title: Metabarcoding 18S rRNA barcode gene V4 region: biofilm
design: A volume of 2-4 ml of each sample was centrifuged at 4C and 11.000 rpg for 5 min. The supernatant was removed and from the remaining pellet the DNA was extracted with the NucleoSpin Soil Kit (Macherey and Nagel) following the manufacturer instructions. Short areas of the hypervariable region V4 of the 18S rRNA gene and the rbcL plastid gene were amplified in separated target PCRs. For the 18S V4 region the Nextera primers DIV4for and DIV4rev3 were used after Zimmermann et al. (2011) with a modification for 300-bp paired-end sequencing for Illumina MiSeq following Visco et al. 2015. The rbcL marker was amplified using an equimolar mix of the modified versions of the Diat_rbcL_708F and R3 primers established by Vasselon et al. (2017). For each sample PCR was repeated for technical replication. Purification of the samples was performed with 25 ml aliquots of the amplicons with HighPrep PCR Clean-up System (Magbio Genomics). Indexing PCR on the purified samples to ligate a unique combination of tags to the 5 end of the primer, DNA quantitation and Illumia MiSeq v3 sequencing (300 bp paired-end reads) with 600 cycles were conducted at the Berlin Center for Genomics in Biodiversity Research (BeGenDiv) of the Berlin Brandenburg Institute of Advanced Biodiversity Research (BBiB).