16S rRNA and ITS Amplicon Sequencing

DNA was extracted from saliva using the Mo Bio Ultraclean kit (Qiagen) and from dental plaque using the MasterPure Gram-Positive Purification kit with an 18-h incubation. DNA from stool samples was extracted using the MagAttract PowerSoil DNA kit on an Eppendorf epMotion 5075 Liquid Handling Workstation.
(iii) Library preparation and sequencing. For bacterial sequencing, we amplified the V3-V4 regions of the 16S rRNA gene using standard primers (48 (link)) with Illumina Nextera adaptors (Illumina). We profiled fungal communities using the ITS1 region (49 (link)), amplified using published primers (forward primer ITS1F and reverse primer ITS2R) (50 (link)) with adaptors for Illumina multiplexing and an adapted cycling protocol (51 (link)). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter) and multiplexed using the Illumina Nextera XT Index kit. Indexed 16S rRNA and ITS libraries were purified using AMPure XP beads and quantified using the Quant-iT Broad Range dsDNA assay kit (Thermo Fisher Scientific). Libraries were normalized and pooled with a 10% PhiX spike and sequenced on an Illumina MiSeq with a v3 kit. Negative controls were included on all sequencing runs, and a Candida glabrata positive control was included in ITS amplification and sequencing.