16s rRNA Seq Human Gut Metagenome
The DNA was extracted from fecal samples using DNA extraction kits with a sample homogenization stage using solid-state microparticles and inhibitor removal (Nobias Technologies, Russia). The quantity of 16S rRNA gene copies in the isolated DNA samples was determined using pre-analytical phase quality control kits for metagenomic studies (Nobias Technologies, Russia). Amplification of the full-length 16S rRNA gene was performed with primers 27F (AGAGTTTGATYMTGGCTCAG) and 1492R (GGTTACCTTGTTAYGACTT) on a CFX96 real-time thermal cycler (Bio-Rad, USA). The resulting PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter Inc., USA). Amplicon quality was evaluated by electrophoresis in 1.5% agarose gel. Library preparation for sequencing employed NEB reagents, including NEBNext FFPE Repair Mix (M6630) for single-strand break and end repair, NEBNext End Repair/dA-Tailing Module (E7546), and NEBNext Quick Ligation Module (E6056) for adapter ligation. All enzyme-mediated intermediate stages of library construction were followed by purification using Agencourt AMPure XP magnetic beads. The concentration of the prepared 16S rRNA gene libraries was quantified with a Qubit fluorometer (Invitrogen, USA) using the dsDNA Quant-iT High-Sensitivity Assay Kit (Thermo Fisher Scientific, USA). Purified libraries were pooled in equimolar ratios according to their measured concentrations. Sequencing was performed with Oxford Nanopore Technologies reagents, including the Ligation Sequencing Kit (SQK-LSK109), Flow Cell Priming Kit (EXP-FLP002), and the Native Barcoding Expansion 96 kit (EXP-NBD196) for PCR-free multiplexing. Data acquisition was carried out on a MinION device equipped with an R9 series flow cell (FLO-MIN106). Basecalling was performed using Guppy software (version 5.1.13).
SUB15541410
H014K