Zymo D6331 - rRNA operon sequencing
The library was prepared using the ONT 1D ligation Sequencing kit (SQK-LSK109, Nanopore) in accordance with the manufacturers protocol. In brief, barcoded DNA amplicons were pooled in equal molar amounts (total about 100~200 fmol) and then repaired using the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-Tailing Module (NEB) at 22C for 30min. End repaired sample was ligated with Nanopore adapter using the NEBNext Quick Ligation Module at RT for 10min. After every enzyme reaction, the DNA samples were purified using AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions. The final library was loaded onto Flongle flow cell (FLO-FLG001, R9.4.1) and sequencing was performed for ~30 h on a MinION MK1b and MinKNOW software (v19.12.5).