Problems with demux command

Hi ,
I made the emp-paired-end-sequences.qza files and am running the demux command.

qiime demux emp-paired --i-seqs emp-paired-end-sequences.qza --m-barcodes-file sample_metadata.csv --m-barcodes-column barcodes_column.tsv --output-dir EMPPairedEndSequences
But qiime reports an error here: :tired_face:

There was an issue with retrieving column 'barcodes_column.tsv' from the metadata:
'barcodes_column.tsv' is not a column in the metadata. Available columns: 'BarcodeSequenceForward', 'BarcodeSequenceReverse', 'LinkerPrimerSequence', 'ReversePrimer'
I validated the sample_metadata file using keemei and there was no problem sample_metadata.csv (682 Bytes)
The barcodes column file is also attached barcodes_column.tsv (145 Bytes)
Can someone kindly help !!!! is it that the names in the 2 tsv files are not matching ....or do I have to keep only one set of barcodes in the sample_metadata file. :sweat: .

If you read the help documentation for that command (qiime demux emp-paired --help) you will see that:

  --m-barcodes-column MetadataColumn[Categorical]
                                  Column from metadata file or artifact
                                  viewable as metadata. The sample metadata
                                  column containing the per-sample barcodes.

So that parameter should be the name of the column in sample_metadata.csv, not the name of a file. The error message also recommends the fix:

So try one of those instead.

I hope that helps!

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:star_struck: Thanks again…so kind of you for your help.

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Hey @Nicholas_Bokulich…all the samples got demultiplexed using the help you rendered…except one where it says: Mismatched sequence ids: M03755:3:000000000-BFMTT:1:1101:22723:1748, M03755:3:000000000-BFMTT:1:1101:22723:1748, and M03755:4:000000000-C48N6:1:1101:18534:1592.
What can I do to remove this error. Kindly help. Do these samples have to be sequenced again ?


Hi @amit,
You definitely do not need to resequence these samples.

This means that those sequence IDs do not have a paired read. You must have done some type of pre-processing that removed the pairs for those reads (since all sequence headers should be present in both the forward and reverse sequence files).

It sounds like this is just a small number causing problems. You could manually remove those sequences before importing.

Good luck!

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