Qiime2-amplicon-2023.9: An error was encountered while running DADA2 in R (return code 255)

Mareike · December 28, 2023, 4:32pm

Hi, I'm new to Qiime2 and this forum.

When following the moving pictures tutorial with this code:

qiime dada2 denoise-single --verbose
--i-demultiplexed-seqs demux.qza
--p-trim-left 0
--p-trunc-len 120
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza
--o-denoising-stats stats-dada2.qza

I encountered the following error:

Running external command line application(s). This may print messages to stdout and/or stderr.
The command(s) being run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.

Command: run_dada.R --input_directory /tmp/qiime2/marei/data/42c8b9ea-bba3-4784-a39f-6a6542afab78/data --output_path /tmp/tmp4zdit9ps/output.tsv.biom --output_track /tmp/tmp4zdit9ps/track.tsv --filtered_directory /tmp/tmp4zdit9ps --truncation_length 120 --trim_left 0 --max_expected_errors 2.0 --truncation_quality_score 2 --max_length Inf --pooling_method independent --chimera_method consensus --min_parental_fold 1.0 --allow_one_off False --num_threads 1 --learn_min_reads 1000000 --homopolymer_gap_penalty NULL --band_size 16

Rscript execution error: No such file or directory
Traceback (most recent call last):
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 220, in _denoise_single
run_commands([cmd])
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/subprocess.py", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/tmp/qiime2/marei/data/42c8b9ea-bba3-4784-a39f-6a6542afab78/data', '--output_path', '/tmp/tmp4zdit9ps/output.tsv.biom', '--output_track', '/tmp/tmp4zdit9ps/track.tsv', '--filtered_directory', '/tmp/tmp4zdit9ps', '--truncation_length', '120', '--trim_left', '0', '--max_expected_errors', '2.0', '--truncation_quality_score', '2', '--max_length', 'Inf', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '1.0', '--allow_one_off', 'False', '--num_threads', '1', '--learn_min_reads', '1000000', '--homopolymer_gap_penalty', 'NULL', '--band_size', '16']' returned non-zero exit status 255.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2cli/commands.py", line 520, in call
results = self._execute_action(
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2cli/commands.py", line 581, in _execute_action
results = action(**arguments)
File "", line 2, in denoise_single
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self.callable_executor(
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/qiime2/sdk/action.py", line 566, in callable_executor
output_views = self._callable(**view_args)
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 244, in denoise_single
return _denoise_single(
File "/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 229, in _denoise_single
raise Exception("An error was encountered while running DADA2"
Exception: An error was encountered while running DADA2 in R (return code 255), please inspect stdout and stderr to learn more.

Plugin error from dada2:

An error was encountered while running DADA2 in R (return code 255), please inspect stdout and stderr to learn more.

See above for debug info.

I searched the forum for the same error and found only one topic with the same return code. I already tried to deactivate and reactivate the environment, but the error will stay.

I am using the qiime2-amplicon-2023.9 version.

Thank you!

lizgehret · December 28, 2023, 5:43pm

Hi @Mareike,

Welcome to the forum

This error indicates that the R script QIIME 2 is using to run DADA2 is unable to be found within the expected location in your conda environment. This can happen when there is an existing version of R on your machine that gets mixed up with the instance of R that's included in your QIIME 2 environment.

While in the same QIIME 2 environment (e.g. qiime2-amplicon-2023.9), can you please run the following commands and share the outputs in your response?

env

which R

Thanks!

Mareike · December 29, 2023, 3:03pm

Thanks, lizgehret, for your responspe!

