Plug in error from demux..

[scho73 (Sungjoon Cho)]

Hello,
I am trying to run a pair-end read analysis using qiime2-2019.10 but have problem in demux step.
I ran following code
qiime demux emp-paired
--m-barcodes-file sample-metadata.tsv
--m-barcodes-column BarcodeSequence
--i-seqs emp-paired-end-sequences.qza
--o-per-sample-sequences demux.qza
--o-error-correction-details demux-details.qza

and results were

Plugin error from demux:
No sequences were mapped to samples. Check that your barcodes are in the correct orientation (see the rev_comp_barcodes and/or rev_comp_mapping_barcodes options).
Debug info has been saved to /var/folders/xx/pghwmr2n773df2n3kq_fl_s00000gn/T/qiime2-q2cli-err-1jtxvq36.log

Interestingly,
If I use following code with "qiime2-2019.1"

Then I got a good result.

I am not sure how to resolve the problem. could you please help me?
Thank you very much in advance for your help!

[ben (benjamin w.)]

Yeah, it maybe something with your barcodes.

Try running it with the options, as suggested:

rev_comp_mapping_barcodes
or
rev_comp_barcodes

[thermokarst]

Hello @scho73 !

@scho73

If I use following code with “qiime2-2019.1”

We added support for Golay error correction of barcodes in 2019.4, you are probably running into issues with the error correction step (which wasn't present in 2019.1, which is why it worked as expected for you).

As @ben mentioned above, you need to check the orientation of your barcode sequences, and the barcode identifiers in your sample metadata, both need to be in the same 5'->3' orientation. Taking a step back - do you have golay barcodes? If not, you should disable the error correction entirely.

:qiime2:

[ben (benjamin w.)]

I am going to sound like I am a broken record, but this support in 2019.4 confused the heck out of me and it changed my multiplexed runs. Once I figured this out, I had to implement rev_comp_mapping_barcodes in all my runs and it fixed all my problems. Ben