Profiling Gut Microbial Diversity Using 16S rRNA Sequencing

DNA was extracted from feces using two different methods of cell lysis and pooled as previously described by Montoya-Ciriaco et al. (2020) (link). DNA quality was verified by electrophoresis through 1% agarose gels. Amplification of the V3–V4 region of the 16S rRNA gene was performed using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-ACHVGGGTATCTAATCC-3′) primers (Herlemann et al., 2011 (link)) modified with adapters for the Illumina sequencing platform. The thermal cycling conditions of PCR were as follows: denaturation at 95°C for 2‍ ‍min, followed by 28 cycles of denaturation at 95°C for 30‍ ‍s, annealing at 55°C for 30‍ ‍s, elongation at 72°C for 30‍ ‍s, and a final extension at 72°C for 5‍ ‍min. A negative control was included in each PCR to detect reagent contamination. PCR was performed in triplicate, pooled, purified using the FastGene Gel/PCR Extraction Kit (Nippon Genetics), quantified using a NanoDrop 3300 fluorospectrometer (Thermo Fisher Scientific) with the PicoGreen dsDNA assay (Invitrogen), and combined at equal molar concentrations. Sequencing was conducted by Macrogen with 300-bp PE MiSeq runs (Illumina). Raw sequence databases are available at the Sequence Read Archive (SRA) from the NCBI under the project number PRJNA816478.