16S rRNA V3-V4 amplicon sequencing of lesional jawbone from MRONJ patient 25
Libraries were prepared following the Illumina 16S Metagenomic Sequencing Library Preparation protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified from jawbone-derived DNA with the 341F and 805R primers carrying Illumina overhang adapters using KAPA HiFi HotStart ReadyMix (Roche). Amplicons were purified with AMPure XP beads (Beckman Coulter) and dual indices were attached with the Nextera XT Index Kit (Illumina). Indexed libraries were quantified on a Qubit fluorometer (Invitrogen) and sequenced on an Illumina MiSeq with the MiSeq Reagent Kit v3 in 2 x 300 bp paired-end mode. Lesional (necrotic) and adjacent macroscopically normal jawbone from the same patient were processed and sequenced as a matched pair.