RESCRIPt plug-in error - deprelicate after extracting reads for amplicon-region specific classifier

Technical Support
1

Hello,

I recently updated to qiime2-amplicon-2026.1 (linux version, installed on a local server).

I am in the process of processing, filtering and training a new Silva classifier for Silva 138.2.

I’ve been following the steps in Processing, filtering, and evaluating the SILVA database (and other reference sequence data) with RESCRIPt pretty much verbatim except with a different primer set (515F-926R).

When I get to the second dereplicate step

qiime rescript dereplicate \
--i-sequences silva-138.2-ssu-nr99-seqs-515f-926r.qza \
--i-taxa silva-138.2-ssu-nr99-tax-derep-uniq.qza \
--p-mode 'uniq' \
--o-dereplicated-sequences silva-138.2-ssu-nr99-seqs-515f-926r-uniq.qza \
--o-dereplicated-taxa silva-138.2-ssu-nr99-tax-515f-926r-derep-uniq.qza

I get the following plugin error.

Plugin error from rescript:

'LN680001.946678.948224'

Any ideas why this might be happening and how I might fix it so can press on with building the classifier? I’m uploading the log file as well. From looking around it seems like this might be because a reference sequence does or does have a matching entry in the taxonomy file.

qiime2-q2cli-err-0he1ds_s.log.txt (5.0 KB)

Here are the exact steps I have run

rescript-2026.1-steps-silva.txt (2.0 KB)

Also as an aside, after updating to this version of qiime2 I keep getting the following message with every command I run.

/home/username/miniconda3/envs/qiime2-amplicon-2026.1/lib/python3.10/site-packages/unifrac/_init_.py:9: UserWarning: pkg_resources is deprecated as an API. See https://setuptools.pypa.io/en/latest/pkg_resources.html. The pkg_resources package is slated for removal as early as 2025-11-30. Refrain from using this package or pin to Setuptools<81.

import pkg_resources

Is there a way that I can fix this to prevent this message from showing up everytime I run anything in qiime2?

Thank you!

1 Like
2

Hi @ctekellogg,

I just copied and pasted the commands you provided on my laptop, and everything worked successfully. So, I am not sure what might have happened on your end.

Is the error repeatable? Perhaps start from the beginning again? The taxonomy can be a super set of the sequences. So, that error often arises when a given taxonomy is not present in the taxonomy file, but present in the sequence file. Make sure there isn't a file mixup somewhere. :thinking:

Perhaps remove the existing QIIME 2 environment, and reinstall anew, then try again?

FYI, if you add --p-threads 8 to the dereplicate command, and --p-n-jobs 8 to the cull-seqs command it will run much quicker. If you'd like to know what options are available add --help to any qiime command.

On another note, if making an amplicon specific classifier I'd recommend:

qiime rescript get-silva-data ...
qiime rescript reverse-transcribe  ...
qiime feature-classifier extract-reads  ...
qiime rescript cull-seqs  ...
qiime rescript dereplicate ...
qiime feature-classifier fit-classifier-naive-bayes ...

That is, there is likely a chance that the extracted region itself is of good quality, compared to the full-length sequences. That is, you might remove "good" amplicon region data when running cull-seqs, and filter-seqs-length-by-taxon prior to extracting the amplicon specific region.

That tutorial, isn't meant to be an SOP, just a bunch of simple examples about what you can do with RESCRIPt. I just used the output from the previous step to keep the tutorial easy to follow. For example filter-seqs-length-by-taxon, is an example of making a more stringent full-length classifier. Feel free to alter the order, and or skip / add commands you think might be best. :slight_smile:

4 Likes
3

Thank you, @SoilRotifer !

I’ll give it a shot again (i hadn’t taken it all the way from the top again… had only tried rerunning that step) and see if everything fixes itself.

And, huge thanks for the recommended adjustments to the workflow for an amplicon specific classifier. I’ll incorporate your suggestions in my next run through.

Cheers!

4

This will be fixed in a future version of Qiime2!

For now, try running this in your terminal:
export PYTHONWARNINGS="ignore:pkg_resources is deprecated"

You can also add this to your .bash_rc or .zsh_rc file so it's persistent.

This will simply silence this warning, like you suggested.

1 Like
5

Ok. Great to know it isn’t an issue on my end. :sweat_smile:

Thanks for the tip on how to silence the warning! (i looked for a post on this but couldn’t find it if there is one. so apologies if there was already a notification about this circulating).

Thanks, @colinbrislawn !

1 Like