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

[shixq]

hi,I have been running dada2 on paired-end sequences, and it ran into a problem as following:
Plugin error from dada2: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and Debug info has been saved to /tmp/qiime2-q2cli-err-3jhw1xz_.log

i have try to truncate any of low quality positions off of my reverse reads, and it does't work

##i gave the following command
2qiime dada2 denoise-paired
3 --i-demultiplexed-seqs paired-end-demux.qza
4 --p-trim-left-f 6
5 --p-trim-left-r 6
6 --p-trunc-len-f 130
7 --p-trunc-len-r 130
8 --o-table table.qza
9 --o-representative-sequences rep-seqs.qza
10 --o-denoising-stats denoising-stats.qza

#Debug log file
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/tmphkc9jaba/forward /tmp/tmphkc9jaba/reverse /tmp/tmphkc9jaba/output.tsv.biom /tmp/tmphkc9jaba/track.tsv /tmp/tmphkc9jaba/filt_f /tmp/tmphkc9jaba/filt_r 150 150 0 0 2.0 2 consensus 1.0 0 1000000

R version 3.4.1 (2017-06-30)
Loading required package: Rcpp
DADA2 R package version: 1.6.0

1. Filtering ..

2. Learning Error Rates
   2a) Forward Reads
   Initializing error rates to maximum possible estimate.
   Sample 1 - 94912 reads in 40002 unique sequences.
   Sample 2 - 139684 reads in 9861 unique sequences.
   selfConsist step 2
   selfConsist step 3
   selfConsist step 4
   selfConsist step 5
   selfConsist step 6
   selfConsist step 7
   selfConsist step 8
   selfConsist step 9
   selfConsist step 10
   Self-consistency loop terminated before convergence.
   2b) Reverse Reads
   Initializing error rates to maximum possible estimate.
   Sample 1 - 94912 reads in 4862 unique sequences.
   Sample 2 - 139684 reads in 10332 unique sequences.
   selfConsist step 2
   selfConsist step 3
   selfConsist step 4
   Convergence after 4 rounds.

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
   In addition: Warning message:
   In is.na(colnames(unqs[[i]])) :
   is.na() applied to non-(list or vector) of type 'NULL'
   Execution halted
   Traceback (most recent call last):
   File "/home/shixq/miniconda3/envs/qiime2-2018.4/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 229, in denoise_paired
   run_commands([cmd])
   File "/home/shixq/miniconda3/envs/qiime2-2018.4/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
   subprocess.run(cmd, check=True)
   File "/home/shixq/miniconda3/envs/qiime2-2018.4/lib/python3.5/subprocess.py", line 398, in run
   output=stdout, stderr=stderr)
   subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmphkc9jaba/forward', '/tmp/tmphkc9jaba/reverse', '/tmp/tmphkc9jaba/output.tsv.biom', '/tmp/tmphkc9jaba/track.tsv', '/tmp/tmphkc9jaba/filt_f', '/tmp/tmphkc9jaba/filt_r', '150', '150', '0', '0', '2.0', '2', 'consensus', '1.0', '0', '1000000']' returned non-zero exit status 1

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/shixq/miniconda3/envs/qiime2-2018.4/lib/python3.5/site-packages/q2cli/commands.py", line 274, in call
results = action(**arguments)
File "", line 2, in denoise_paired
File "/home/shixq/miniconda3/envs/qiime2-2018.4/lib/python3.5/site-packages/qiime2/sdk/action.py", line 231, in bound_callable
output_types, provenance)
File "/home/shixq/miniconda3/envs/qiime2-2018.4/lib/python3.5/site-packages/qiime2/sdk/action.py", line 366, in callable_executor
output_views = self._callable(**view_args)
File "/home/shixq/miniconda3/envs/qiime2-2018.4/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 244, 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.
#Additional information

It would be great if anyone could guide me.
The qiime 2 version that am using is qiime2 2018.6 and my i ran in linux(redhat)

[ebolyen (Evan Bolyen)]

Hi @shixq,

I don’t think I’ve seen the chimera filtering step fail in that way before. I don’t suppose you wouldn’t be able to send a DM with your demultiplexed QZA file?

(pinging @benjjneb in case he’s seen this before)

[thermokarst]

I don’t think I’ve seen the chimera filtering step fail in that way before.

We have a handful of examples of this on the forum — my understanding is this error happens when there are no SVs at this step — usually an indication of insufficient overlap or incorrect trim/trunc params. @shixq - can you share your quality plots from the demux summarize command?

[shixq]

@thermokarst, @ebolyen thanks a lot ，following is quality plots from the demux summarize command：

@thermokarst You are right, I just learned from squencing company that the overlap only 7-9bp, and I found some other topics in qiime2 forum which said a minimum of 20bp overlap was need for proper merging, is this correct？does it means the data can't be Processing in dada2? apart from deblur in qiime2, any suggestions for the analyse? @thermokarst, @ebolyen, thanks again!
Looking forward to hearing from you soon! -S

[ebolyen (Evan Bolyen)]

You should be able to use denoise-single instead in that case, as it will only consider the forward reads.

That being said, from the quality profile you’ve shared, those scores seem artificial. Is this data by chance from Mr. DNA?

[shixq]

Hello! Thanks for the new command. I had tried denoise-single before, and it real worked, but isn’t that reverse information lost, Is this a good choice? I got those two samples data from Sequencing company, how do I judge whether those scores are artificial data?

thanks for your patient！
shixq

[thermokarst]

how do I judge whether those scores are artificial data

Check out this post for a good example of a “typical” Illumina run. When we see very uniform Q-score distributions, it is a pretty good indication that some kind of post-processing has occurred. Talk to your sequencing center and ask them for more details.