error with dada2 - paired end sequences 2x250 bp

Hi, i was running dada2 in qiime2 and i’ve got this error:

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/tmpd8tc0ndt/forward /tmp/tmpd8tc0ndt/reverse /tmp/tmpd8tc0ndt/output.tsv.biom /tmp/tmpd8tc0ndt/track.tsv /tmp/tmpd8tc0ndt/filt_f /tmp/tmpd8tc0ndt/filt_r 0 0 0 0 2.0 2 consensus 1.0 20 1000000

R version 3.5.1 (2018-07-02)
Loading required package: Rcpp
DADA2: 1.10.0 / Rcpp: 1.0.1 / RcppParallel: 4.4.2

  1. Filtering .
  2. Learning Error Rates
    32778867 total bases in 131219 reads from 1 samples will be used for learning the error rates.
    32778867 total bases in 131219 reads from 1 samples will be used for learning the error rates.
  3. Denoise remaining samples .
  4. Remove chimeras (method = consensus)
    Error in isBimeraDenovoTable(unqs[[i]], …, verbose = verbose) :
    Input must be a valid sequence table.
    Calls: removeBimeraDenovo -> isBimeraDenovoTable
    Execution halted
    Traceback (most recent call last):
    File “/illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 231, in denoise_paired
    run_commands([cmd])
    File “/illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 36, in run_commands
    subprocess.run(cmd, check=True)
    File “/illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/subprocess.py”, line 418, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command ‘[‘run_dada_paired.R’, ‘/tmp/tmpd8tc0ndt/forward’, ‘/tmp/tmpd8tc0ndt/reverse’, ‘/tmp/tmpd8tc0ndt/output.tsv.biom’, ‘/tmp/tmpd8tc0ndt/track.tsv’, ‘/tmp/tmpd8tc0ndt/filt_f’, ‘/tmp/tmpd8tc0ndt/filt_r’, ‘0’, ‘0’, ‘0’, ‘0’, ‘2.0’, ‘2’, ‘consensus’, ‘1.0’, ‘20’, ‘1000000’]’ returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File “/illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/site-packages/q2cli/commands.py”, line 311, in call
results = action(**arguments)
File “</illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/site-packages/decorator.py:decorator-gen-451>”, line 2, in denoise_paired
File “/illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 231, in bound_callable
output_types, provenance)
File “/illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 365, in callable_executor
output_views = self._callable(**view_args)
File “/illumina/anasantos/software/miniconda31/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 246, 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.

Anyone knows what it means?

The sample in question had this table:

151878 (input) 131219(filtered) 127552(denoised) 0(merged) 0 (non-chimeric)

thanks

Hi @annsantos!

The error message is a bit confusing, but basically we see this message when DADA2 is unable to join any reads. Can you please provide the command you ran, and some more info about the target amplicon, region, primers, etc?

1 Like

Sure,
The command was:

qiime dada2 denoise-paired
–i-demultiplexed-seqs rawdata-paired-end.qza
–p-trunc-len-f 240
–p-trunc-len-r 240
–o-table table.qza
–o-representative-sequences rep-seqs.qza
–o-denoising-stats denoising-stats.qza \

I used 342F and 806R primers for V3-V4 region, that is about 460 bp.

Thanks for sharing! So, truncating both fwd and rev at 240, and accounting for DADA2’s 20nt min overlap, that leaves you with 240+240-20=460nts. My guess is that this is a bit too tight for 342F-806R (464nts). Any chance you can loosen up the truncation values? Feel free to share a demux summarize visualization if you want us to take a peek. Thanks! :t_rex: