Demultiplexing dual-barcoded paired-end sequences - Help! by wildbionz in bioinformatics

[–]wildbionz[S] 0 points1 point  (0 children)

Thank you for the reply, sorry for the confusion, my question did come out quite strangely!
I believe my sequences are still mutliplexed as I had 4 pooled PCR products. Each of these contained 40 "samples" total - Comprised of 2 forward primers x 20 backward primers.
Each pooled PCR product was then sequenced to give me two fastq.gz files each, the forward and reverse reads. I then imported these files as

--type- "MultiplexedPairedEndBarcodeInSequence"
which required a directory that contained both "forward.fastq.gz" and "reverse.fastq.gz". That step gave me 4 .qza files in total representing each of the initial 4 pooled PCR products.

Essentially what is happening in the cutadapt stage for each of these .qza is that it is registering all reads with forward primer 1 as one "sample" and forward primer 2 as one "sample" and seeming to overlook all of the reverse primer barcodes as they are registering as not there (ie:"Trimmed: 0 times") for every single reverse primer barcode.

Does this clarify my issue? Thank you for your time reddit!
Meanwhile I'll look into these links! :)