An error was encountered while running DADA2 in R (return code 1), please inspect stout and stderr to learn more (Single end data)

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Hello

I received a fasta file with single end demultiplexed sequences and no quality file. Since I was interested in performing diversity analysis with qiime2, i converted it to fastq with galaxy online using Combine Fasta and Qual and later using fastq Groomer using the sanger and illumina 1.8+ option (theoretically should give a quality of 33). after that I made a Manifest with this exact data:
sample-id absolute-filepath
Data $PWD/qiime/Datos.fastqsanger

And I imported it using the following command since it wasn't one of the other formats in the tutorial:
qiime tools import --type SampleData[SequencesWithQuality]
--input-path qiime/manifest.txt
--output-path qiime/demux.qza --input-format SingleEndFastqManifestPhred33V2

the visualization data shows no bars on the graph because not all samples are on the correct phred offset, however changing the importing format to SingleEndFastqManifestPhred64V2 gives the exact same result:


Captura de Pantalla 2020-06-08 a la(s) 13.15.20
Captura de Pantalla 2020-06-08 a la(s) 13.15.28

The problems comes when I try to use dada2 to clean my file a bit. the exact command I use is:
qiime dada2 denoise-single
--i-demultiplexed-seqs qiime/demux.qza
--p-trim-left 0
--p-trunc-len 165
--o-representative-sequences rep-seqs.qza
--o-table table.qza
--o-denoising-stats stats.qza

The following error shows up:
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-g1fpiisb.log

There are two articles into this problem (im attaching both
Latest one Original one), and both proved to not be helpful in my case.

When i run the command with --verbose this comes up:

qiime dada2 denoise-single --i-demultiplexed-seqs qiime/demux.qza --p-trim-left 0 --p-trunc-len 165 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats 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_single.R /tmp/qiime2-archive-pkvh0ea0/8c508633-92cc-48e4-9e73-b89af7540eda/data /tmp/tmp0s1wsefe/output.tsv.biom /tmp/tmp0s1wsefe/track.tsv /tmp/tmp0s1wsefe 165 0 2.0 2 Inf consensus 1.0 1 1000000 NULL 16

R version 3.5.1 (2018-07-02)
Loading required package: Rcpp
DADA2: 1.10.0 / Rcpp: 1.0.2 / RcppParallel: 4.4.3

  1. Filtering .
  2. Learning Error Rates
    351615 total bases in 2131 reads from 1 samples will be used for learning the error rates.
    Error in err[c(1, 6, 11, 16), ] <- 1 :
    incorrect number of subscripts on matrix
    Execution halted
    Traceback (most recent call last):
    File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 154, in _denoise_single
    run_commands([cmd])
    File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
    subprocess.run(cmd, check=True)
    File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/subprocess.py", line 418, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command '['run_dada_single.R', '/tmp/qiime2-archive-pkvh0ea0/8c508633-92cc-48e4-9e73-b89af7540eda/data', '/tmp/tmp0s1wsefe/output.tsv.biom', '/tmp/tmp0s1wsefe/track.tsv', '/tmp/tmp0s1wsefe', '165', '0', '2.0', '2', 'Inf', 'consensus', '1.0', '1', '1000000', 'NULL', '16']' returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/q2cli/commands.py", line 327, in call
results = action(**arguments)
File "</hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/decorator.py:decorator-gen-457>", line 2, in denoise_single
File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/qiime2/sdk/action.py", line 240, in bound_callable
output_types, provenance)
File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/qiime2/sdk/action.py", line 383, in callable_executor
output_views = self._callable(**view_args)
File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 189, in denoise_single
band_size='16')
File "/hpcfs/apps/anaconda/3.7/envs/qiime2-2019.7/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 165, in _denoise_single
" 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.

My guess is that it has to somehow with the import format but I'm not really an expert with computers so i need help.

Can you confirm or correct my suspicions?

Thanks in advance :smiley_cat:

J

Hello @jd.lozano. I believe what you are experiencing here is similar to this Doing taxonomy analysis and getting abundancies with manifests. It would appear as though dada2 requires real quality scores in order to work because it expects a range of different scores for different reads not every score being identical.

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