DADA2 plugin error during paired-end denoising (R return code 1)

[Pelin_Kaplan]

After running the qiime dada2 denoise-paired command with truncation lengths set to 250 bp for both forward and reverse reads, the analysis fails with the following error:

Plugin error from dada2: An error was encountered while running DADA2 in R (return code 1). Please inspect stdout and stderr to learn more.

Here is the exact command I executed:

qiime dada2 denoise-paired \
  --i-demultiplexed-seqs paired-end-demux.qza \
  --p-trim-left-f 21 \
  --p-trim-left-r 20 \
  --p-trunc-len-f 250 \
  --p-trunc-len-r 250 \
  --p-n-threads 8 \
  --o-table 16S-table.qza \
  --o-representative-sequences 16S-rep-seqs.qza \
  --o-denoising-stats 16S-denoising-stats.qza \
  --verbose

QIIME 2 version: 2024.10
Installation method: Conda environment (qiime2-amplicon-2024.10)

Could you please help me understand why this error occurs and how I can resolve it?

[lizgehret]

Hi @Pelin_Kaplan,

Welcome to the forum

Could you please copy and paste the entire traceback from your terminal (including your full command)? This will help us better determine where the issue lies. Thanks!

[Pelin_Kaplan]

(qiime2-amplicon-2024.10) pelin@LAPTOP-45GH5C0T:~$ cd denemey
(qiime2-amplicon-2024.10) pelin@LAPTOP-45GH5C0T:~/denemey$ qiime dada2 denoise-paired
--i-demultiplexed-seqs paired-end-demux.qza
--p-trim-left-f 21
--p-trim-left-r 20
--p-trunc-len-f 250
--p-trunc-len-r 250
--p-n-threads 8
--o-table 16S-table.qza
--o-representative-sequences 16S-rep-seqs.qza
--o-denoising-stats 16S-denoising-stats.qza
--verbose
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/tmpeca7klbu/forward --input_directory_reverse /tmp/tmpeca7klbu/reverse --output_path /tmp/tmpeca7klbu/output.tsv.biom --output_track /tmp/tmpeca7klbu/track.tsv --filtered_directory /tmp/tmpeca7klbu/filt_f --filtered_directory_reverse /tmp/tmpeca7klbu/filt_r --truncation_length 250 --truncation_length_reverse 250 --trim_left 21 --trim_left_reverse 20 --max_expected_errors 2.0 --max_expected_errors_reverse 2.0 --truncation_quality_score 2 --min_overlap 12 --pooling_method independent --chimera_method consensus --min_parental_fold 1.0 --allow_one_off False --num_threads 8 --learn_min_reads 1000000

R version 4.3.3 (2024-02-29)
Loading required package: Rcpp
DADA2: 1.30.0 / Rcpp: 1.0.13.1 / RcppParallel: 5.1.9
2) Filtering Error in names(answer) <- names1 :
'names' attribute [4] must be the same length as the vector [3]
4: mcmapply(fastqPairedFilter, mapply(c, fwd, rev, SIMPLIFY = FALSE),
mapply(c, filt, filt.rev, SIMPLIFY = FALSE), MoreArgs = list(truncQ = truncQ,
truncLen = truncLen, trimLeft = trimLeft, trimRight = trimRight,
maxLen = maxLen, minLen = minLen, maxN = maxN, minQ = minQ,
maxEE = maxEE, rm.phix = rm.phix, rm.lowcomplex = rm.lowcomplex,
orient.fwd = orient.fwd, matchIDs = matchIDs, id.sep = id.sep,
id.field = id.field, n = n, OMP = OMP, qualityType = qualityType,
compress = compress, verbose = verbose), mc.cores = ncores,
mc.silent = TRUE)
3: filterAndTrim(unfilts, filts, unfiltsR, filtsR, truncLen = c(truncLen,
truncLenR), trimLeft = c(trimLeft, trimLeftR), maxEE = c(maxEE,
maxEER), truncQ = truncQ, rm.phix = TRUE, multithread = multithread)
2: withCallingHandlers(expr, warning = function(w) if (inherits(w,
classes)) tryInvokeRestart("muffleWarning"))
1: suppressWarnings(filterAndTrim(unfilts, filts, unfiltsR, filtsR,
truncLen = c(truncLen, truncLenR), trimLeft = c(trimLeft,
trimLeftR), maxEE = c(maxEE, maxEER), truncQ = truncQ,
rm.phix = TRUE, multithread = multithread))
Traceback (most recent call last):
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_dada2/_denoise.py", line 353, in denoise_paired
run_commands([cmd])
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_dada2/_denoise.py", line 38, in run_commands
subprocess.run(cmd, check=True)
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/subprocess.py", line 526, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/tmp/tmpeca7klbu/forward', '--input_directory_reverse', '/tmp/tmpeca7klbu/reverse', '--output_path', '/tmp/tmpeca7klbu/output.tsv.biom', '--output_track', '/tmp/tmpeca7klbu/track.tsv', '--filtered_directory', '/tmp/tmpeca7klbu/filt_f', '--filtered_directory_reverse', '/tmp/tmpeca7klbu/filt_r', '--truncation_length', '250', '--truncation_length_reverse', '250', '--trim_left', '21', '--trim_left_reverse', '20', '--max_expected_errors', '2.0', '--max_expected_errors_reverse', '2.0', '--truncation_quality_score', '2', '--min_overlap', '12', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '1.0', '--allow_one_off', 'False', '--num_threads', '8', '--learn_min_reads', '1000000']' returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2cli/commands.py", line 530, in call
results = self._execute_action(
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2cli/commands.py", line 602, in _execute_action
results = action(**arguments)
File "", line 2, in denoise_paired
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/qiime2/sdk/action.py", line 299, in bound_callable
outputs = self.callable_executor(
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/qiime2/sdk/action.py", line 570, in callable_executor
output_views = self._callable(**view_args)
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_dada2/_denoise.py", line 366, in denoise_paired
raise Exception("An error was encountered while running DADA2"
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

Plugin error from dada2:

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

See above for debug info.
(qiime2-amplicon-2024.10) pelin@LAPTOP-45GH5C0T:~/denemey$ qiime dada2 denoise-paired \d
--i-demultiplexed-seqs paired-end-demux.qza
--p-trim-left-f 21
--p-trim-left-r 20
--p-trunc-len-f 260
--p-trunc-len-r 220
--p-n-threads 8
--o-table 16S-table.qza
--o-representative-sequences 16S-rep-seqs.qza
--o-denoising-stats 16S-denoising-stats.qza
--verbose
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/tmp1dixpgzs/forward --input_directory_reverse /tmp/tmp1dixpgzs/reverse --output_path /tmp/tmp1dixpgzs/output.tsv.biom --output_track /tmp/tmp1dixpgzs/track.tsv --filtered_directory /tmp/tmp1dixpgzs/filt_f --filtered_directory_reverse /tmp/tmp1dixpgzs/filt_r --truncation_length 260 --truncation_length_reverse 220 --trim_left 21 --trim_left_reverse 20 --max_expected_errors 2.0 --max_expected_errors_reverse 2.0 --truncation_quality_score 2 --min_overlap 12 --pooling_method independent --chimera_method consensus --min_parental_fold 1.0 --allow_one_off False --num_threads 8 --learn_min_reads 1000000

