PacBio 16S rRNA gene amplicon of lake water metagenome
Water samples were collected on Sterivex filters (EMD Millipore, Darmstadt, Germany) and extracted using QIAamp DNA mini kit (Qiagen, Hilden, Germany) while sediment samples were extracted directly using the NucleoSpin Soil kit (Macherey Nagel, Dren, Germany). The whole 16S rRNA genes was amplified using 27F (5-AGRGTTYGATYMTGGCTCAG-3) and 1492R (5-RGYTACCTTGTTACGACTT-3) primers using MyFiTM Mix (Bioline, London, UK) and the following PCR steps: 95C for 3 min, 25 cycles of 95C for 30 s, 57C for 30 s and 72C for 60 s with a final elongation step at 72C for 3 min. Concentration and quality of products were determined using a TapeStation 4200. Samples were purified with the Agencourt AMPure XP kit (Beckman Coulter, USA), sequencing libraries were prepared using the SMRTbell Template Prep Kit 1.0SPv3. The Sequel Binding Kit 2.0 (Pacific Biosciences, USA) was used to bind DNA template libraries to the Sequel polymerase 2.0. Sequencing data was collected in Sequel SMRT Cells 1M v2 with 600 min movie time on the Sequel system I (Pacific Biosciences, USA).
W_St_2_W2016_bc55