2.6 | Diet composition computation
The analytic protocols of OTUs refinement were performed by way of referring to the description in Shutt et al. (2020). First, only OTUs belonging to the plant kingdom were considered as possible food items. Uncorrelated nonfood OTUs (e.g., fungi, bacteria, or Metazoa) were removed. All OTUs identified as environmental contamination (e.g., algae) were removed. Then, all OTU reads with fewer than 0.01% of the total were removed as possible false positives. The above steps reduced the number of sequence reads from 2,463,964 to 1,426,517 containing 92 OTUs. Finally, all remaining OTUs belonging to the same best-match taxon (at the genus or species level) were merged (remaining n = 56).
Plant taxa and their respective numbers of sequences in each sample were summarized. Samples from the stall-feeding treatment were excluded from analyses. Multiple metrics were used to interpret the diets of Tan sheep. Those considered here are Occurrence data and Read abundance data (Deagle et al., 2019).
Occurrence Data, including percent frequency of occurrence (%FOO), percent of occurrence (POO), and weighted percent of occurrence (wPOO), were calculated as
\begin{equation} {\%FOO}_{i}=\frac{1}{S}\sum_{k=1}^{S}{I_{i,k}\times 100\%},\nonumber \\ \end{equation}\begin{equation} \text{POO}_{i}=\frac{\sum_{k=1}^{S}I_{i,k}}{\sum_{i=1}^{T}{\sum_{k=1}^{S}I_{i,k}}},\nonumber \\ \end{equation}\begin{equation} \text{wPOO}_{i}=\ \frac{1}{S}\sum_{K=1}^{S}{\frac{I_{i,k}}{\sum_{i=1}^{T}I_{i,k}},}\nonumber \\ \end{equation}
where T is the number of plant items, S is the number of samples, and I is an indicator function such that \(I_{i,k}=1\)if plant item i is present in sample k , and 0 otherwise. FOO analysis was based on a 0.01% threshold (Alberdi, Aizpurua, Gilbert, & Bohmann, 2018).
Read abundance data using the sequence counts were used to calculate relative read abundance \((\text{RRA}_{i})\) for plant item i as follows:
\begin{equation} \text{RRA}_{i}=\frac{1}{S}\sum_{k=1}^{S}\frac{n_{i,k}}{\sum_{i=1}^{T}n_{i,k}}\times 100\%,\nonumber \\ \end{equation}
where \(n_{i,k}\) is the number of sequences of plant item i in sample k .