FIG 1.

Construction and evaluation of FL-ASV and FL-OTU expanded reference databases. (a) Preparation of FL-ASVs and FL-OTUs. Samples were collected from WWTPs and ADs, and DNA and RNA were extracted. Purified DNA or RNA was used for the preparation of primer-based and “primer-free” full-length 16S rRNA libraries, respectively. They were sequenced and processed bioinformatically to produce the FL-ASVs and FL-OTUs. (b) Mapping of V1-V3 amplicon data to the FL-ASV reference database, the FL-OTU expanded database, and commonly applied universal reference databases. ASVs were obtained from activated sludge and anaerobic digester samples and filtered based on their relative abundance before the analyses to uncover how well the databases cover the rare biosphere. The fraction of the microbial community represented by the remaining ASVs after the filtering (coverage) is shown as the mean ± standard deviation across plants. (c) Mapping of V1-V3 ASVs from Dutch WWTPs based on raw data from Gonzalez-Martinez et al. (36). For details, see Fig. S2a. (d) Mapping of V3-V5 ASVs from Canadian WWTPs, based on raw data from Isazadeh et al. (35). For details, see Fig. S2b.