18S_freshwater_>5um
The 16S rRNA genes for prokaryote diversity and the 18S rRNA genes for microeukaryote diversity were amplified via PCR. For eukaryote 18S rRNA genes the primer pair V8f (ATAACAGGTCTGTGATGCCCT) and 1510R (CCTTCYGCAGGTTCACCTAC) modified after Bradley et al. (2016) was used. For prokaryote 16S rRNA gene amplification the primer pair F319 (5-ACTCCTACGGGAGGCAGCAG-3) and R806 (5-GGACTACHVGGGTWTCTAAT-3) (Fadrosh et al., 2014) targeting the V3-V4 region was used. To increase read quality we used a dual multiplexing approach and a heterogeneity spacer of 210 bp length between the Illumina adapter and the forward/reverse primer sequence. PCR reactions were carried out in duplicates for each sample using the Q5 High-Fidelity polymerase (New England Biolabs, Ipswich, USA) according to the user manual and a final primer concentrating of 0.2 M and 15 ng DNA per reaction. Cycling conditions for 18S rRNA gene amplification were 98C 2 min [98C 15 s, 67C 20 s, 72C 20 s] x22, 72C 20 s and for 16S rRNA gene amplification 98C 2 min [98C 15 s, 63C 15 s, 72C 20 s] x22, 72C 20 s. In a second step PCR the Illumina tags were added in 15 cycles. Sequencing was performed on an Illumina MiSeq platform and a v2 500 cycles kit were used to sequence the PCR libraries by the company Microsynth, Switzerland.