Error when running q2-picrust2 in qiime2-2019.10

Hi everyone
I am trying to use PICRUSt for the first time. I have installed PICRUSt and the qiime plugin. I started following the tutorial in https://github.com/picrust/picrust2/wiki/q2-picrust2-Tutorial
The first time I ran the full pipeline command (copied and pasted from the tutorial) I got the followin notification:

Missing the following tools from your $PATH variable: epa-ng and gappa. Note that this QIIME2 command is for running the default PICRUSt2 pipeline, which includes steps for running sequence placement with EPA-NG. If you do not want to install the missing programs you can run sequence placement with q2-fragment-insertion and input this tree to the “qiime picrust2 custom_tree_pipeline” command

I have then tried to installed epa-ng using the following code:

#Epa-ng
tar -xzf epa-ng-0.2.1-beta-dev.tar.gz
cd epa-ng-0.2.1-beta-dev
make
ln -s $PWD/bin/epa-ng /usr/local/bin

And this is what I got back:

5 warnings and 2 errors generated.

make[3]: *** [libs/genesis/lib/genesis/CMakeFiles/genesis_lib_static.dir/build.make:63: libs/genesis/lib/genesis/CMakeFiles/genesis_lib_static.dir/////genesis_unity_sources/lib/all.cpp.o] Error 1

make[3]: Leaving directory ‘/Users/cota/picrust2-2.0.3-b/placement_tools/epa-ng-0.2.1-beta-dev/build’

make[2]: *** [CMakeFiles/Makefile2:956: libs/genesis/lib/genesis/CMakeFiles/genesis_lib_static.dir/all] Error 2

make[2]: Leaving directory ‘/Users/cota/picrust2-2.0.3-b/placement_tools/epa-ng-0.2.1-beta-dev/build’

make[1]: *** [Makefile:95: all] Error 2

make[1]: Leaving directory ‘/Users/cota/picrust2-2.0.3-b/placement_tools/epa-ng-0.2.1-beta-dev/build’

make: *** [Makefile:12: run_make] Error 2

I assume that the problem is with the genesis lib. I was not able to solve this issue so I decided to do a conda installation for both epa-ng and grappa:

conda install -c bioconda epa-ng
conda install -c bioconda grappa

After the installation, I ran again the picrust pipeline command:

qiime picrust2 full-pipeline --i-table mammal_biom.qza --i-seq mammal_seqs.qza --output-dir q2-picrust2_output --p-threads 1 --p-hsp-method pic --p-max-nsti 2 --verbose

Initially was looking good but I ended up with the following:

gappa - Genesis Applications for Phylogenetic Placement Analysis
INFO Converting given FASTA file to BFAST format…
INFO Resulting bfast file was written to: /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out/study_seqs_hmmalign.fasta.bfast
INFO Selected: Output dir: /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out/
INFO Selected: Query file: /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out/study_seqs_hmmalign.fasta.bfast
INFO Selected: Tree file: /Users/cota/picrust2-2.0.3-b/default_files/prokaryotic/reference.tre
INFO Selected: Reference MSA: /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/ref_seqs_hmmalign.fasta
INFO Selected: Automatic switching of use of per rate scalers
INFO Selected: Preserving the root of the input tree
INFO Selected: Specified model: GTR+G
INFO Rate heterogeneity: GAMMA (4 cats, mean), alpha: 1 (ML), weights&rates: (0.25,0.136954) (0.25,0.476752) (0.25,1) (0.25,2.38629)
Base frequencies (ML): 0.25 0.25 0.25 0.25
Substitution rates (ML): 0.5 0.5 0.5 0.5 0.5 1
INFO Selected: Reading queries in chunks of: 5000
INFO Selected: Using threads: 1
INFO

INFO Output file: /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out/epa_result.jplace

INFO 371 Sequences done!

INFO Time spent placing: 165s

INFO Elapsed Time: 172s

The following arguments were not expected: /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out --out-dir --fully-resolve /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out/epa_result.jplace --jplace-path

Run with --help for more information.

Error running this command:

gappa analyze graft --jplace-path /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out/epa_result.jplace --fully-resolve --out-dir /var/folders/0s/wsp9m4k901z87rljn7xs6p340000gv/T/tmplwxhia78/picrust2_out/intermediate/place_seqs/epa_out

Could some help me to understand why am I getting the unexpected argument and the error at the end? Thanks!

1 Like

Hi @Cotissima,
I am cc:ing @gmdouglas to see if he can help troubleshoot this issue with q2-picrust. Thanks for your patience!

Hey @Cotissima,

The issue is that specific versions of EPA-NG (v0.3.5) and GAPPA (v0.4.0) are required for the current version of PICRUSt2. These are meant to be installed automatically with conda - how did you install PICRUSt2 and did you run into any particular errors when doing so?

You could also check to see what versions of EPA-NG and gappa you installed to see if the version numbers match what I posted above - it’s possible that installing the appropriate versions with separate conda commands like you did before would resolve the problem.

Best,

Gavin

1 Like