Where am I going wrong - filter features output empty (greengenes2)

[rosew]

Hi Daniel,
sorry for the slow reply!
switching to using the md5 version of the file did fix the issue - I had a bug in my pipeline that meant it wasn't using the correct output from that step. Because I was re-running a previous job it was using outputs from the previous run instead.
thanks again for your help!

[rosew]

@wasade
Hi Daniel,
sorry to disturb again but I've managed to get stuck again.
I would like to improve species level classification and my initial focus is on Bacteroides, Bifidobacteria and Phocaeicola.
I wanted to use fragment-insertion to add additional species to the phylogeny tree but it seems I need an artifact of type SeppReferenceDatabase.
From searching the forum I found this reply from you: "We are examining right now what is necessary to facilitate users placing their own fragments, but we do not have an ETA yet. That said, the current V4 representation in Greengenes2 at 90, 100, and 150 nucleotides in length is appreciable."

Is that referring to the issue I have or am I misunderstanding? If so, is that statement still current?

Is there another way forward to curate the sequence DB further while using the greengenes2 phylogeny rather than a pre-trained (or my own trained) classifier?

thank you!

[wasade (Daniel McDonald)]

Hi @rosew,

If the intent is to place additional ASVs, then yes the noted comment is correct.

It may not be possible to get reliable species level classification for the ASVs of interest. One thing that can be done is to examine the full length records of the enclosing clade in the phylogeny for the current genera / species of interest, and excise the corresponding variable region, and test whether the regions are sufficiently unique. We've done that, somewhat, on greengenes2.ucsd.edu for 515F/806R which will be noted if looking for an ASV or a species so that information may be readily usable here. But, it is important to note that even if the variable region appears unique in Greengenes2 2022.10 for a given species, that cannot be assumed to be definitive due to taxon sampling.

Best,
Daniel