Plugin error from demux no sequences were mapped check your barcodes

The barcodes are in Golay. Any thoughts on why this is popping up? (qiime2-2020.8) Kyles-MBP:~ kyleharris$ qiime demux emp-paired \

–m-barcodes-file /Users/kyleharris/Desktop/CFMB2020/MetadataMicrositesandToxicology.txt \

–m-barcodes-column BarcodeSequence \

–i-seqs /Users/kyleharris/Desktop/CFMB2020/emp-paired-end-sequences.qza \

–o-per-sample-sequences demux.qza \

–o-error-correction-details demux-details.qza

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). If barcodes are NOT Golay format set golay_error_correction to False.

Debug info has been saved to /var/folders/6w/6zjxtzz12y5dkn40sxjg9mbm0000gn/T/qiime2-q2cli-err-butgz6vy.log

Hi @mudbugecology,

The error message has a suggestion,

I would try playing with those two flags to see what makes things align.

Best,
Justine

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Thank you, Justine. Do you mean to add these options both as parameters? I see something on this here: https://docs.qiime2.org/2019.10/plugins/available/demux/emp-paired/ --p-rev-comp-barcodes / --p-no-rev-comp-barcodes
If provided, the barcode sequence reads will be
reverse complemented prior to demultiplexing.
[default: False]
–p-rev-comp-mapping-barcodes / --p-no-rev-comp-mapping-barcodes
If provided, the barcode sequences in the sample
metadata will be reverse complemented prior to
demultiplexing. [default: False] Thank you for your help.

Hi @mudbugecology,

I mean I don’t know how your barcodes are sequenced, so my suggestion is that you try each alone, and then the two together until you find one configuration that works.

You could also try asking the people who sequenced for you what configuration they use for their golay barcodes.

Best,
Justine

1 Like

That worked when I tried the --p-rev-comp-maping-barcodes, but not with the --p-rev-comp-barcodes. Thank you for pointing me in the right direction and helping me interpret what the error is asking me to do. (qiime2-2020.8) Kyles-MBP:~ kyleharris$ qiime demux emp-paired --m-barcodes-file /Users/kyleharris/Desktop/CFMB2020/MetadataMicrositesandToxicology.txt --m-barcodes-column BarcodeSequence --i-seqs /Users/kyleharris/Desktop/CFMB2020/emp-paired-end-sequences.qza --p-rev-comp-mapping-barcodes --o-per-sample-sequences demux.qza --o-error-correction-details demux-details.qza
Saved SampleData[PairedEndSequencesWithQuality] to: demux.qza
Saved ErrorCorrectionDetails to: demux-details.qza

2 Likes

I’m glad you got it sorted out!

Best,
Justine

1 Like

Would there be a good reason to run the demux with both of these suggested parameters? --p-rev-comp and --p-rev-comp-mapping?

Hi @mudbugecology,

It depends on how your sequencing and metadata are set up. So, if you have reverse complemented barcodes and they’re reverse complemented in your metadata, then yes. I think UCSD had that set up for a while. In general, demultiplexing is one of the hardest places for people who don’t have access to the data and sequencing center to predict parameters because it really is sequencing center dependent.

Best,
Justine

Thank you, Justine. We used dfci.ilab for the sequencing and I have asked if they could also demux and check the sequencing reads with illumina software to compare with what I am seeing in our Q2 demux file since we should have more reads. I am running it again right now with both of the suggested parameters to see how it looks. demux.qzv (318.4 KB) This was created with the rev-comp-mapping-barcodes.

I’ll chime in here - ignoring Golay barcodes first - in order to match a read to a given sample, the barcode sequence has to exactly match the barcode in the sample metadata. If the barcodes are Golay barcodes, then there is a preliminary “error correction” step. The error correction only works when the barcode sequence is in the 5’-3’ orientation. So, if the barcode sequence is Golay, and 3’-5’, you’ll first have to reverse complement it in order to error correct (--p-rev-comp). Now, if you reoriented your barcode sequence, you’ll have to reorient your barcode sample metadata, in order to match them up (--p-rev-comp-mapping).

As @jwdebelius mentioned, there are many variations on this out there in the wild, but as long as you’re able to coordinate with your sequencing center to ascertain things like “what orientation are the barcode sequences in?” and “are my barcodes Golay barcodes?” then you should be set.

:qiime2:

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Thank you both. I reached out to the sequencing lab to verify, but using both parameters has provided a demux file that looks correct. Here is what I used:

(qiime2-2020.8) Kyles-MBP:~ kyleharris$ qiime demux emp-paired   --m-barcodes-file /Users/kyleharris/Desktop/CFMB2020/MetadataMicrositesandToxicology.txt   --m-barcodes-column BarcodeSequence   --i-seqs /Users/kyleharris/Desktop/CFMB2020/emp-paired-end-sequences.qza --p-rev-comp-barcodes   --p-rev-comp-mapping-barcodes   --o-per-sample-sequences demux.qza   --o-error-correction-details demux-details.qza
Saved SampleData[PairedEndSequencesWithQuality] to: demux.qza
Saved ErrorCorrectionDetails to: demux-details.qza
(qiime2-2020.8) Kyles-MBP:~ kyleharris$ qiime demux summarize  --i-data /Users/kyleharris/Desktop/CFMB2020/demux.qza --o-visualization demux.qzv
Saved Visualization to: demux.qzv
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