Fecal 16S rRNA Gene Sequencing Analysis

DNA was extracted using the PowerSoil^® DNA Isolation Kit (MO BIO, Carlsbad, CA) according to the manufacturer’s protocol. DNA quantification was performed with PicoGreen (Invitrogen, Carlsbad, CA) and DNA quality was evaluated using a Nanodrop spectrophotometer. Each sequenced sample was prepared according to the Illumina 16S Metagenomic Sequencing Library protocols. The 16S rRNA genes were amplified using the 16S V3–V4 primers: 16S Amplicon PCR Forward Primer 341F (5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and 16S Amplicon PCR Reverse Primer 805R (5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) (Herlemann et al., 2011).

gDNA (2 ng) was amplified using PCR and 5x reaction buffer, 1 mM of dNTP mix, 500 nM each of the universal forward and reverse PCR primer, and Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA). Cycle conditions for the first PCR were as follows: 3 min at 95°C for heat activation, 25 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C, followed by 5 min of final extension at 72°C. The PCR product was purified with AMPure beads (Agencourt Bioscience, Beverly, MA), and then 2 μL of the product was amplified using PCR for final library construction with the Nextera XT Index Kit v2 (Illumina, San Diego, USA). The cycle conditions for the second PCR were the same as those of the first PCR except that only 10 cycles were performed. The PCR product was purified with AMPure beads and the final purified product was then quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for IlluminaSequecing platforms) and evaluated for quality using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Paired-end (2×300 bp) sequencing was performed using the MiSeq™ platform (Illumina, San Diego, USA). In the preprocessing step, the adapter trimming, read assembly and OTU clustering were performed using fastp (v.0.19.3), FLASH (v.1.2.11) and CD-HIT-OTU, respectively (Magoc and Salzberg, 2011; Li and Chang, 2017; Chen et al., 2018). The final assembly was annotated by searching against the NCBI 16S ribosomal RNA sequence (Bacteria and Archaea) database using BLASTn (v.2.4.0) (Altschul et al., 1990; Sayers et al., 2021).