Hi,
I have multiplexed paired-end data for 384 samples generated by mixing two primers: ITS1 and the P6 loop of plastid DNA. Both the forward and reverse primers of each type were tagged with the same set of unique barcodes before PCR. Therefore, my sequences contain a combination of primer, barcode sequence, and DNA, which together make them unique for demultiplexing.
However, when running the code, I encountered the error: “Plugin error from cutadapt: The following samples have duplicate barcode pairs.” This makes sense, as both primers used the same set of barcode sequences.
Can anyone please suggest how I can demultiplex this type of data?
Am I right assuming that now you have barcode file in which for each unique barcode you have two samples listed?
If it is so, I would demultiplex all the samples by unique barcode (in the barcode file, there should be one sample for one barcode). Then, after demultiplexing, I would run cutadapt with enabled option "discard untrimmed" two times on the demultiplexed samples, one for each primers pair. Each time, sequences with no primers would be discarded, leaving only targeted sequences.