dada2 denoise-paired R

Hello everyone!

I am a predoctoral student and I am using QIIME2 to analyze results. I have been used qiime2 without any problem until I used another dataset. The data is formed by 12 paired-end sequences with were previously demultiplexed. I imported then with

«qiime tools import » command with the flags --type as ‘SampleData[PairedEndSequencesWithQuality]’ and the --input-format with Casava paired end option. The import didn’t show problems.

However, when I tried to run the «qiime dada2 denoise-paired» command (that I had used before without any problems),

qiime dada2 denoise-paired --i-demultiplexed-seqs demux-16sdic2019.qza --p-trunc-len-f 215 --p-trunc-len-r 215 --o-table table16sdic2019.qza --o-representative-sequences reps-seq-16sdic2019.qza --o-denoising-stats denoising-stats.qza --verbose

The shell showed the next message:

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/tmpxg8hi8t5/forward /tmp/tmpxg8hi8t5/reverse /tmp/tmpxg8hi8t5/output.tsv.biom /tmp/tmpxg8hi8t5/track.tsv /tmp/tmpxg8hi8t5/filt_f /tmp/tmpxg8hi8t5/filt_r 215 215 0 0 2.0 2.0 2 12 independent consensus 1.0 1 1000000

R version 4.0.3 (2020-10-10)
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.6 / RcppParallel: 5.1.4

  1. Filtering Error in validObject(.Object) :
    invalid class “SRFilterResult” object: superclass “Mnumeric” not defined in the environment of the object’s class
    Ejecución interrumpida
    Traceback (most recent call last):
    File “/home/elsa/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_dada2/_denoise.py”, line 266, in denoise_paired
    run_commands([cmd])
    File “/home/elsa/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_dada2/_denoise.py”, line 36, in run_commands
    subprocess.run(cmd, check=True)
    File “/home/elsa/miniconda3/envs/qiime2-2021.4/lib/python3.8/subprocess.py”, line 516, in run
    raise CalledProcessError(retcode, process.args,
    subprocess.CalledProcessError: Command ‘[‘run_dada_paired.R’, ‘/tmp/tmpxg8hi8t5/forward’, ‘/tmp/tmpxg8hi8t5/reverse’, ‘/tmp/tmpxg8hi8t5/output.tsv.biom’, ‘/tmp/tmpxg8hi8t5/track.tsv’, ‘/tmp/tmpxg8hi8t5/filt_f’, ‘/tmp/tmpxg8hi8t5/filt_r’, ‘215’, ‘215’, ‘0’, ‘0’, ‘2.0’, ‘2.0’, ‘2’, ‘12’, ‘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/elsa/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2cli/commands.py”, line 329, in call
results = action(**arguments)
File “”, line 2, in denoise_paired
File “/home/elsa/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py”, line 244, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File “/home/elsa/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py”, line 390, in callable_executor
output_views = self._callable(**view_args)
File “/home/elsa/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_dada2/_denoise.py”, line 279, 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.

Anyone knows how can I resolve it? I have installed RESCRIPT library, but I didn’t change anything else. I also looked for other solutions in the forum (because Casava is really specific about file’s format name), but any of them worked.

Thank you so much for your help!

Elsa

Hi there @elsamdea!

This is a very rare error, we’ve only seen it here once or twice before. I don’t have any immediate advice, but it would be helpful to us if you confirmed some information about your QIIME 2 env. Can you please activate the conda environment that produced the failing command above, then run the following, and copy-and-paste the complete results:

conda list '^r-matrix$'

After that command, please run the following (also in the active QIIME 2 conda env):

conda list --revisions

Thanks!

2 Likes

Hi @thermokarst!

Thank you so much for you quick reply!

When I run the first command you wrote:

conda list '^r-matrix

The results are:

packages in environment at /home/elsa/miniconda3/envs/qiime2-2021.4:

Name Version Build Channel

r-matrix 1.3_2 r40he454529_0 conda-forge

And with the second one:

