Incorrect version of dada2 Installed

User Support
1

Hi,
I think my issues with denoising demultiplexed paired-end reads relates to this thread.
I keep getting an error after ~2 hours that the program has run, only using 2 samples now, tried as well to use multithreading (3), and I have 8GB RAM…
Any suggestions?

The command + verbose

qiime dada2 denoise-paired   --i-demultiplexed-seqs demux-paired-end_Sam_V4.qza   --o-table table-Sam-V4   --o-representative-sequences rep-seqs-Sam-V4   --p-trunc-len-f 200   --p-trunc-len-r 200 --p-n-threads 3 --verbose --p-n-reads-learn 1000
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/tmpci4clnuh/forward /tmp/tmpci4clnuh/reverse /tmp/tmpci4clnuh/output.tsv.biom /tmp/tmpci4clnuh/filt_f /tmp/tmpci4clnuh/filt_r 200 200 0 0 2.0 2 3 1000

R version 3.3.2 (2016-10-31) 
Loading required package: Rcpp
DADA2 R package version: 1.0.3 
1) Filtering ..
2) Learning Error Rates
2a) Forward Reads
Initial error matrix unspecified. Error rates will be initialized to the maximum possible estimate from this data.
Initializing error rates to maximum possible estimate.
Sample 1 - 350072 reads in 85402 unique sequences.
   selfConsist step 2 
   selfConsist step 3 
   selfConsist step 4 
   selfConsist step 5 


Convergence after  5  rounds.
Warning message:
In dada(drpsF, err = NULL, selfConsist = TRUE, multithread = multithread) :
  multithread is not a valid DADA option.
2b) Reverse Reads
Initial error matrix unspecified. Error rates will be initialized to the maximum possible estimate from this data.
Initializing error rates to maximum possible estimate.
Sample 1 - 350072 reads in 112056 unique sequences.
   selfConsist step 2 
   selfConsist step 3 
   selfConsist step 4 
   selfConsist step 5 


Convergence after  5  rounds.
Warning message:
In dada(drpsR, err = NULL, selfConsist = TRUE, multithread = multithread) :
  multithread is not a valid DADA option.

3) Denoise remaining samples .Warning messages:
1: In dada(drpF, err = errF, multithread = multithread) :
  multithread is not a valid DADA option.
2: In dada(drpR, err = errR, multithread = multithread) :
  multithread is not a valid DADA option.

The sequences being tabled vary in length.
4) Remove chimeras
Error in isBimeraDenovo(unqs[[i]], ..., verbose = verbose) : 
  unused argument (multithread = 3)
Calls: removeBimeraDenovo -> isBimeraDenovo
Execution halted
Traceback (most recent call last):
  File "/home/rahel/miniconda3/envs/qiime2-2017.2/lib/python3.5/site-packages/q2cli-2017.2.0-py3.5.egg/q2cli/commands.py", line 217, in __call__
    results = action(**arguments)
  File "<decorator-gen-133>", line 2, in denoise_paired
  File "/home/rahel/miniconda3/envs/qiime2-2017.2/lib/python3.5/site-packages/qiime2-2017.2.0-py3.5.egg/qiime2/sdk/action.py", line 171, in callable_wrapper
    output_types, provenance)
  File "/home/rahel/miniconda3/envs/qiime2-2017.2/lib/python3.5/site-packages/qiime2-2017.2.0-py3.5.egg/qiime2/sdk/action.py", line 248, in _callable_executor_
    output_views = callable(**view_args)
  File "/home/rahel/miniconda3/envs/qiime2-2017.2/lib/python3.5/site-packages/q2_dada2-2017.2.0-py3.5.egg/q2_dada2/_denoise.py", line 154, in denoise_paired
    run_commands([cmd])
  File "/home/rahel/miniconda3/envs/qiime2-2017.2/lib/python3.5/site-packages/q2_dada2-2017.2.0-py3.5.egg/q2_dada2/_plot.py", line 26, in run_commands
    subprocess.run(cmd, check=True)
  File "/home/rahel/miniconda3/envs/qiime2-2017.2/lib/python3.5/subprocess.py", line 708, in run
    output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmpci4clnuh/forward', '/tmp/tmpci4clnuh/reverse', '/tmp/tmpci4clnuh/output.tsv.biom', '/tmp/tmpci4clnuh/filt_f', '/tmp/tmpci4clnuh/filt_r', '200', '200', '0', '0', '2.0', '2', '3', '1000']' returned non-zero exit status 1

Plugin error from dada2:

  Command '['run_dada_paired.R', '/tmp/tmpci4clnuh/forward',
  '/tmp/tmpci4clnuh/reverse', '/tmp/tmpci4clnuh/output.tsv.biom',
  '/tmp/tmpci4clnuh/filt_f', '/tmp/tmpci4clnuh/filt_r', '200', '200',
  '0', '0', '2.0', '2', '3', '1000']' returned non-zero exit status 1

See above for debug info.
2

Hi @rahel_park, it looks like you are running an old version of dada2 (that doesn't support multithreaded operation). From the output you posted above:

DADA2 R package version: 1.0.3

Did you install dada2 using the instructions on the QIIME 2 install page? You should be able to follow the instructions there to either upgrade or perform a fresh install (the directions are the same either way) of dada2 in that conda environment (don't forget to activate your conda environment with qiime2 installed in it first). Please let us know if that works for you, or if you get stuck on anything. Thanks!

