Plugin error from demux:

Hello I've run the following for de-multiplexing sequences:

qiime demux emp-single \
  --i-seqs emp-single-end-sequences.qza \
  --m-barcodes-file sample-metadata_old.tsv \
  --m-barcodes-column BarcodeName \
  --o-per-sample-sequences demux.qza \
  --p-no-golay-error-correction \
  --p-rev-comp-mapping-barcodes \
  --o-error-correction-details demux-details.qza

And this is the error I got:

Plugin error from demux:

 Invalid characters in sequence: ['1', '3', 'F', 'P', 'i', 'l']. 
 Valid characters: ['C', 'M', 'G', 'W', '-', 'H', 'T', 'K', 'S', 'Y', 'B', 'A', 'N', 'D', 'V', 'R', '.']
 Note: Use `lowercase` if your sequence contains lowercase characters not in the sequence's alphabet.

Debug info has been saved to /tmp/qiime2-q2cli-err-hux310iu.log

The log report:

Traceback (most recent call last):
  File "/home/kalonji/miniconda3/envs/qiime2/lib/python3.6/site-packages/q2cli/commands.py", line 329, in __call__
    results = action(**arguments)
  File "<decorator-gen-488>", line 2, in emp_single
  File "/home/kalonji/miniconda3/envs/qiime2/lib/python3.6/site-packages/qiime2/sdk/action.py", line 245, in bound_ca$
    output_types, provenance)
  File "/home/kalonji/miniconda3/envs/qiime2/lib/python3.6/site-packages/qiime2/sdk/action.py", line 390, in _callabl$
    output_views = self._callable(**view_args)
  File "/home/kalonji/miniconda3/envs/qiime2/lib/python3.6/site-packages/q2_demux/_demux.py", line 255, in emp_single
    barcodes, rev_comp_mapping_barcodes)
  File "/home/kalonji/miniconda3/envs/qiime2/lib/python3.6/site-packages/q2_demux/_demux.py", line 229, in _make_barc$
    barcode = str(skbio.DNA(barcode).reverse_complement())
  File "/home/kalonji/miniconda3/envs/qiime2/lib/python3.6/site-packages/skbio/sequence/_grammared_sequence.py", line$
    self._validate()
  File "/home/kalonji/miniconda3/envs/qiime2/lib/python3.6/site-packages/skbio/sequence/_grammared_sequence.py", line$
    list(self.alphabet)))
ValueError: Invalid characters in sequence: ['1', '3', 'F', 'P', 'i', 'l'].
Valid characters: ['M', 'G', '.', 'S', 'B', 'D', 'K', 'R', 'W', 'C', 'T', 'A', '-', 'N', 'H', 'V', 'Y']
Note: Use `lowercase` if your sequence contains lowercase characters not in the sequence's alphabet.

Any assistance will be highly appreciated.

Hi @kalonji_08,

Thanks for your patience here!

To better understand where this issue is coming from, can you please provide the following:

  1. The version of QIIME 2 you are using
  2. Run the following command and copy/paste the output:
    qiime tools validate emp-single-end-sequences.qza
  3. A snippet of the first few lines of your single end sequences, to make sure the file formatting isn't malformed in any way

Thanks! :qiime2:

Hi Liz Gehret
Sorry for the late response

1.The version of QIIME 2 you are using
q2cli version 2020.8.0
2. Run the following command and copy/paste the output:
qiime2ITS1$ qiime tools validate emp-single-end-sequences.qza
Result emp-single-end-sequences.qza appears to be valid at level=max.
3. A snippet of the first few lines of your single end sequences, to make sure the file formatting isn't malformed in any way

Thank you very much for your assistance

Hi @kalonji_08,

Thanks for providing those details! It does look like you are using an older version of QIIME 2 - I'd recommend installing our latest version, just to ensure that problems aren't arising from using an older release version. If you'd like to do that, you can follow the installation instructions here.

Can you also provide me with the first few lines of your barcode sequences? After looking over the traceback you provided, this is the error message that stands out (which seems to be related to the barcodes within your file):

Thanks!

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Hi @lizgehret
Thank you very much i will try that and let you know how it goes. Thank you so much for your time and assistance

1 Like

Hi @lizgehret
I hope you are well
i have installed a new version of Qiime2 (2021.8)
I'm still getting the same error

i have attached a screen shot of the Barcodes
Thanks

Hi @kalonji_08 - @lizgehret will get back to you soon, thanks for your patience.

2 Likes

Hi @thermokarst Thank you very much

1 Like

Hi @kalonji_08,

Thanks for your patience, and for providing those follow up details! Glad you were able to update your version of QIIME 2 successfully. The screenshot of your barcodes looks normal (no strange characters, etc) - I'm wondering if there is potentially a row (or rows) somewhere within your barcodes file where all of the columns have somehow been mixed up. Please see the error message from your original post below:

This essentially means there were invalid characters found within your barcodes file. If possible, I'd do a quick search within that file for the characters included in the error message above (1, 3, F, P, i, l) to see if there are any rows where your data has somehow been malformed or the data has been placed in the wrong columns. Let me know what you find!

2 Likes

Hi @lizgehret I have message the with a link to download the barcode and have a look if possible.
I tried going through the file but i didn't see anything.
Thank you for your time and assistance

Hi @kalonji_08,

Thanks for sharing that data with me - I think I've found the source of the problem here. I took your sample-metadata.tsv file and ran qiime metadata tabulate, and then viewed the output.qzv file in QIIME 2 View, just to get a better idea of what your barcodes file looked like. Here is the visualization of first row of data:

You've used BarcodeName as your barcodes column, which doesn't contain valid barcode sequence characters. I suspect you may be meaning to use the BarcodeSequence column, which contains the actual sequences, vs those names/identifiers.

I'd try re-running qiime demux emp-single with --m-barcodes-column set to BarcodeSequence - this should address that error message you've been running into!

Cheers :lizard:

1 Like