Fig. 3

a Schematic benchmarking of the main steps of metabarcoding pipelines using an RMQS subsample. For all pipelines, purple color represents the chimera removal step, green represents the clustering step (or the ASV definition for BIOCOM-PIPE ASV), and dark orange represents ASV/OTU richness after rarefying the number of sequences for each sample to the smallest sample size. For BIOCOM-PIPE, the light orange color represents the specific post-clustering step. For FROGS, the light blue color represents a specific filtering step. For mothur, the red color represents the pre-processing step. Finally, the dark gray color represents the sequences to be removed for the next step. b Distribution of singletons defined with FROGS and mothur into OTUs defined with BIOCOM-PIPE. All singletons defined with FROGS (in pink) and mothur (in blue) were checked to define to which class of OTU they were associated with BIOCOM-PIPE. Six classes of OTUs were defined (dominant > 0.5% of the total sequencing depth; 0.5% > medium ≥ 0.05%; 0.05% > minor ≥ 0.005%; 0.005% > rare ≥ 0.0005%; rarest > 0.00005%; and singletons with one sequence into one OTU)