Trim & trunc, and rarefaction depth

Hi,

I am analyzing four datasets from different organisms and comparing them against each other for the same treatment, so in order to account for unequal sequencing depth, I was wondering if the same values should be used for trimming and truncating the sequences in each data set? in that case, should I choose the values based on the worst quality dataset?
Additionally, when I need to sub-sample for diversity analysis, should it then be the same depth for all datasets?

Hi @Parix,

If you’re comparing ASVs fromt he same primers/same region, you need to use the same trunc length and the same trimming. I would use the worst values.

If you’re not using the same primer pair, you need to do close reference OTU picking to be able to make something like a reasonable comparison.

And then, as far as rarefaction goes, yes, you should choose the same depth. My suggestion there would be to calculate diversity on a merged table. It’s easy and quick to filter a distance matrix but a lot more time consuming to generate a new one.

Best,
Justine

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