QIIME2 2021.11 importing & demultiplexing files with forward & reverse barcodes

I am running QIIME2 2021.11 in a conda enviroment on Unbuntu. I am trying to import and demultpliex Illumina reads that have forward & reverse reads (R1 & R2) and two index (barcode) files (I1 and I2) because my sequences have both a forward and a reverse barcode - I am not sure if the barcodes are still in sequence, but even if they are not, I do not understand how to import the data into QIIME2 with two barcode files.

I've already tried importing this data into a MultiplexedPairedEndBarcodeInSequence file, and realized by reading this forum that demultiplexing this type of file with both forward and reverse barcodes is... a trial. My lab pipeline, for which I have only the barest of bones (not even a copy-paste script, which I will be building after this ordeal!) uses the EMPPairedEndSequences format, so it's possible... but how? I'm very much a novice, so thank you for any help you provide.

I don't have any code to report because I'm still stuck at step 0.5. Any help getting to step 1 (importing data) is appreciated.

Hey @carrollk2015,

Welcome to the :qiime2: forum!

The route you'll need to take to import/demultiplex these data will depend on a couple of things - I'd recommend reaching out to your sequencing provider and asking them the following questions:

  1. Are the barcodes combinatorial or redundant/unique?

    • If the barcodes are redundant/unique, this will be easier to deal with in QIIME 2 - you can simply ignore one of your index files and use the EMP protocol for importing your data.
  2. Are the barcodes in sequence?

    • If so, cutadapt can be used for demultiplexing.

Once you have those details, we can discuss the best approach for importing/demultiplexing your data! :lizard:

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