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Old 06-16-2015, 06:56 AM   #4
Location: USA

Join Date: Mar 2015
Posts: 30

Thanks for the suggestions. I agree and my goal is quite simple:
I have a knock-out mouse with a special gut microbiota-associated phenotype. So I'd like to know the difference (bacterial species and abundance) of fecal microbiota between wild-type mice and my knock-out mice.
If there is no need to do de novo assemble, how would you suggest me to start (NGS coverage, depth and mouse numbers)? The host (mouse) DNA content will be excluded, I think.

Originally Posted by Brian Bushnell View Post
Metagenomes often have an exponential coverage distribution which makes it difficult to determine how much depth you need (other than "more"). Your calculation is fine if the species distribution is perfectly even, but it won't be. Of course, whether you do shotgun or 16s depends on what you are trying to accomplish... as does the number of replicates. Perhaps you could elaborate on your goal? If you just want to quantify abundances for various conditions, there's no reason to do denovo assemblies each time; just assemble once and use mapping to calculate abundance subsequently. It's possible that mouse gut bacteria already have good assemblies, in which case you wouldn't need to assemble at all unless you notice a large amount of reads not mapping to known gut bacteria.

For assembly, I'd suggest starting with a HiSeq lane (~100Gbp) at minimum. For mapping, you could use a tiny fraction of that.
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