Need/How/when to separate 16S and ITS on the same run (MiSeq)

Hello all,
My samples were run both 16S and ITS on the same run (MiSeq). I have imported the data (paired-end read) and got a .qza file. Also, I have created two different metadata files, one for 16S and another one for ITS.
I started demultiplexing for the 16S data, so I used the .tsv for metadata16S, and the .qza (which includes ITS data).
I wanted to check if I am doing the right thing, or if I need separate the data before importing it on Qiime2?
Thank you!

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Hi @helenaax2r! Assuming you used different barcodes for the 16S and ITS data, your strategy of demultiplexing with separate mapping files should work. After demultiplexing your 16S and ITS data, I recommend running qiime demux summarize on both of the .qza files to see how many sequences were successfully demultiplexed for each of your samples (it’ll also show you some quality score plots to help determine trimming parameters, e.g. for DADA2).

Let us know how it goes!


An off-topic reply has been split into a new topic: Demultiplexing multiple marker genes with overlapping barcodes

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Thank you, @jairideout! I did get the two different .qza files.
I will continue with the analysis. I do not expect any challenges regarding the data. I will let you know if I do!
Thank you so much for your help!

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