16S rRNA seq day 223
Genomic DNA was extracted from the biomass pellets using the DNeasy PowerSoil Pro Kit (Qiagen, Germany) according to the manufacturer's protocol. The quality of the extracted DNA was verified on 2% agarose gel by electrophoresis at 100V for 30 min. 10l genomic DNA extract was shipped to LGC genomics GmbH (Berlin, Germany) for library preparation and sequencing on an Illumina Miseq platform with v3 chemistry with the primers 341F (5-CCT ACG GGN GGC WGC AG -3) and 785Rmod (5-GAC TAC HVG GGT ATC TAA KCC-3) (Klindworth et al., 2013). The Divisive Amplicon Denoising Algorithm 2 (DADA2) R package was used to process the amplicon sequence data according to the pipeline tutorial (Callahan et al., 2016). In a first quality control step, the primer sequences were removed and reads were truncated at a quality score cut-off (truncQ=2). Besides trimming, additional filtering was performed to eliminate reads containing any ambiguous base calls or reads with high expected errors (maxEE=2,2). After dereplication, unique reads were further denoised using the DADA error estimation algorithm and the selfConsist sample inference algorithm (with the option pooling =TRUE). The obtained error rates were inspected and after approval, the denoised reads were merged. Finally, the ASV table obtained after chimera removal was used for taxonomy assignment using the Naive Bayesian Classifier and the DADA2 formatted Silva v138.1 (Quast et al., 2013).