title: 16S rRNA gene seq. of human oral plaque: T2DM_P19
design: Genomic DNA was extracted using the QIAamp PowerFecal Pro DNA Kit (QIAGEN) and bead-beating. DNA quality (A260/280 >1.8) was assessed by NanoDrop and Qubit. Integrity was verified on a 0.8% agarose gel with SYBR Safe. The V3-V4 region of 16S rRNA was amplified using Illumina-adapted primers. Amplicons were generated in a two-step PCR using KAPA HiFi HotStart ReadyMix and dual indexing with Nextera XT Index kit. PCR conditions were 95C (3 min), 25 cycles of 95C (30 s), 55C (30 s), 72C (30 s), and final extension at 72C (5 min). Indexed amplicons were purified with AMPure XP beads, quantified using Qubit, and pooled. Fragment size (~630 bp) was confirmed on a TapeStation. Libraries were sequenced on Illumina MiSeq (v2 500-cycle kit, 2  300 bp).
library_name: T2DM_P19/2024/00820