2.3.3. Fecal DNA Extraction and NGS Analysis
LYSIS
Fecal samples were collected from participants using the fecal collection tube at week 0 and week 12, and immediately stored at −80 °C. Bacterial DNA was extracted from the fecal samples, which were warmed up to room temperature, using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) with certain modifications to the standard protocol. The fecal sample underwent centrifugation at 13,200 rpm for 10 min to eliminate the storage buffer, followed by lysis using Inhibit EX buffer. After homogenization, proteinase K and ethanol were added to the sample to obtain the processed supernatant. Subsequently, the supernatant was subjected to washing with a QIAamp spin column and elution with elution buffer. The concentration of extracted DNA was assessed using NanoDrop 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA), and a 10× dilution was performed with elution buffer.The gut microbiome library was generated using the standard V3–V4 region of the 16S rRNA gene. Polymerase chain reaction (PCR) amplification was performed with KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), and the resulting PCR products were purified using AMPure XP magnetic beads (Beckman Coulter Inc., Brea, CA, USA). The quality and quantity of the amplified PCR products were assessed using a Fragment Analyzer (Advanced Analytical, Orangeburg, NY, USA) and a Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), respectively. Subsequently, the constructed library was sequenced on a MiSeq platform (Illumina, San Diego, CA, USA) with paired-end reads (2 × 301 nt). Each sample was sequenced to generate at least 100,000 reads.Following data acquisition, the raw paired-end reads were subjected to trimming in accordance with the GreenGene database Version 13.8. Subsequently, the reads that successfully met the quality filters were allocated to operational taxonomic units (OTUs) based on a 97% similarity threshold. Sequences that appeared as singletons or failed to map to the database were removed from the dataset. Taxonomic classification was then performed following the annotation of OTUs. Alpha diversity, beta diversity, and top 7 phyla were employed. Alpha diversity (Chao1) analysis assessed the species complexity within each sample, while beta diversity (Bray–Curtis) analysis evaluated the distinctions among microbial communities.