16S rRNA gene seq human saliva 38
The 27F and 1492R universal primer set was used. The KAPA HiFi Hot Start DNA Polymerase (KAPA Biosystems) was used to perform 27 cycles of PCR amplification, with denaturing at 95 ?C for 30 sec, annealing at 57 ?C for 30 sec and extension at 72 ?C for 60 sec. Equimolar concentration were pooled and processed with the SMRTbell Express Template Prep Kit 2.0 (PacBio, USA), following the Procedure & Checklist - Amplification of Full-Length 16S Gene with Barcoded Primers for Multiplexed SMRTbell Library Preparation and Sequencing protocol (Part Number 101-599-700 Version 04, PACBIO). Primer annealing and polymerase binding steps were carried out, using the Sequel II Binding Kit 2.1 and the Sequel II DNA Internal Control Complex 1.0 (PacBio, USA). Sequencing was carried out using the Sequel II Sequencing Kit 2.0 (PacBio, USA) on the Sequel II PacBio system.