Metabarcoding of biofilm eukaryotic communities
DNA was extracted from 0.2 g of biofilm using the DNeasy PowerBiofilm Kit (Qiagen, Hilden, Germany) following the manufacturers instructions. DNA quantity and quality were determined photometrically with a NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). To assess the prokaryotic diversity, the hypervariable V4 region of the 16S rRNA gene was targeted using the primer pair 515Fm (5-GTGYCAGCMGCCGCGGTAA-3) and 806Rm (5-GGACTACNVGGGTWTCTAAT-3, Walters et al., 2016). The eukaryotic communities were investigated using the hypervariable V4 region of the 18S rRNA gene as metabarcode and the primer pair V4-TAR-EUK_F (5CCAGCASCYGCGGTAATTCC-3) and V4-TAR-Euk_R (5- CAGACTTTCGTTCTTGATYRA-3, (Stoeck et al., 2010). Both PCR protocols employed an initial activation step at 98C for 30 s, followed by 26 cycles consisting of 98C for 10 s, 63C for 30 s and 72C for 30 s, and a final extension for 5 min at 72C. To minimize PCR bias, three individual PCR reactions for each of the samples were prepared and pooled afterwards. Library preparation followed Next Ultra DNA Library Prep Kit for Illumina (NEB, United States). The quality of the final libraries was assessed with an Agilent Bioanalyzer 2100 system. Libraries were sequenced on an Illumina MiSeq platform, generating 2  250 bp paired-end reads by SeqIT, Kaiserslautern, Germany.