I have been trying to process sequences generated from Argonne national lab, which should run the EMP. I had no issues importing as EMP-Paired-End, but demultiplexing either a) doesn’t work (generates the ‘can’t map samples’) or works, but after DADA2, examining the rep-seqs file reveals that the Linker Primer Sequence remains, but in the middle of the sequences? I tried using cutadapt after demultiplexing to remove the linker primer sequences, but this did not work, I assume because the Linker is in the middle and not on the ends of the seqs.
The person who emailed us the sequences said “Note that you no longer need to reverse complement the reads to demultiplex”. But when I leave these options out, the demux step does not work. I have tried the “no-golay-error-correction” as well as both rev-comp settings (mapping and non mapping) individually and together with no luck, the same result occurs each time. Again, since the Linker is in the middle of the sequences I believe this is a demultiplexing issue… Please provide advice as to how to proceed. Thanks, and below is an example of my code
qiime demux emp-paired --m-barcodes-file argonne_metadata.tsv --m-barcodes-column BarcodeSequence --p-rev-comp-barcodes --i-seqs argonne_jan2020.qza --o-per-sample-sequences argonne_demux_test.qza --o-error-correction-details argonne_demux-details_test.qza
I have tried
-rev-comp-barcodes and -rev-comp-mapping-barcodes
Here’s an example of a barcode - ATCAGGTAAACA
Here is what all the linker primer sequences look like- GTGTGYCAGCMGCCGCGGTAA
Oh I also tried cutadapt on the demultiplexed sequences (cutadapt demux paired) and put the same metadata column for forward and reverse barcodes, this also generated the same rep-seq result after DADA2.