How can I Know if my forward and reverse reads are demultiplexed or not?

Hi everyone!

I'm working with 16s sequences (i have forward and reverse in fastq.gz) time ago I imported the files to qiime2 with a manifest in phred 33v2, but I have a doubt because a don't know if my reads are demultiplexed or not, and i don't know if when I imported the data, there where demultiplexed.
(Photos below)

What i did exactly?

I had forward in format fastq.gz and reverse too, but I unzip reverse to only fastq (due to format "PairedEndFastqManifestPhred33V2" in tutorial says forward in format fastq.gz and reverse in fastq and i had asked before in forum ). Then I made a manifest file with sample Id, filepath for forward and filepath for reverse. After that I ran qiime tools import command an i generate a .qza file, then I ran qiime demux summarize command and i got the .qzv file,i guess the next step is THE denoising and merged with DADA2 but I think is missing the demultiplex step (I'm wrong? :thinking:)

Thanks for your help

Example of a raw sequence:

Hi @kevin_SalOrt,

Thanks for providing details on your analysis pipeline! There isn't a demultiplexing step in the commands you've included - I'll break down what you've done below and recommend some next steps:

  • Creating your manifest file and running qiime tools import provides you with a QIIME 2 Artifact that you can then use in subsequent commands. This is great! You'll want to use this .qza file when demultiplexing your reads.

  • Running qiime demux summarize will provide you with counts per sample for all of your samples, and generate interactive positional quality plots based on n randomly selected sequences. This should be run on data that has already been demultiplexed, so this won't do anything for you just yet.

You can either use q2-demux or q2-cutadapt for demultiplexing your reads (this will depend on whether or not your sequence data contains your barcodes). You'll want to use one of the paired end commands in the appropriate plugin for your data, since you are working with paired end reads. The docs pages for these two plugins can be found here for your reference:


Hope this helps! Cheers :lizard:

That's my doubt, I have forward files with barcodes on the header and reverse files with barcodes on the header too, but I don have a file with solo barcodes, as far as i understand, if you have forward, reverse and barcodes files, then the data is multiplexed, but if you have only forward and reverse files, then the data is demultiplexed and i can skip demultiplexing process. I'm i right?

So much thanks for your help :+1:

Hi @kevin_SalOrt,

Your reads can still be multiplexed if you have barcodes in sequence. q2-cutadapt can be used for demultiplexing reads with adapters in sequence (i.e. barcodes, primers, etc). You could run a quick test to see if your barcodes are still in your sequences - just doing a quick search within a handful of your files for the barcodes on your header.

Ultimately though, if you are unsure of whether or not your reads have been demultiplexed prior to any analysis steps on your end, you should contact your sequencing provider to confirm! :nerd_face:

Cheers :lizard:

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