Demultiplexing multiple marker genes with overlapping barcodes

[Stream_biofilm (Danny W)]

I have a similar data set however each sample has an individual barcode but not each amplicon (16S and 18S). I can use the original primer used to amplify the gene that is still on the sequences to parse them out, but I’m not sure how to do this with the demultiplexing tools already in place.

[jairideout (Jai Ram Rideout)]

Hi @Stream_biofilm! Are all of your data in a single sequencing run? If so, we don’t have QIIME 2 tools to separate your sequences by marker gene if the barcodes are re-used across marker genes. You’ll probably need to do some custom scripting/coding to separate your data using the primers. Perhaps there’s a tool that already accomplishes this – it might be possible with QIIME 1 but I’m not certain (you might try checking on the QIIME 1 Forum).

[jairideout (Jai Ram Rideout)]

Just noticed this is a duplicate topic. Locking this topic and moving further support here:

Multi-gene amplicon sequences with dada2

Hello, We amplified four gene markers (16S, 18S, 23S, rbcL) for each sample and combined all four markers before adding the barcode and sequencing. I now have demultiplexed sequences but each sample still contains sequences of all four amplicons. Do I need to create individual files for each sample and marker before processing with dada2? Each amplicon is a different size but I figure I can use the primer to parse them out. Is there anything in place to assist me with this? Thanks, Danny