Concatenating data from the same individual with different barcodes

Hi, I have a 16s dataset where we sampled all individuals twice, and then prepped these with different barcodes. I assume it’s not OK to analyzed these as independent samples, but now that we have our data back it’s unclear to me how to concatenate the data from the same individual, and at what step.

Is it after or during de-multiplexing? Can I simply give the same sample ID to multiple barcodes in the metadata file?

Thanks for the help!

Hi @Ben_Titus,
You can merge these replicates into single samples after denoising/clustering sequences. Use the group action to group these.

So demultiplex separately (I am assuming these were sequenced on a single run, but let me know if not), then denoise, then group replicates before proceeding with other analyses. Before grouping you will probably want to run some other analyses anyway just as a sanity check that your replicates are yielding similar results (e.g., just running beta diversity to see if they cluster more or less together is a good sanity check before grouping).

You will need to have two sample metadata files. First, one to demultiplex and group replicates, which will look like this:

#SampleID BarcodeSequence Sample
s1A ACGT s1
s1B AAGT s1
s2A AGGT s2
s2B AAAT s2

You will group on the “Sample” column in that file (which you can name whatever you want, just specify the name of the column to qiime feature-table group). Your samples will then be grouped, and the merged samples will be labeled with whatever the labels in the “Sample” column are: in this example, s1 and s2.

So then you will need to make a new sample metadata file that has those sample IDs as the row labels, and contains the rest of the sample metadata that you actually care about for analysis. Something like this:

#SampleID Species Time
s1 Human 1
s2 Toad 2
s3 Peanut 3

I hope that helps!

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