Repository of code for Frontiers manuscript:

https://doi.org/10.5061/dryad.v6wwpzh2b

We designed an experiment to quantify the potential bias of sample age (i.e. time since defecation) on fecal microbiota of a North American ungulate, where sampling takes place in late winter/early spring conditions, typically within a 2-4 day window. The purpose was to assess potential community compositional changes through time that might confound experimental observations and interpretation when samples come from an unknown timeline. We sampled fecal pellets from four Rocky Mountain elk (Cervus canadensis) non-invasively, but at the time of defecation, near Yellowstone National Park, Montana in March 2016. The longitudinal component of this fecal microbiota experiment was conducted in a forested plot near Evaro, MT beginning on the day of collection after transporting the fecal pellet samples in sterile whirl-pak bags on ice. In our design, sample age is controlled by repeatedly subsampling elk fecal pellets originally sampled from the same individual across day 0, 1, 3, 7, and 14, in triplicate. Thus, our hierarchical block experimental design captures compositional fecal microbiota change through time within four elk biological replicates (blocks) and estimates the variation within each block using three technical replicates for each sample/time combination.