2.5. Decontamination pipeline
Contaminant species were detected and removed using the decontam R package.19 We used the most stringent hyperparameter value (P * = 0.5) for both frequency-based and prevalence-based contaminant identification of the isContaminant function. DNA concentrations for the frequency method were measured by qPCR and were obtained during library preparation. The scores from the frequency and prevalence methods were combined using the “minimum” approach. We performed decontamination in batches and the species identified as contaminants in any batch were removed. The species not identified as contaminants with decontam were further excluded if they met one of the following criteria: (1) The relative abundance of a species show an inverse correlation with DNA concentration20 (ρ < -0.2, P < 0.05, Spearman’s correlation); (2) The relative abundance of a species > 0.01% in at least one negative blank control.