conda list --revisions
2021-06-05 13:21:45 (rev 0)
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+q2-composition-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-cutadapt-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-dada2-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-deblur-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-demux-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-diversity-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-diversity-lib-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-emperor-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-feature-classifier-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-feature-table-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-fragment-insertion-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-gneiss-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-longitudinal-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-metadata-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-mystery-stew-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-phylogeny-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-quality-control-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-quality-filter-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-sample-classifier-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-taxa-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-types-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2-vsearch-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2cli-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2galaxy-2021.4.0 (qiime2/label/r2021.4/linux-64)
+q2templates-2021.4.0 (qiime2/label/r2021.4/linux-64)
+qiime2-2021.4.0 (qiime2/label/r2021.4/linux-64)
+qt-5.12.9 (conda-forge/linux-64)
+r-assertthat-0.2.1 (conda-forge/noarch)
+r-backports-1.2.1 (conda-forge/linux-64)
+r-base-4.0.3 (conda-forge/linux-64)
+r-bh-1.75.0_0 (conda-forge/noarch)
+r-bitops-1.0_6 (conda-forge/linux-64)
+r-brio-1.1.1 (conda-forge/linux-64)
+r-callr-3.6.0 (conda-forge/noarch)
+r-cli-2.4.0 (conda-forge/noarch)
+r-cluster-2.1.1 (conda-forge/linux-64)
+r-colorspace-2.0_0 (conda-forge/linux-64)
+r-crayon-1.4.1 (conda-forge/noarch)
+r-desc-1.3.0 (conda-forge/noarch)
+r-diffobj-0.3.3 (conda-forge/linux-64)
+r-digest-0.6.27 (conda-forge/linux-64)
+r-ellipsis-0.3.1 (conda-forge/linux-64)
+r-evaluate-0.14 (conda-forge/noarch)
+r-fansi-0.4.2 (conda-forge/linux-64)
+r-farver-2.1.0 (conda-forge/linux-64)
+r-formatr-1.9 (conda-forge/noarch)
+r-futile.logger-1.4.3 (conda-forge/noarch)
+r-futile.options-1.0.1 (conda-forge/noarch)
+r-ggplot2-3.3.3 (conda-forge/noarch)
+r-glue-1.4.2 (conda-forge/linux-64)
+r-gtable-0.3.0 (conda-forge/noarch)
+r-hwriter-1.3.2 (conda-forge/noarch)
+r-isoband-0.2.4 (conda-forge/linux-64)
+r-jpeg-0.1_8.1 (conda-forge/linux-64)
+r-jsonlite-1.7.2 (conda-forge/linux-64)
+r-labeling-0.4.2 (conda-forge/noarch)
+r-lambda.r-1.2.4 (conda-forge/noarch)
+r-lattice-0.20_41 (conda-forge/linux-64)
+r-latticeextra-0.6_29 (conda-forge/noarch)
+r-lifecycle-1.0.0 (conda-forge/noarch)
+r-magrittr-2.0.1 (conda-forge/linux-64)
+r-mass-7.3_53.1 (conda-forge/linux-64)
+r-matrix-1.3_2 (conda-forge/linux-64)
+r-matrixstats-0.58.0 (conda-forge/linux-64)
+r-mgcv-1.8_34 (conda-forge/linux-64)
+r-munsell-0.5.0 (conda-forge/noarch)
+r-nlme-3.1_152 (conda-forge/linux-64)
