Plugin error from dada2 in R

User Support
1

Hi,
I have faced such kind of problem after the denoising step:
Plugin error from dada2:

An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

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

The command I use was:
qiime dada2 denoise-single \

--i-demultiplexed-seqs paired-end-demux.qza
--p-trim-left 0
--p-trunc-len 248
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza
--o-denoising-stats stats-dada2.qza

The detailed error was:
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_single.R /tmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-10c1gi27 /tmp/tmponmavjt7/output.tsv.biom /tmp/tmponmavjt7/track.tsv /tmp/tmponmavjt7 248 0 2.0 2 Inf independent consensus 1.0 1 1000000 NULL 16

R version 4.0.2 (2020-06-22)
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.6 / RcppParallel: 5.0.2

  1. Filtering ......................................................................................................
  2. Learning Error Rates

I have no idea how to encounter this problem. I am very much appreciated it if anyone could guide me.

2

Hello! Welcome to the forum!
You are passing

paired data to

Dada2 plugin for single end data.
Here is the link to Dada2 paired plugin

1 Like
3

Hi@timanix!

Thank you for your answer.

However, after I changed my code to

qiime dada2 denoise-paired \

--i-demultiplexed-seqs paired-end-demux.qza
--p-trim-left-f 0
--p-trunc-len-f 248
--p-trunc-len-r 248
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza
--o-denoising-stats stats-dada2.qza

The same problem still exists.
Command: run_dada_paired.R /tmp/tmpotlnvtne/forward /tmp/tmpotlnvtne/reverse /tmp/tmpotlnvtne/output.tsv.biom /tmp/tmpotlnvtne/track.tsv /tmp/tmpotlnvtne/filt_f /tmp/tmpotlnvtne/filt_r 248 248 0 0 2.0 2.0 2 independent consensus 1.0 1 1000000

R version 4.0.2 (2020-06-22)
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.6 / RcppParallel: 5.0.2

  1. Filtering ......................................................................................................
  2. Learning Error Rates

Thank you!

5

Hi @Yuki - we need the entire error output in order to help - there appears to be quite a bit missing right after "2. Learning Error Rates"

Please re-run with the --verbose flag and copy and paste everything here. Thanks!

:qiime2:

7

Hi!

Thank you for your message.

If I re-run the code:

qiime dada2 denoise-paired \

--i-demultiplexed-seqs paired-end-demux.qza
--p-trim-left-f 0
--p-trunc-len-f 247
--p-trunc-len-r 248
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza
--o-denoising-stats stats-dada2.qza
--verbose

The error message is:
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_paired.R /tmp/tmpy3sel6wj/forward /tmp/tmpy3sel6wj/reverse /tmp/tmpy3sel6wj/output.tsv.biom /tmp/tmpy3sel6wj/track.tsv /tmp/tmpy3sel6wj/filt_f /tmp/tmpy3sel6wj/filt_r 247 248 0 0 2.0 2.0 2 independent consensus 1.0 1 1000000

R version 4.0.2 (2020-06-22)
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.6 / RcppParallel: 5.0.2

  1. Filtering
    ......................................................................................................
  2. Learning Error Rates
    200758142 total bases in 812786 reads from 102 samples will be used for learning the error rates.
    Error: cannot allocate vector of size 95.4 Mb
    Execution halted
    Warning message:
    system call failed: Cannot allocate memory
    Traceback (most recent call last):
    File "/home/qiime2/miniconda/envs/qiime2-2021.2/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 264, in denoise_paired
    run_commands([cmd])
    File "/home/qiime2/miniconda/envs/qiime2-2021.2/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
    subprocess.run(cmd, check=True)
    File "/home/qiime2/miniconda/envs/qiime2-2021.2/lib/python3.6/subprocess.py", line 438, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmpy3sel6wj/forward', '/tmp/tmpy3sel6wj/reverse', '/tmp/tmpy3sel6wj/output.tsv.biom', '/tmp/tmpy3sel6wj/track.tsv', '/tmp/tmpy3sel6wj/filt_f', '/tmp/tmpy3sel6wj/filt_r', '247', '248', '0', '0', '2.0', '2.0', '2', 'independent', 'consensus', '1.0', '1', '1000000']' returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/qiime2/miniconda/envs/qiime2-2021.2/lib/python3.6/site-packages/q2cli/commands.py", line 329, in call
results = action(**arguments)
File "", line 2, in denoise_paired
File "/home/qiime2/miniconda/envs/qiime2-2021.2/lib/python3.6/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
output_types, provenance)
File "/home/qiime2/miniconda/envs/qiime2-2021.2/lib/python3.6/site-packages/qiime2/sdk/action.py", line 390, in callable_executor
output_views = self._callable(**view_args)
File "/home/qiime2/miniconda/envs/qiime2-2021.2/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 279, in denoise_paired
" and stderr to learn more." % e.returncode)
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

If possible, could you please give me some suggestions?
Actually I have 100 samples, but if I run several or about 25 samples for test, this code goes very well; if I run 100 samples, it will have error (the details are above). I first thought this might be lack of computer memory of my computer, but I tried run this code through my university's cluster, it still have bugs.

Thank you.

8

Hi there @Yuki, I'm so sorry for the slow reply.

That thought is right, please check these lines of the error message:

My next suggestion is to run on your institutional cluster, but you mentioned you did that already and it didn't work - can you please provide us with the command you ran and the error you observed? Please note, we can't really help you unless we have those details, otherwise there are a million different things that could be implicated. Thanks!

:qiime2:

1 Like
9

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