High proportion of Unmapped Uniref90 reads (and very few KOs after regroup)

Hi all,

I’m a novice user of software like this and I’m trying to figure out why I’m getting such a low proportion of my reads assigned to Uniref90 / Metacyc pathways and also why when I regroup the genefamilies I end up with extremely few KEGG orthologs.

I started with DNA isolated from mouse stool, and had the sequences generated with a HiSeq (150bp single). I also filtered out reads from mice using kneaddata. After using Humann2, My Unmapped reads are somewhere around 88%. (I would love to post a portion of my data but I don’t know how to do that in-line with the text).

Any advice would be very helpful, thank you so much,

Sorry for the delayed response. HUMAnN 2.0’s defaults are probably too stringent for working with mouse stool. You could either 1) use HUMAnN 2.0 in UniRef50 mode (which will map more reads via translated search, but with unclassified taxonomy) or 2) use HUMAnN 3.0 alpha, which has better support out of the box for the murine gut.