title: Freshwater metagenome: 16S and 18S amplicon
design: The 16S and 18S regions were PCR-amplified, resulting in 464 bp and 576 bp fragments for the 16S and 18S, respectively. The primer sequences are listed in Supplementary Table S1 (35,36) and include frameshifts by random nucleotides to enhance the sequencing performance on Illumina platforms. The PCRs were performed for 16S amplicons as follows: 1x QIAGEN Multiplex PCR Master Mix, (catalogue no. 206145) and 0.5 M of each primer (the framshifted primers mixed in equimolar amounts). Thermocycling conditions consisted of an initial denaturation of 95 C for 15 min; 33 cycles of 95 C for 30 s, 60 C for 30 s, 72 C for 30s; and extension of 72 C for 10 min. The PCRs were performed for 18S amplicons as follows: 1x Phusion HF buffer (catalogue no. M0535L), 0.2 mM dNTPs (Promega catalogue no. U151B), 0.4 M of each primer and 0.5 U Phusion polymerase (catalogue no. M0535L). Thermocycling conditions consisted of an initial denaturation of 98 C for 30 s; 33 cycles of 98 C for 5 s, 60 C for 30 s, 72 C for 45 s; and extension of 72 C for 5 min. PCR products were cleaned using AMPure XP beads in a ratio of 0.8x per sample. The samples were indexed using the standard Illumina Nextera XT index kit and sequenced on an Illumina MiSeq machine in a single flow cell in paired-end mode with 300 bp-read-lengths at the Genetic Diversity Center (GDC), ETH Zrich.