Hello,
New QIIME2 user here 
! I am running across issues when attempting to demultiplex my sequences. I have been looking around the forum to see if anyone has had similar issues, but it looks like many people have the opposite problem, so I am hoping for some guidance as to what I am doing wrong. I'll try to keep this as brief as possible while giving all the details.
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I am running qiime2-2019.4 on a mac via conda.
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I was provided 3 files 1 forward, 1 reverse, and 1 barcode
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I have imported my files as EMP-paired end sequences following the protocol from the "Atacama Soil microbiome"
using the command:
qiime tools import **
--type EMPPairedEndSequences **
--input-path non-swapped **
--output-path end-sequences.qza -
I am able to import the files no problem, however, I am coming across an issue while demultiplexing.
I originally demultiplexed following the exact commands:
qiime demux emp-paired
--m-barcodes-file bbmetadata.tsv
--m-barcodes-column BarcodeSequence
--p-rev-comp-mapping-barcodes
--i-seqs end-sequences.qza
--o-per-sample-sequences bbdemux.qza
--o-error-correction-details bbdemux-details.qza
qiime demux summarize
--i-data bbdemux.qza
--o-visualization bbdemux.qzv
and received no error and all of my samples were accounted for, however, when looking at my demux.qzv file all of my samples had extremely low sequence counts <1,000...of course I got pretty worried.
I reached out to the lab that did the illumina Miseq library for me and they were able to demultiplex the samples with their illumina cassava program and all of my samples had above 40k sequences which of course a huge relief and fantastic news!
They confirmed that I should be importing as EMP-paired, however, that their barcodes are now forward read and I should eliminate -rev-comp-mapping-barcode from the demux command as the updated EMP primer set no longer requires it, however, when I do that I get the following error:
qiime demux emp-paired
--m-barcodes-file bbmeta.tsv
--m-barcodes-column BarcodeSequence
--i-seqs end-sequences.qza
--o-per-sample-sequences nonsw-demux.qza
--o-error-correction-details demux-details.qza
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).
Which is why originally I had assumed it needed the -rev-comp-mapping-barcode.....
To troubleshoot:
- I made sure my forward and reverse files were both equal in size ( just to be sure they downloaded correctly)
- I re-downloaded and re-imported to ensure I hadn't swapped files around, but that didn't work.
- I tried swapping the froward and reverse files incase they had been named incorrectly that also didn't work.
I have the demultiplexed files from the lab now, however, I would like to be able to troubleshoot this so I can handle this on my own in the future.
Additionally, I was using the demultiplexing step to subsample from the sequences by creating 3 different metadata files as we had 3 different projects all on one lane and it seemed like the easiest way to separate them out for analysis.
Where could I be going wrong?
Thanks in advance for your help and insight!
S
! I feel I should have caught that.
.