Error message while running qiime fragment-insertion sepp

I am using the PICRUSt tutorial mentioned here
For qiime fragment-insertion sepp command I had given my aligned-rep-seq.qza file and 99 otus aligned which I have downloaded from here: I have imported this database using the commands mentioned here, where I have used the import command for importing aligned sequences. This is the command line I have given in PICRUSt plugin for placing the ASV:

qiime fragment-insertion sepp --i-representative-sequences aligned-rep-seqs.qza
–p-threads 20 --i-reference-alignment 99_otus.qza
–i-reference-phylogeny rooted-tree.qza \
–output-dir tutorial_placed_out
I am getting the following error after I ran this command:
Parameter ‘representative_sequences’ received an argument of type FeatureData[AlignedSequence]. An argument of subtype FeatureData[Sequence] is required.

Following is the error log report:

Traceback (most recent call last):
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/q2cli/”, line 274, in call
results = action(**arguments)
File “”, line 2, in sepp
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/qiime2/sdk/”, line 199, in bound_callable
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/qiime2/core/type/”, line 301, in check_types
name, kwargs[name].type, spec.qiime_type))
TypeError: Parameter ‘representative_sequences’ received an argument of type FeatureData[AlignedSequence]. An argument of subtype FeatureData[Sequence] is required.

Could you please help me to resolve this issue.

Hi there,

The error means that the input sequences need to be unaligned - you are currently inputting sequences that are already aligned.

In addition, you need to place your study sequences into the reference tree / alignment specified in the tutorial otherwise you will run into downstream errors with q2-picrust2. In other words, the reference files you’re specifying are different from the reference files used by PICRUSt2. The commands to download the PICRUSt2 reference files are specified in the tutorial.



I already finished the tutorial this is my own dataset. So can i use the reference files mentioned in the tutorial to my data, Even with unaligned data I am getting this error:
Parameter ‘reference_alignment’ received an argument of type FeatureData[Sequence]. An argument of subtype FeatureData[AlignedSequence] is required.

I am having problem with PICRUSt. Based on the previous suggestions I have used the following commands:

qiime fragment-insertion sepp --i-representative-sequences rep-seqs.qza
–p-threads 20 --i-reference-alignment reference.fna.qza
–i-reference-phylogeny reference.tre.qza
–output-dir tutorial_placed_out

This worked fine. Now I am running the qiime picrust2 custom-tree-pipeline command. The following commands where used:

qiime picrust2 custom-tree-pipeline --i-table tutorial_placed_out/table.qza
–i-tree tutorial_placed_out/tree.qza
–output-dir q2-picrust2_output
–p-threads 25 --p-hsp-method pic
–p-max-nsti 2

While running this command, following error message came:
Error running this command: -i 16S -t /tmp/tmpslpe8dsa/placed_seqs.tre -p 1 -n -o /tmp/tmpslpe8dsa/picrust2_out/16S_predicted -m pic

Then the commands was re-ran using --verbose command this was the message:

File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/bin/”, line 4, in
import(‘pkg_resources’).run_script(‘PICRUSt2==2.0.3b0’, ‘’)
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/pkg_resources/”, line 661, in run_script
self.require(requires)[0].run_script(script_name, ns)
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/pkg_resources/”, line 1441, in run_script
exec(code, namespace, namespace)
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/PICRUSt2-2.0.3b0-py3.5.egg/EGG-INFO/scripts/”, line 149, in
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/PICRUSt2-2.0.3b0-py3.5.egg/EGG-INFO/scripts/”, line 115, in main
check_files_exist([args.tree, trait_table])
File “/home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/PICRUSt2-2.0.3b0-py3.5.egg/picrust2/”, line 326, in check_files_exist
raise ValueError("This input file was not found: " + missing_files[0])
ValueError: This input file was not found: /home/gene/sankar/miniconda3/envs/qiime2-2018.11/lib/python3.5/site-packages/PICRUSt2-2.0.3b0-py3.5.egg/default_files/prokaryotic/16S.txt.gz
Error running this command: -i 16S -t /tmp/tmp_qq5_hrr/placed_seqs.tre -p 1 -n -o /tmp/tmp_qq5_hrr/picrust2_out/16S_predicted -m pic

I understood the file 16S.txt gz is missing. Should I have to download this file separately? As PICRUSt installation was done without any errors I really couldn’t understand what is the actual problem?. Could you kindly help me to resolve this issue. If I have to post this as a new post kindly inform me.

Please can anyone help me with this problem. I am really stuck at this point. I have read many qiime2 forums but was not able to find the solution.

Hi can you help me with this error. I have posted this error in the qiime forum. I got a reply from Gavin Douglas. But nobody is replying my second error which I have posted in the same post as a second reply regarding another issue with same plugin. I am really stuck at this point.


This error is for a different parameter than what you posted about above:

Your first error was about representative_sequences, while the second was about reference_alignment. Either way, @gmdouglas has given you some really valuable advise! Please double check that everything is in order on your end. Thanks.

PS - I made your DM public - we are not able to provide private support.

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Thank you for the reply. yes both are different errors. first error i rectified. But the second error i could not figure out what is the actual problem. i need to resolve the second error, regarding the can you please give me some suggestions.


Hi Sree,

If you installed PICRUSt2 with bioconda I think this problem will be resolved if you follow the instructions on the qiime2 library page instead:

There is an issue with the bioconda installation currently which I’m working to resolve.

If you have already followed the instructions on the QIIME2 library page then I would recommend re-installing the tool and running “pytest” to test the installation after you have finished.



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Thank you for the reply. I have installed Picrust as metioned in the qiime2 library page as mentioned above. Should I have to re-install based on installation procedure mentioned here ? or shall I use bioconda and install the Picrust in qiime2-2018.11 environment? If I did like that will the qiime files work with Picrust2?


Hi Gavin,
This was the test results. I think everything looks good. I will re-ran my commands and update you.

tests/ … [ 20%]
tests/ … [ 47%]
tests/ … [ 55%]
tests/ … [100%]

============================================================================== 40 passed in 45.03 seconds



Hi Gavin,
Thank you for the suggestions. After reinstalling it worked.

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