QIIME2 - ITSx docker container or CyVerse App

[Charles_Hauser]

Hi

We are interested in running fungal analysis using qiime2 & extracting ITS using ITSx
We have used the QIIME2 app on Cyverse, but need to add ITSx - how?
Alternatively, should we be building a docker container w/ qiime2 & itsx?

We are at undergrad institution, this is primarily for undergrad research projects

Charles

[thermokarst]

Hi @Charles_Hauser, I'm not familiar with Cyverse, and the QIIME 2 team isn't involved with their QIIME 2 app - have you reached out to the Cyverse folks to see what they recommend doing?

@Charles_Hauser

Alternatively, should we be building a docker container w/ qiime2 & itsx?

Sure! Check out this community tutorial:

https://forum.qiime2.org/t/installing-community-plugins-in-docker/7106/4

Hope that helps :qiime2:

[Nicholas_Bokulich]

Hi @Charles_Hauser,
This doesn't answer your installation question, but just to make sure you know it exists, the q2-itsxpress plugin offers a way to integrate ITS extraction into a QIIME 2 workflow:

Q2-ITSxpress: A tutorial on a QIIME 2 plugin to trim ITS sequences

ITSxpress: a QIIME 2 plugin to trim ITS sequences Adam R. Rivers - USDA Agricultural Research Service Sveinn V. Einarsson - Dep. Microbiology and Cell Science, U. Florida & USDA Agricultural Research Service Background The internally transcribed spacer (ITS) region a widely used phylogenetic marker for fungi and other taxa. Previous work by Nilsson et al. (2009) showed that removing the conserved regions around the ITS results in more accurate taxonomic classification. An existing program, ITS…

You could install that in a docker container following the tutorial that @thermokarst shared.

Good luck!

[Charles_Hauser]

Thanks to you both for suggestions.
I can successfully implement adding the plugin q2_perc_norm to the qiime2/core:2018.11 docker container as described here:

Installing Community Plugins in Docker

Installing Community Plugins (https://library.qiime2.org) is a breeze with Docker! First, let's launch an interactive Docker container: docker run --rm -it --name my-custom-container qiime2/core:2018.11 /bin/bash Next, pick a plugin to install: I will try out @cduvallet's q2-perc-norm. Running the current install command: # This plugin is just an example, please follow the directions for # the plugin (or plugins) you wish to install! conda install -c cduvallet q2_perc_norm -y qiime dev refre…

However, when I attempt to follow the same steps but add itsxpress
conda install -c bioconda itsxpress
pip install q2-itsxpress

itsxpress installs w/o error, but when I run:

docker run --rm -it
-u (id -u):(id -g)
-v $(pwd):/data
chauser-qiime2/2018.11-with-q2-perc-norm
qiime itsxpress

I get the error:

(base) charleshauser@~/qiime2_docker => ./runit_qiime_itsx_docker

Usage: qiime [OPTIONS] COMMAND [ARGS]...

Try "qiime --help" for help.

Error: No such command "itsxpress".

I did run 'qiime dev refresh-cache' after installing itsxpress.

This is my first foray into docker containers, so I suspect it is something on my end.
However, I'm not sure why I can get perc_norm to run, but not itsxpress

Suggestions?

Charles

[thermokarst]

@Charles_Hauser

I can successfully implement adding the plugin q2_perc_norm to the qiime2/core:2018.11 docker

Great news!

@Charles_Hauser

chauser-qiime2/2018.11-with-q2-perc-norm

I think I know what the problem is - you built this using an extremely old version of QIIME 2 (2018.11). On versions that old, you need to run qiime dev refresh-cache before finalizing the docker container. If you re-run the docker steps using the latest version of QIIME 2, you would be able to skip that qiime dev refresh-cache step, which should work just fine.

Give that a shot and let us know how it goes!

[Charles_Hauser]

Hi Matt

Understood.
FYI, their app ii just a docker container running qiime2 & itsxpress (itsxpress: 1.8.0).
Running stadalone itsxpress on a fastq file works, it's just an issue with single-end reads runn with qiime itsxpress

Charles

[thermokarst]

I'm sorry @Charles_Hauser - I don't follow, can you please elaborate?

[Charles_Hauser]

Apologies for lack of clarity

re: itsxpress
if I demultiplex single-end sequences in qiime (qiime demux emp-single), and then run itsxpress (qiime itsxpress trim-single) I get the following error:

Plugin error from itsxpress:

Command '['vsearch', '--cluster_size', '/tmp/itsxpress_fp0p5hbq/seq.fq.gz', '--centroids', '/tmp/itsxpress_fp0p5hbq/rep.fa', '--uc', '/tmp/itsxpress_fp0p5hbq/uc.txt', '--strand', 'both', '--id', '0.995', '--threads', '1']' returned non-zero exit status 1.

Debug info has been saved to /tmp/qiime2-q2cli-err-oopcu8c_.log

Looking in the log file, the error arises from vsearch

vsearch v2.7.0_linux_x86_64, 251.8GB RAM, 32 cores

GitHub - torognes/vsearch: Versatile open-source tool for microbiome analysis

Fatal error: File too small
subprocess.CalledProcessError: Command '['vsearch', '--cluster_size', '/tmp/itsxpress_fp0p5hbq/seq.fq.gz', '--centroids', '/tmp/itsxpress_fp0p5hbq/rep.fa', '--uc', '/tmp/itsxpress_fp0p5hbq/uc.txt', '--strand', 'both', '--id', '0.995', '--threads', '1']' returned non-zero exit status 1.

Checking the dir w/ tmp filles:
ls -l /tmp/itsxpress_fp0p5hbq/
total 6872
-rw-r--r-- 1 qiime2 qiime2 0 Mar 19 16:18 rep.fa
-rw-r--r-- 1 qiime2 qiime2 20 Mar 19 16:18 seq.fq.gz
-rw-r--r-- 1 qiime2 qiime2 3524967 Mar 19 16:18 seq_r1.fq.gz
-rw-r--r-- 1 qiime2 qiime2 3502500 Mar 19 16:18 seq_r2.fq.gz
-rw-r--r-- 1 qiime2 qiime2 0 Mar 19 16:18 uc.txt

I'm not sure why htere is a .r2 seq file for silngle-end method, is this the basis for the vsearch error?

Hope htat helps

Charles

[thermokarst]

Hi @Charles_Hauser - I will repeat my request from earlier today

Please do not cross-post - this is a violation of the QIIME 2 Forum Code of Conduct. Administration of these kinds of issues takes away moderator time from being able to help you and others with their actual questions - we appreciate your cooperation on this matter.

You first posted this question to:

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: vsea…

Please keep the discussion to one topic at a time. Thanks!

[Charles_Hauser]

Sorry - understood

Charles