+r-permute-0.9_5 (conda-forge/noarch)
+r-pillar-1.6.0 (conda-forge/noarch)
+r-pkgconfig-2.0.3 (conda-forge/noarch)
+r-pkgload-1.2.1 (conda-forge/linux-64)
+r-plyr-1.8.6 (conda-forge/linux-64)
+r-png-0.1_7 (conda-forge/linux-64)
+r-praise-1.0.0 (conda-forge/noarch)
+r-processx-3.5.1 (conda-forge/linux-64)
+r-ps-1.6.0 (conda-forge/linux-64)
+r-r6-2.5.0 (conda-forge/noarch)
+r-rcolorbrewer-1.1_2 (conda-forge/noarch)
+r-rcpp-1.0.6 (conda-forge/linux-64)
+r-rcppparallel-5.1.2 (conda-forge/linux-64)
+r-rcurl-1.98_1.3 (conda-forge/linux-64)
+r-rematch2-2.1.2 (conda-forge/noarch)
+r-reshape2-1.4.4 (conda-forge/linux-64)
+r-rlang-0.4.10 (conda-forge/linux-64)
+r-rprojroot-2.0.2 (conda-forge/noarch)
+r-rstudioapi-0.13 (conda-forge/noarch)
+r-scales-1.1.1 (conda-forge/noarch)
+r-snow-0.4_3 (conda-forge/noarch)
+r-stringi-1.5.3 (conda-forge/linux-64)
+r-stringr-1.4.0 (conda-forge/noarch)
+r-testthat-3.0.2 (conda-forge/linux-64)
+r-tibble-3.1.1 (conda-forge/linux-64)
+r-utf8-1.2.1 (conda-forge/linux-64)
+r-vctrs-0.3.7 (conda-forge/linux-64)
+r-vegan-2.5_7 (conda-forge/linux-64)
+r-viridislite-0.4.0 (conda-forge/noarch)
+r-waldo-0.2.5 (conda-forge/noarch)
+r-withr-2.4.2 (conda-forge/noarch)
+r-zeallot-0.1.0 (conda-forge/noarch)
+raxml-8.2.12 (bioconda/linux-64)
+readline-8.1 (conda-forge/linux-64)
+requests-2.25.1 (conda-forge/noarch)
+samtools-1.12 (bioconda/linux-64)
+scikit-bio-0.5.6 (conda-forge/linux-64)
+scikit-learn-0.24.1 (conda-forge/linux-64)
+scipy-1.6.2 (conda-forge/linux-64)
+seaborn-0.11.1 (conda-forge/noarch)
+seaborn-base-0.11.1 (conda-forge/noarch)
+sed-4.8 (conda-forge/linux-64)
+send2trash-1.5.0 (conda-forge/noarch)
+sepp-4.3.10 (bioconda/linux-64)
+setuptools-49.6.0 (conda-forge/linux-64)
+six-1.15.0 (conda-forge/noarch)
+sortmerna-2.0 (bioconda/linux-64)
+sqlite-3.35.4 (conda-forge/linux-64)
+statsmodels-0.12.2 (conda-forge/linux-64)
+sysroot_linux-64-2.12 (conda-forge/noarch)
+tbb-2020.2 (conda-forge/linux-64)
+terminado-0.9.4 (conda-forge/linux-64)
+testpath-0.4.4 (conda-forge/noarch)
+threadpoolctl-2.1.0 (conda-forge/noarch)
+tk-8.6.10 (conda-forge/linux-64)
+tktable-2.10 (conda-forge/linux-64)
+toml-0.10.2 (conda-forge/noarch)
+tornado-6.1 (conda-forge/linux-64)
+traitlets-5.0.5 (conda-forge/noarch)
+typing_extensions-3.7.4.3 (conda-forge/noarch)
+tzlocal-2.1 (conda-forge/noarch)
+unifrac-0.20.2 (bioconda/linux-64)
+urllib3-1.26.4 (conda-forge/noarch)
+vsearch-2.7.0 (bioconda/linux-64)
+wcwidth-0.2.5 (conda-forge/noarch)
+webencodings-0.5.1 (conda-forge/noarch)
+wheel-0.36.2 (conda-forge/noarch)
+widgetsnbextension-3.5.1 (conda-forge/linux-64)
+xopen-1.1.0 (conda-forge/linux-64)
+xorg-kbproto-1.0.7 (conda-forge/linux-64)
+xorg-libice-1.0.10 (conda-forge/linux-64)
+xorg-libsm-1.2.3 (conda-forge/linux-64)
+xorg-libx11-1.7.0 (conda-forge/linux-64)
+xorg-libxau-1.0.9 (conda-forge/linux-64)
+xorg-libxdmcp-1.1.3 (conda-forge/linux-64)
+xorg-libxext-1.3.4 (conda-forge/linux-64)
+xorg-libxrender-0.9.10 (conda-forge/linux-64)
+xorg-libxt-1.2.1 (conda-forge/linux-64)
+xorg-renderproto-0.11.1 (conda-forge/linux-64)
+xorg-xextproto-7.3.0 (conda-forge/linux-64)
+xorg-xproto-7.0.31 (conda-forge/linux-64)
+xz-5.2.5 (conda-forge/linux-64)
+yaml-0.2.5 (conda-forge/linux-64)
+zeromq-4.3.4 (conda-forge/linux-64)
+zipp-3.4.1 (conda-forge/noarch)
+zlib-1.2.11 (conda-forge/linux-64)
+zstd-1.4.9 (conda-forge/linux-64)

