title: Metabarcoding of prokaryotic biofilm communities
design: DNA was extracted from freeze-dried biofilms using the DNeasy PowerBiofilm extraction kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The DNA amount in each extract was quantified using a Qubit fluorimeter (Qubit Fluorometer Invitrogen) and its quality being checked by a Nanodrop (NanoDrop ND-1000 Spectrophotometer peqlab). Aliquots of the DNA extracts (see above) were used to study the diversity, composition and structure of the biofilm communities by metabarcoding. For this purpose, 50 ng of DNA was used to amplify the hypervariable V4-region of the microeukaryotic 18S and prokaryotic 16S rRNA gene using the primer sets V4-TAR-EUK_F (5CCAGCASCYGCGGTAATTCC-3) and V4-TAR-Euk_R (5- CAGACTTTCGTTCTTGATYRA-3, (Stoeck et al., 2010), and 515Fm (5-GTGYCAGCMGCCGCGGTAA-3) and 806Rm (5-GGACTACNVGGGTWTCTAAT-3, Walters et al., 2016), respectively. Both PCR protocols started with an 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 amplification bias, three independent PCR amplifications were performed per sample and subsequently pooled prior to library preparation. Libraries were constructed using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, USA) following the manufacturers protocol. Library quality and fragment size distribution were assessed using the Agilent 2100 Bioanalyzer system. Paired-end sequencing (2  250 bp) was carried out on an Illumina MiSeq platform by StarSeq (Mainz, Germany).