title: 16S rRNA sequencing of chewing gum sample (week 9)
design: The conserved regions V3 and V4 (459 bp) of the 16S rRNA gene were then amplified using forward and reverse primers: 5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG 3 and 5- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C -3, respectively. Amplification was carried out using the KAPA HiFi HotStart ReadyMix PCR kit (KK2602) and the following PCR cycle: initial denaturation at 95 C for 3 min; 25 cycles of amplification (30 s at 95 C, 30 s at 55 C, 30 s at 72 C); and 5 min of extension at 72 C. Amplicons were mixed with Illumina sequencing adaptors and dual-index barcodes (Nextera XT index kit v2, FC-131-2001). Libraries were normalized and merged before the sequencing. Then, the pool containing indexed amplicons was loaded onto the MiSeq reagent cartridge v3 (MS-102-3003), spiked with 10 % PhiX control to enhance the quality of the sequencing. Finally, paired-end sequencing (2x300 bp) was carried out on the Illumina MiSeq sequencing system