2021-06-05 14:01:18 (rev 1)
ca-certificates {2020.12.5 (conda-forge/linux-64) → 2021.5.30 (conda-forge/linux-64)}
certifi {2020.12.5 (conda-forge/linux-64) → 2021.5.30 (conda-forge/linux-64)}
+xmltodict-0.12.0 (conda-forge/noarch)

2021-06-06 13:09:28 (rev 2)
certifi {2021.5.30 (conda-forge/linux-64) → 2021.5.30 (defaults/linux-64)}
+q2-metabolomics-0.0.6 (mwang87/noarch)

2021-06-06 13:10:39 (rev 3)
ca-certificates {2021.5.30 (conda-forge/linux-64) → 2021.5.25 (defaults/linux-64)}
openssl {1.1.1k (conda-forge/linux-64) → 1.1.1k (defaults/linux-64)}

2021-06-06 14:40:48 (rev 4)
ca-certificates {2021.5.25 (defaults/linux-64) → 2021.5.30 (conda-forge/linux-64)}
certifi {2021.5.30 (defaults/linux-64) → 2021.5.30 (conda-forge/linux-64)}
openssl {1.1.1k (defaults/linux-64) → 1.1.1k (conda-forge/linux-64)}
pip {21.0.1 (conda-forge/noarch) → 21.1.2 (conda-forge/noarch)}
+appdirs-1.4.4 (conda-forge/noarch)
+cobra-0.22.0 (conda-forge/noarch)
+colorama-0.4.4 (conda-forge/noarch)
+commonmark-0.9.1 (conda-forge/noarch)
+depinfo-1.5.4 (conda-forge/noarch)
+diskcache-5.2.1 (conda-forge/noarch)
+glpk-4.65 (conda-forge/linux-64)
+gmp-6.2.1 (conda-forge/linux-64)
+gmpy2-2.1.0b1 (conda-forge/linux-64)
+h11-0.12.0 (conda-forge/noarch)
+h2-4.0.0 (conda-forge/linux-64)
+hpack-4.0.0 (conda-forge/noarch)
+httpcore-0.13.3 (conda-forge/noarch)
+httpx-0.18.1 (conda-forge/linux-64)
+hyperframe-6.0.1 (conda-forge/noarch)
+importlib_resources-5.1.4 (conda-forge/noarch)
+libflint-2.7.1 (conda-forge/linux-64)
+mpc-1.1.0 (conda-forge/linux-64)
+mpfr-4.0.2 (conda-forge/linux-64)
+mpmath-1.2.1 (conda-forge/noarch)
+optlang-1.5.2 (conda-forge/noarch)
+pipdeptree-1.0.0 (conda-forge/noarch)
+pydantic-1.8.2 (conda-forge/linux-64)
+python-libsbml-5.19.0 (conda-forge/linux-64)
+python-symengine-0.7.2 (conda-forge/linux-64)
+rfc3986-1.5.0 (conda-forge/noarch)
+rich-10.2.2 (conda-forge/linux-64)
+ruamel.yaml-0.17.7 (conda-forge/linux-64)
+ruamel.yaml.clib-0.2.2 (conda-forge/linux-64)
+sniffio-1.2.0 (conda-forge/linux-64)
+swiglpk-5.0.3 (conda-forge/linux-64)
+symengine-0.7.0 (conda-forge/linux-64)
+sympy-1.8 (conda-forge/linux-64)
+typing-extensions-3.7.4.3 (conda-forge/noarch)

2021-06-07 14:12:07 (rev 5)
certifi {2021.5.30 (conda-forge/linux-64) → 2021.5.30 (defaults/linux-64)}

2021-06-07 14:27:48 (rev 6)
certifi {2021.5.30 (defaults/linux-64) → 2021.5.30 (conda-forge/linux-64)}
+colormath-3.0.0 (conda-forge/noarch)
+coverage-5.5 (conda-forge/linux-64)
+epa-ng-0.3.8 (bioconda/linux-64)
+gappa-0.7.1 (bioconda/linux-64)
+picrust2-2.0.3_b (bioconda/noarch)
+pytest-cov-2.12.1 (conda-forge/noarch)
+r-castor-1.6.7 (conda-forge/linux-64)
+r-naturalsort-0.1.3 (conda-forge/noarch)
+r-nloptr-1.2.2.2 (conda-forge/linux-64)
+r-rcppeigen-0.3.3.9.1 (conda-forge/linux-64)
+r-rspectra-0.16_0 (conda-forge/linux-64)
+spectra-0.0.11 (conda-forge/noarch)

Maybe the problem is related to pycrust2 installation? I had completed it succesfully, but it didn’t work and didn’t appear as qiime plugging when I run

qiime

The results are:

Usage: qiime [OPTIONS] COMMAND [ARGS]…

QIIME 2 command-line interface (q2cli)

To get help with QIIME 2, visit https://qiime2.org.

To enable tab completion in Bash, run the following command or add it to
your .bashrc/.bash_profile:

  source tab-qiime

To enable tab completion in ZSH, run the following commands or add them to
your .zshrc:

  autoload -Uz compinit && compinit
  autoload bashcompinit && bashcompinit
  source tab-qiime

Options:
–version Show the version and exit.
–help Show this message and exit.