Here are the outsputs for

$ env
SHELL=/bin/bash
WSL2_GUI_APPS_ENABLED=1
CONDA_EXE=/home/marei/miniconda3/bin/conda
_CE_M=
WSL_DISTRO_NAME=Ubuntu
PYTHONNOUSERSITE=/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/python*/site-packages/
JAVA_HOME=/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/jvm
JAVA_LD_LIBRARY_PATH=/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/jvm/lib/server
XML_CATALOG_FILES=file:///home/marei/miniconda3/envs/qiime2-amplicon-2023.9/etc/xml/catalog file:///etc/xml/catalog
NAME=LMMibi
PWD=/home/marei
GSETTINGS_SCHEMA_DIR=/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/share/glib-2.0/schemas
LOGNAME=marei
CONDA_PREFIX=/home/marei/miniconda3/envs/qiime2-amplicon-2023.9
GSETTINGS_SCHEMA_DIR_CONDA_BACKUP=
RSTUDIO_WHICH_R=/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/bin/R
MOTD_SHOWN=update-motd
HOME=/home/marei
LANG=C.UTF-8
WSL_INTEROP=/run/WSL/608_interop
LS_COLORS=rs=0:di=01;34:ln=01;36:mh=00:pi=40;33:so=01;35:do=01;35:bd=40;33;01:cd=40;33;01:or=40;31;01:mi=00:su=37;41:sg=30;43:ca=30;41:tw=30;42:ow=34;42:st=37;44:ex=01;32:.tar=01;31:.tgz=01;31:.arc=01;31:.arj=01;31:.taz=01;31:.lha=01;31:.lz4=01;31:.lzh=01;31:.lzma=01;31:.tlz=01;31:.txz=01;31:.tzo=01;31:.t7z=01;31:.zip=01;31:.z=01;31:.dz=01;31:.gz=01;31:.lrz=01;31:.lz=01;31:.lzo=01;31:.xz=01;31:.zst=01;31:.tzst=01;31:.bz2=01;31:.bz=01;31:.tbz=01;31:.tbz2=01;31:.tz=01;31:.deb=01;31:.rpm=01;31:.jar=01;31:.war=01;31:.ear=01;31:.sar=01;31:.rar=01;31:.alz=01;31:.ace=01;31:.zoo=01;31:.cpio=01;31:.7z=01;31:.rz=01;31:.cab=01;31:.wim=01;31:.swm=01;31:.dwm=01;31:.esd=01;31:.jpg=01;35:.jpeg=01;35:.mjpg=01;35:.mjpeg=01;35:.gif=01;35:.bmp=01;35:.pbm=01;35:.pgm=01;35:.ppm=01;35:.tga=01;35:.xbm=01;35:.xpm=01;35:.tif=01;35:.tiff=01;35:.png=01;35:.svg=01;35:.svgz=01;35:.mng=01;35:.pcx=01;35:.mov=01;35:.mpg=01;35:.mpeg=01;35:.m2v=01;35:.mkv=01;35:.webm=01;35:.webp=01;35:.ogm=01;35:.mp4=01;35:.m4v=01;35:.mp4v=01;35:.vob=01;35:.qt=01;35:.nuv=01;35:.wmv=01;35:.asf=01;35:.rm=01;35:.rmvb=01;35:.flc=01;35:.avi=01;35:.fli=01;35:.flv=01;35:.gl=01;35:.dl=01;35:.xcf=01;35:.xwd=01;35:.yuv=01;35:.cgm=01;35:.emf=01;35:.ogv=01;35:.ogx=01;35:.aac=00;36:.au=00;36:.flac=00;36:.m4a=00;36:.mid=00;36:.midi=00;36:.mka=00;36:.mp3=00;36:.mpc=00;36:.ogg=00;36:.ra=00;36:.wav=00;36:.oga=00;36:.opus=00;36:.spx=00;36:.xspf=00;36:
WAYLAND_DISPLAY=wayland-0
CONDA_PROMPT_MODIFIER=(qiime2-amplicon-2023.9)
R_LIBS_USER=/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/lib/R/library/
JAVA_LD_LIBRARY_PATH_BACKUP=
LESSCLOSE=/usr/bin/lesspipe %s %s
TERM=xterm-256color
_CE_CONDA=
LESSOPEN=| /usr/bin/lesspipe %s
USER=marei
CONDA_SHLVL=2
DISPLAY=:0
SHLVL=1
MPLBACKEND=Agg
CONDA_PYTHON_EXE=/home/marei/miniconda3/bin/python
XDG_RUNTIME_DIR=/run/user/1000/
CONDA_DEFAULT_ENV=qiime2-amplicon-2023.9
WSLENV=
JAVA_HOME_CONDA_BACKUP=
XDG_DATA_DIRS=/usr/local/share:/usr/share:/var/lib/snapd/desktop
PATH=/home/marei/bin:/home/marei/miniconda3/envs/qiime2-amplicon-2023.9/bin:/home/marei/miniconda3/condabin:/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin:/usr/games:/usr/local/games:/usr/lib/wsl/lib:/mnt/c/Program Files (x86)/Common Files/Oracle/Java/javapath:/mnt/c/Windows/system32:/mnt/c/Windows:/mnt/c/Windows/System32/Wbem:/mnt/c/Windows/System32/WindowsPowerShell/v1.0:/mnt/c/Windows/System32/OpenSSH:/mnt/c/Program Files/dotnet:/mnt/c/Program Files (x86)/NVIDIA Corporation/PhysX/Common:/mnt/c/Users/marei/AppData/Local/Microsoft/WindowsApps:/snap/bin
DBUS_SESSION_BUS_ADDRESS=unix:path=/run/user/1000/bus
HOSTTYPE=x86_64
CONDA_PREFIX_1=/home/marei/miniconda3
PULSE_SERVER=unix:/mnt/wslg/PulseServer
_=/usr/bin/env

$ which R
/home/marei/bin/R

lizgehret · December 29, 2023, 7:57pm

Hi @Mareike,

Thanks for providing those details!

It's interesting because within your conda environment, the R-related environment variables are in the expected locations. But, the which R command does show that you do in fact have another version of R outside of your QIIME 2 environment, which is most likely what's causing this issue.

Is this your first time installing and using QIIME 2 on this machine?

Mareike · December 31, 2023, 2:47pm

Hi @lizgehret,

yes, it is the first time installing and using QIIME 2 on this machine. Is there any way to fix this issue, for example by deleting the other R version outside of the QIIME 2 environment?

Thank you!

colinbrislawn · January 2, 2024, 2:30am

Yes, that should work!

I only install R using conda for this exact reason; I don't want 'extra' copies of R on my machine to be confused for the R needed by each of my conda environments.

Mareike · January 2, 2024, 4:38pm

Thanks, @colinbrislawn, that worked.

When I now type $ which R, the output is: /home/marei/miniconda3/envs/qiime2-amplicon-2023.9/bin/R

And the dada2 command runs without any problems.

Thanks again for the help @lizgehret and @colinbrislawn!

system · February 2, 2024, 10:38pm

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