title: 16S metagenomic sequencing of water sample
design: The 27F: AGRGTTTGATYNTGGCTCAG and 1492R: TASGGHTACCTTGTTASGACTT universal primer set was used to amplify the full-length 16S rRNA gene from the genomic DNA extracted from each sample. Both the forward and reverse 16S primers were tailedwith sample-specific PacBio barcode sequences to allow for multiplexed sequencing. We chose to use barcoded primers because this reduces chimera formation as compared to the alternative protocol in which primers are added in a second PCR reaction. The KOD One PCR Master Mix (TOYOBOLife Science) was used to perform 25 cycles of PCR amplification, with initial denaturation at 95 C for 2 min, followed by 25 cycles of denaturation at 98 C for 10 s, annealing at 55 C for 30 s, and extension at 72 C for 1 min 30 s, and a final step at 72 C for 2 min. The total of PCR amplicons were purified with Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN) and quantified using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, USA). After the individual quantification step, amplicons were pooled in equal amounts. SMRTbell libraries were prepared from the amplified DNA by SMRTbell Express Template Prep Kit 2.0 according to the manufacturers instructions (Pacific Biosciences). Purified SMRTbell libraries from the pooled and barcoded samples were sequenced on a single PacBio Sequel II 8M cell using the Sequel II Sequencing kit 2.0.