2.4. Analysis of simulated and observed coefficient of variants (CVs)

Fastq data from the 10 repeated replicates of each sample were merged, randomly re-sampled according to the original data sizes (total number of reads), and analyzed by the CLARK-based pipeline. The CVs were calculated based on the read numbers mapped to each microbe within each re-sampled replicate. This above process was repeated 10 times to obtain a total of 10 simulated CVs for each microbe. The average of these simulated CVs represented the CV derived from variations in data size. A linear regression model was used to evaluate the contribution of these CVs to the observed overall CVs. In addition, we used a linear mixed model to further evaluate whether the sequencing platform, library method, and class of microorganism affected the observed CV. The formula of the linear mixed model was defined as:

Cv_observed ∼ Cv_datasize + Library Prep + Microbial Class + Platform + (1+Cv_observed|Center) where Center was a random effect, and the read depth CV (Cv_datasize), library preparation method (Library Prep), microbial class, and sequencing platform (Platform) were fixed effects.