Dual Index Demultiplexing Workaround

Hi all,

I have paired-end dual indexed reads that I am wanting to demultiplex - 4 files total (read1, read2, index1, index2). From my understanding, Qiime2 does not support dual index demultiplexing, but Qiime1 does. From the Qiime1 documentation under “Two index/barcode reads and two fast reads” in the “Preparing raw Illumina data in different formats for use with Qiime” section, the following script will output a single barcode file:

extract_barcodes.py --input_type barcode_paired_end -f index1.fastq -r index2.fastq --bc1_len 6 --bc2_len 6 -o parsed_barcodes/

It goes on to say that the output barcodes.fastq files can be used for downstream processing, and that the reads1 and reads2 files can be ignored. Am I to interpret this as meaning I can use the single barcode file output from Qiime1 in Qiime2 like I would with non dual-indexed files for demultiplexing? I’m not sure how to interpret the part about reads1 and reads2 files being ignored because it is in the context of downstream processing. Not sure if this is the right place to post this, but any help or advice would be greatly appreciated! Thank you!

Hi @jrede!

I’m not 100% certain, but I think the answer is “yes.” I’m not sure what that QIIME 1 script outputs specifically, so you might need to experiment with this a bit - keep us posted. FWIW, QIIME 2 does have support for certain kinds of DI setups - when the barcode is inline in the sequencing data you can use q2-cutadapt to demux.

Keep us posted!