Commands:
info Display information about current deployment.
tools Tools for working with QIIME 2 files.
dev Utilities for developers and advanced users.
alignment Plugin for generating and manipulating alignments.
composition Plugin for compositional data analysis.
cutadapt Plugin for removing adapter sequences, primers, and
other unwanted sequence from sequence data.

dada2 Plugin for sequence quality control with DADA2.
deblur Plugin for sequence quality control with Deblur.
demux Plugin for demultiplexing & viewing sequence quality.
diversity Plugin for exploring community diversity.
diversity-lib Plugin for computing community diversity.
emperor Plugin for ordination plotting with Emperor.
feature-classifier Plugin for taxonomic classification.
feature-table Plugin for working with sample by feature tables.
fragment-insertion Plugin for extending phylogenies.
gneiss Plugin for building compositional models.
longitudinal Plugin for paired sample and time series analyses.
metabolomics Plugin for the creation of a biom feature table for
metabolomics data.

metadata Plugin for working with Metadata.
micom Plugin for metabolic modeling of microbial communities.
phylogeny Plugin for generating and manipulating phylogenies.
quality-control Plugin for quality control of feature and sequence data.
quality-filter Plugin for PHRED-based filtering and trimming.
rescript Pipeline for reference sequence annotation and curation.
sample-classifier Plugin for machine learning prediction of sample
metadata.

taxa Plugin for working with feature taxonomy annotations.
vsearch Plugin for clustering and dereplicating with vsearch.

And after that qiime dada2 problems started.

Thank you so much!

Regards,

Elsa

Hi,

I’ve been having the same problem and so had a look on the dada2 issues pages (Error when running fastqPairedFilter · Issue #212 · benjjneb/dada2 · GitHub). It seems like the problem is to do with the version of Matrix.

conda list --revisions showed:
+r-matrix-1.3_2 (conda-forge/linux-64)

However, when inside R and using the packageVersion("Matrix") command, it was actually version 1.3.3.

To resolve this I had to do the following, as specified in the earier link:

remove.packages("Matrix")
install.packages("devtools")
devtools::install_version("Matrix", version = "1.3.2", repos = "http://cran.us.r-project.org")

I also needed to install conda install -c conda-forge r-gert too, as for some reason I couldn’t install this inside of R!

Hope this helps,
Sam

2 Likes

Oh thanks for pointing this out @SamMc, this is a really useful piece of information! I’m not a fan of removing and reinstalling via R in this case though, since now it is kind of ignoring the root of the issue (the environmental mismatch) that was already present, rather than dealing with it, but if it works for you, great!

My guess is that there are additional entries in your r libpath that is driving the mismatch, lets check (@elsamdea can you run this too):

# run this _inside_ the activated offending conda env
R -e ".libPaths()"

:qiime2:

1 Like

Just the one -

“/home/smcgreig/miniconda2/envs/qiime2-2021.2/lib/R/library”

Oh, thank you @SamMc!!

I will wait for @thermokarst reply. If he thinks that this is a good solution at the end, I will follow your instructions!

Hi @thermokarst

Sorry for the late reply. When I run this command inside conda qiime2-2021.4 env

R -e ".libPaths()"

The reply is:

R version 4.0.3 (2020-10-10) -- "Bunny-Wunnies Freak Out"
Copyright (C) 2020 The R Foundation for Statistical Computing
Platform: x86_64-conda-linux-gnu (64-bit)

R es un software libre y viene sin GARANTIA ALGUNA.
Usted puede redistribuirlo bajo ciertas circunstancias.
Escriba 'license()' o 'licence()' para detalles de distribucion.

R es un proyecto colaborativo con muchos contribuyentes.
Escriba 'contributors()' para obtener más información y
'citation()' para saber cómo citar R o paquetes de R en publicaciones.

Escriba 'demo()' para demostraciones, 'help()' para el sistema on-line de ayuda,
o 'help.start()' para abrir el sistema de ayuda HTML con su navegador.
Escriba 'q()' para salir de R.

> .libPaths()
[1] "/home/elsa/miniconda3/envs/qiime2-2021.4/lib/R/library"

Hmm, thanks @SamMc & @elsamdea - it looks like both of you have nice clean R paths… That’s strange, I’m not sure where the contamination is coming from…

@elsamdea, can you run the following, inside your conda env with QIIME 2 in it, and share the output:

R -e 'packageVersion("Matrix")'

Sure @thermokarst!

The output of that command is:

> packageVersion("Matrix")
[1] ‘1.3.4’