16S rRNA Sequencing of Human Gut Microbiota
Genomic DNA was extracted from 200 mg of feces using the ZymoBIOMICS kit and assessed for purity  and integrity (1% agarose gel). The V4 region of the 16S rRNA gene (311 bp) was amplified with V4-520F/V4-701R primers, purified (AMPure XP), and dual-indexed (8 PCR cycles) using Nextera XT v2. Libraries were quantified (Qubit), size-checked (Bioanalyzer 2100; 388 bp), normalized to 1 nM, and sequenced on an Illumina iSeq100 (150x2).