How to set metadata file for dual-barcode demultiplexing

Hi thermokarst,

I would like to know if the dual-indexed sequences were demultiplexed by the bcl2fastq, for each sample, R1 reads have one index, and R2 reads have another index, so how should I fill in the index information into the sample-metadata? I will employ the Miseq PE300 for my samples. the V3V4 region is about 460 bp, R1 and R2 read are about 300 bp with one primer and one-index, respectively.

Hi @Decen,
I’m not sure if you are specifically asking about bcl2fastq or just looking for a way to demultiplex dual barcoded reads. If the latter, the most recent release supports this now through cutadapt.

Hi Mehrbod,

Thanks for your reply. I not looking for a demultiplexing method, I wanna know “how should I fill in the index information into the sample-metadata”, since for the dual-indexed suequences, the index 1 (in read 1) and index (in read 2) are different in sequence, for example, index 1 is ATCTGC, while index 2 is TTGCAG,and the QIIME2 uses the index 1: index 2 in sample-metadata.tsv to recognize the corresponding sequence. here I would like to know whether I need to fill the both indice in the sample-metadata. In the example of qiime tutorial, even if the sequences are dual-indexed, but the dual-index are the same, so in the sample-metadata, only one index sequence was filled out. Thanks a lot

Hi @Decen,
Sorry I misunderstood your original question. I’ve also moved this topic to its own thread.

If you follow the cutadapt link above you’ll see that the requirements are as follows

Parameters:
  --m-forward-barcodes-file METADATA
  --m-forward-barcodes-column COLUMN  MetadataColumn[Categorical]
                          The sample metadata column listing the per-sample
                          barcodes for the forward reads.           [required]
  --m-reverse-barcodes-file METADATA
  --m-reverse-barcodes-column COLUMN  MetadataColumn[Categorical]
                          The sample metadata column listing the per-sample

You’ll need to set these 4 parameters for dual-index demultiplexing. The metadata file can be any metadata file you are using however it will have to have at least 3 columns, your sample-id, forward-index, and reverse-index. The column names for your indices can be whatever you want as long as it it matches the requirements here. so for example:

sample-id forward-index reverse-index
example1 AATTCC… CCTTGG…

Hope that helps!

Hi Mehrbod,

Grazie!Thanks a million!

Cheers,

Decen

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