Full-length 16S sequencing of JonstD2
Amplicons for the V1  V9 region of the bacterial 16S rRNA gene were generated with universal bacterial primers S-D-Bact-0008-c-S-20 (27F) (5-AGRGTTYGATYMTGGCTCAG-3) and S-D-Bact-1492-a-A-22 (1492R) (5-TACGGYTACCTTGTTACGACTT-3). Each PCR contained 9.75 l MQ water, 12.5 l 2x Terra PCR Direct Buffer, 0.5 l/0,625 U Terra PCR Direct Polymerase Mix (Takara Bio USA Inc.), 0.625 l of both forward and reverse primers (10 M) and 1 l DNA template. The PCR was performed with an initial denaturation at 98C for 2 minutes, 35 cycles of 95C for 1 min, 60C for 1 min and 68C for 3 min and a final extension at 68C for 10 minutes. Amplicons from each PCR reaction were inspected visually with agarose gel electrophoresis, purified with 1,8x Agencourt AMPure XP magnetic beads (Beckman Coulter Inc. USA) and quantified with Qubit 2.0 dsDNA Assay (Thermo Fisher Scientific, USA). Library preparation and sequencing was performed with protocol for Native barcoding amplicons SQK-LSK109 with expansions EXP-NBD104 and EXP-NBD114 according to the instructions from the supplier and loaded on MinION R9.4 flow cell (Oxford Nanopore Technology, Oxford, UK).