This question is good and interesting but at the same time no so practical to answer. There are some points which you can give thought for..

1) You should have consider this before extracting the DNA. You could have separated prokaryotic and eukaryotic cells by may be filteration. Ans sequence both separately.

2) your concern about the sequence depth is correct but it will just reflect the proportion you have in your samples.

3) I suppose, no one can answer your question about " % of my sequences would be bacterial, and/or what proportion bacteria make up in freshwater communities ", since its your sample. And this proportion is not universal that all samples have some kind of proportion. You may be estimate the proportion by counting the cell in cytometer (just a suggestion).

If you have samples left, you may consider this.

Hey Abhijeet,

thanks for your answer, highly appreciate it.

I actually didn't extract the DNA yet, I'm just in the planning process for my project. I am already planning to filter water samples to separate prokaryotic and eukaryotic cells, but thank you for supporting that idea!

But you're definitely right, maybe my main goal should be to reflect the proportion in my samples rather than covering prokaryotes and eukaryotes equally.

I'll think about it, thanks again!

Hello Christopher,

If you would like to co-extract the DNA from both prokaryotic and eukaryotic communities, I would suggest to determine the ratio of prokaryotes:eukaryotes by qPCR, for example. One reaction with specific primers for bacteria and another one for eukaryotes, should do the trick, I believe. At least, in theory.

The filtration and separation of prokaryotes from eukaryotes before DNA extraction might be technically possible but it will depended on the questions you want to answer on your project. If you want to address community structure, you might find some biases. However, if you're only interested in community composition and their genomes, it should be fine.

Good luck,

Marco

Thanks Marco for your answer,

determining the ratio of prokaryotes:eukaryotes using qPCR is definitely a smart way to figure out what's going on in your sample, I haven't considered that yet! That's a very valuable advice that I will keep in mind once I'm at that point of my project. Thanks again!