16S rRNA amplicon sequencing of a freshwater microbial community
Filtering of water samples and extraction of DNA using the standard protocol for Dneasy PowerWater Kit (Qiagen, Germany). Duplicate amplification of the 16S rRNA gene region V4: [515FB] GTGYCAGCMGCCGCGGTAAand [806RB] GGACTACNVGGGTWTCTAAT (Apprill et al., 2015) with the following conditions were pooled: Initial denaturation at 95 C for 2 min, 30 cycles of amplification (95 C for 15 s, 55 C for 15 s, 72 C for 50s) and a final elongation at 72 C for 5 min. After the second PCR, sequencing was performed using a MiSeq Reagent kit v3 (Illumina, USA)
MQ190412-49