Problem in demux

Hi!

I have an issue in demux process.

I used following flags:

qiime demux emp-paired \
>   --m-barcodes-file sample-metadata.tsv \
>   --m-barcodes-category BarcodeSequence \
>   --i-seqs emp-paired-end-sequences.qza \
>   --o-per-sample-sequences demux

which give me demux.qza of only 324 KB (FYI, emp-paired-end-sequences.qza is 5GB).

If I make qzv file from the file using following flags,

qiime demux summarize \
>   --i-data demux.qza \
>   --o-visualization demux.qzv

There is error message saying,

Plugin error from demux:

  len() of unsized object

Debug info has been saved to /var/folders/xx/pghwmr2n773df2n3kq_fl_s00000gn/T/qiime2-q2cli-err-08dao5a2.log.

Could you please let me know how to solve the problem?
Thank you very much in advance for your help.

Have a great day

Hi @scho73!

I suspect demux wasn’t able to match the barcodes. It’s likely that they are just reverse complemented, so you should be able to add this flag:

--p-rev-comp-barcodes

Let me know if that works!

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Hi ebolyen!
Thanks for kind response.

I tried reverse complemented flag as followings,
qiime demux emp-paired \

–m-barcodes-file sample-metadata.tsv
–m-barcodes-category BarcodeSequence
–i-seqs emp-paired-end-sequences.qza
–o-per-sample-sequences demux
–p-rev-comp-mapping-barcodes

But, the resultant demux.qza is 18KB which is even smaller than previous qza file.
And If I use following flags for creating qzv file,

qiime demux summarize \

–i-data demux.qza
–o-visualization demux.qzv

it still gives same error messages saying

Plugin error from demux:

len() of unsized object

Debug info has been saved to /var/folders/xx/pghwmr2n773df2n3kq_fl_s00000gn/T/qiime2-q2cli-err-fp_krdda.log.

I also checked the validity of metadata using keemei and it said my metadata is fine.

For your information, I downloaded the sequencing data (i.e, barcode, forward and reverse sequence data) from our collaborator’s cloud and I changed file names to “barcodes.fastq.gz”, “forward,fastq.gz” and “reverse.fastq.gz” so that I can exactly follow atacama soil tutorial.

Please let me know if you have any idea to solve the problem.
Thank you very much for your help!

Hi,

Never mind.
I solved the problem.
That was because my sample name has underscore which interrupt the demux.
I removed the underscore and retried it and it worked!

Anyway, thanks for your kind help.
Thank you!

Glad you were able to get it working!

I was fairly certain we had squashed that :bug:, Are you using QIIME 2017.8 (or 2017.9 for that matter)?

Hi, I was using QIIME 2017.6.
But after that, I updated it to QIIME 2017.9 and have not tried with underscored sample name yet.

1 Like

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