R version 4.3.3 (2024-02-29)
Loading required package: Rcpp
DADA2: 1.30.0 / Rcpp: 1.0.13.1 / RcppParallel: 5.1.9
2) Filtering Error in names(answer) <- names1 :
'names' attribute [4] must be the same length as the vector [3]
4: mcmapply(fastqPairedFilter, mapply(c, fwd, rev, SIMPLIFY = FALSE),
mapply(c, filt, filt.rev, SIMPLIFY = FALSE), MoreArgs = list(truncQ = truncQ,
truncLen = truncLen, trimLeft = trimLeft, trimRight = trimRight,
maxLen = maxLen, minLen = minLen, maxN = maxN, minQ = minQ,
maxEE = maxEE, rm.phix = rm.phix, rm.lowcomplex = rm.lowcomplex,
orient.fwd = orient.fwd, matchIDs = matchIDs, id.sep = id.sep,
id.field = id.field, n = n, OMP = OMP, qualityType = qualityType,
compress = compress, verbose = verbose), mc.cores = ncores,
mc.silent = TRUE)
3: filterAndTrim(unfilts, filts, unfiltsR, filtsR, truncLen = c(truncLen,
truncLenR), trimLeft = c(trimLeft, trimLeftR), maxEE = c(maxEE,
maxEER), truncQ = truncQ, rm.phix = TRUE, multithread = multithread)
2: withCallingHandlers(expr, warning = function(w) if (inherits(w,
classes)) tryInvokeRestart("muffleWarning"))
1: suppressWarnings(filterAndTrim(unfilts, filts, unfiltsR, filtsR,
truncLen = c(truncLen, truncLenR), trimLeft = c(trimLeft,
trimLeftR), maxEE = c(maxEE, maxEER), truncQ = truncQ,
rm.phix = TRUE, multithread = multithread))
Traceback (most recent call last):
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_dada2/_denoise.py", line 353, in denoise_paired
run_commands([cmd])
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_dada2/_denoise.py", line 38, in run_commands
subprocess.run(cmd, check=True)
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/subprocess.py", line 526, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/tmp/tmp1dixpgzs/forward', '--input_directory_reverse', '/tmp/tmp1dixpgzs/reverse', '--output_path', '/tmp/tmp1dixpgzs/output.tsv.biom', '--output_track', '/tmp/tmp1dixpgzs/track.tsv', '--filtered_directory', '/tmp/tmp1dixpgzs/filt_f', '--filtered_directory_reverse', '/tmp/tmp1dixpgzs/filt_r', '--truncation_length', '260', '--truncation_length_reverse', '220', '--trim_left', '21', '--trim_left_reverse', '20', '--max_expected_errors', '2.0', '--max_expected_errors_reverse', '2.0', '--truncation_quality_score', '2', '--min_overlap', '12', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '1.0', '--allow_one_off', 'False', '--num_threads', '8', '--learn_min_reads', '1000000']' returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2cli/commands.py", line 530, in call
results = self._execute_action(
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2cli/commands.py", line 602, in _execute_action
results = action(**arguments)
File "", line 2, in denoise_paired
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/qiime2/sdk/action.py", line 299, in bound_callable
outputs = self.callable_executor(
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/qiime2/sdk/action.py", line 570, in callable_executor
output_views = self._callable(**view_args)
File "/home/pelin/miniconda3/envs/qiime2-amplicon-2024.10/lib/python3.10/site-packages/q2_dada2/_denoise.py", line 366, in denoise_paired
raise Exception("An error was encountered while running DADA2"
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

Plugin error from dada2:

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

See above for debug info.

[colinbrislawn (Colin J Brislawn)]

@Pelin_Kaplan

2. Filtering Error in names(answer) <- names1 :
   'names' attribute [4] must be the same length as the vector [3]

Denoising error, names attribute must be the same length as vector

Hi @Roger_Huerlimann, Congratulations for running into one of the more cryptic error messages that dada2 can throw at you. This is effectively a memory error even though it does not look like one — or more specifically a multithreading issue that can be fixed by reducing the number of threads used. See here for some discussion: I think the dada2 version was upgraded somewhere in there, so it could be a version issue, but it could also be coincidence. Reduce the number of threads you are usi…

---

Thanks for posting all of that. Try rerunning with --p-n-threads 4 or maybe even --p-n-threads 2. That will take longer, but reduce the memory footprint so it can finish.

And while I'm giving advice, install the new version of Qiime2!

@Pelin_Kaplan

QIIME 2 version: 2024.10