The 454 SOP recommends that we subsample after generating a shared file but before classifying our OTUs. I don’t see mention of subsampling in the MiSeq SOP. After generating a share file, I used count.groups to check it out and the number of sequences in each group is different. Should I subsample before using classify.otu as in the 454 SOP?
If yes, I will need a subsampled list and count file, right?
I ran this command:
mothur > sub.sample(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.an.unique_list.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.pick.count_table, size=13355)
It generated several list files…should I use the one at 0.03?
Thanks.
Because the steps after forming clusters is the same for MiSeq and 454 we refer users to the 454 once they are ready for those steps.
See the section on “Shared file analysis” in the MiSeq SOP:
“Once you have a shared file you can do pretty much anything else you want in mothur, R, or whatever. While we work on re-organizing our SOP pages, why don’t you head over to the 454 SOP page and see where to go from here with alpha and beta diversity and population-based analyses?”
I saw that note about going to the 454 SOP but it seems to suggest to pick up at the point of alpha/beta diversity analyses. This is after the user has subsampled the shared file in the 454 SOP so it wasn’t clear to me if I should still subsample…?
Sure. The subsampled file is used in the metastats / lefse analyses.