Golay Barcodes not being detected with demux

Technical Support
1

Hello everyone,

I am trying to demux a new dataset and am getting the following error:

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.

I have confirmed that I do NOT need to use the reverse complement options and if I set "golay_error_correction" to "False", the sequences DO demultiplex. Where I am confused, is that these should be Golay barcodes as they are the EMP 16S V4 primers/indexes. For example here is a barcode sequence from by fastq file which is the Plate 3 G5 index:

@VL00838:14:AAGG27FM5:1:2404:59018:56394 1:N:0:CCGGCCGCGTGC
CCGGCCGCGTGC
+
IIIIIIIIIIII

Does anyone know what might the issue be?

2

Hi Zach,

Can you post the full Qiime2 command you ran?

3

Sure. it was the following:

qiime tools import \
   --type EMPPairedEndSequences \
   --input-path fastq/ \
   --output-path import/emp-paired-end-sequences.qza

qiime demux emp-paired \
  --m-barcodes-file metadata/SR1_updated_barcodes.txt \
  --m-barcodes-column GolayBarcode \
  --i-seqs import/emp-paired-end-sequences.qza \
  --o-per-sample-sequences import/demux-full.qza \
  --o-error-correction-details import/demux-details.qza
4

Maybe it's related to this?

Yeah, that's surprising to me too! I wonder if this is a hold over from the original EMP methods. They are pretty old so there could be some surprises in there!

Could you try this on your data? Hit'em with the double reverse, and report back!

  --p-rev-comp-barcodes
  --p-rev-comp-mapping-barcodes
4 Likes
5

The double reverse worked like a charm! Interesting quirk!

3 Likes