16S rRNA full-gene sequencing the oral microbiome in adolescents in relation to smoking: buccal swabs
Buccal swabs were collected with sterile and DNA-free cotton swabs from the inside of each cheek. DNA was extracted with a customized protocol under DNA-free conditions using the QIAamp Blood Mini Kit with an additionl mechanical cell disruption step. Bacterial full-length 16S rDNA genes were amplified using the bacteria-specific primer pair 27F (AGRGTTYGATYMTGGCTCAG) and 1492R (RGYTACCTTGTTACGACTT). Preparation for sequencing, including barcode ligation, followed the PacBio protocol Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Overhang Adapters for Multiplexing Amplicons (version 06, Jan 2021) using chemicals of the SMRTbell Express Template Prep Kit 2.0. Samples were sequenced using PacBio Sequel CCS technology. Sequencing was performed with the Sequel Binding and Sequencing kits 3.0 according to PacBio-recommendation for full-length 16S sequencing.