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:
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. .
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 ?
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.