1 Like
3

Hi @thermokarst , I followed the instructions, but that resulted in DADA2 version 1.0.3 (see below)
Using the tar.gz file I got the version 1.2.0 and now it looks that everything worked fine :slight_smile:

$ CDPATH= R -e 'source("https://bioconductor.org/biocLite.R"); biocLite("dada2")'

R version 3.3.2 (2016-10-31) -- "Sincere Pumpkin Patch"
Copyright (C) 2016 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu (64-bit)

R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.

  Natural language support but running in an English locale

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.

> source("https://bioconductor.org/biocLite.R"); biocLite("dada2")
Bioconductor version 3.3 (BiocInstaller 1.22.3), ?biocLite for help
A newer version of Bioconductor is available for this version of R,
  ?BiocUpgrade for help
BioC_mirror: https://bioconductor.org
Using Bioconductor 3.3 (BiocInstaller 1.22.3), R 3.3.2 (2016-10-31).
Installing package(s) ‘dada2’
**trying URL 'https://bioconductor.org/packages/3.3/bioc/src/contrib/dada2_1.0.3.tar.gz'**
Content type 'application/x-gzip' length 1271470 bytes (1.2 MB)
==================================================
downloaded 1.2 MB

* installing *source* package ‘dada2’ ...
** libs
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c RcppExports.cpp -o RcppExports.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c Rmain.cpp -o Rmain.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c chimera.cpp -o chimera.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c cluster.cpp -o cluster.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c error.cpp -o error.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c evaluate.cpp -o evaluate.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c filter.cpp -o filter.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c misc.cpp -o misc.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c nwalign_endsfree.cpp -o nwalign_endsfree.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c nwalign_vectorized.cpp -o nwalign_vectorized.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c pval.cpp -o pval.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c strmap.cpp -o strmap.o
g++ -I/usr/share/R/include -DNDEBUG   -I"/home/rahel/R/x86_64-pc-linux-gnu-library/3.3/Rcpp/include"   -fpic  -g -O2 -fstack-protector-strong -Wformat -Werror=format-security -Wdate-time -D_FORTIFY_SOURCE=2 -g  -c taxonomy.cpp -o taxonomy.o
g++ -shared -L/usr/lib/R/lib -Wl,-Bsymbolic-functions -Wl,-z,relro -o dada2.so RcppExports.o Rmain.o chimera.o cluster.o error.o evaluate.o filter.o misc.o nwalign_endsfree.o nwalign_vectorized.o pval.o strmap.o taxonomy.o -L/usr/lib/R/lib -lR
installing to /home/rahel/R/x86_64-pc-linux-gnu-library/3.3/dada2/libs
** R
** data
*** moving datasets to lazyload DB
** inst
** preparing package for lazy loading
** help
*** installing help indices
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (dada2)

The downloaded source packages are in
	‘/tmp/RtmpLNoKwk/downloaded_packages’
Old packages: 'ade4', 'annotate', 'ape', 'chron', 'cluster', 'colorspace',
  'curl', 'data.table', 'DBI', 'digest', 'evaluate', 'ggplot2', 'git2r',
  'htmlwidgets', 'jsonlite', 'knitr', 'lattice', 'Matrix', 'mgcv', 'networkD3',
  'nlme', 'openssl', 'R6', 'Rcpp', 'reshape2', 'rmarkdown', 'RSQLite',
  'scales', 'shiny', 'shinythemes', 'stringi', 'stringr', 'survival', 'vegan',
  'XML', 'yaml', 'cluster', 'lattice', 'Matrix', 'mgcv', 'nlme', 'survival'
2 Likes
4

Hi @rahel_park, glad to hear that tar.gz worked for you. Looking at your logs, it appears that you aren’t actually installing this in a conda environment (your installation paths are picking up a user-level install of R, instead of an environment-level install of R). Unfortunately, this means you just installed dada2 version 1.2.0 into your user-level installation of R, which can impact any other non-qiime2 analyses you may run with dada2 (if you needed that old version for some particular reason). We are currently testing out a simpler installation method of dada2, using conda for everything, so hopefully this problem goes away in the future. I just wanted to give you a head’s-up. Thanks!

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