your data so I can see if I can reproduce and fix the error?

2 Likes
11

ITSxpress developer !! So happy of that. Thanks very much @colinbrislawn.

You said: single-end, clustered data?? The Artifiact i used to perform the extraction step does not contain clustered data, may be this is the point??
I used the artifact that contains the de-multiplexed sequences, and they are not clustered. This is the only SampleData[SequencesWithQuality] i have.
Excuse me @Adam_Rivers , which type of qiime artifact you did try the plugin with? From which step you obtain that artifact??
I do not How could i send you the data??

1 Like
12

The ITS1 sequences were generated using Ion torrent PGM.

Marwa

13

ITSxpress temporarily de-replicates or clusters data and runs hmmer on the dereplicated/clustered data to speed up search with real no loss of sensitivity. The procedure is explained in the paper: https://f1000research.com/articles/7-1418

By setting cluster_id to less than 1 you requested clustering.

The test code checks all relevant artifact types including SampleData[SequencesWithQuality] , SampleData[JoinedSequencesWithQuality] and SampleData[PairedEndSequencesWithQuality]

This sounds like some peculiarity with your data but I can take a look. You can put your demultiplexed qza file on something like Google Drive and PM me the link.

2 Likes
14

Hi Adam,

I am also passing through similar issue right now. Are there some updates? Thank you!

Yiming

2 Likes